Abstract
Over the last several decades, investigations of Earth’s subsurface and other extremely low-biomass systems have refined our understanding of the environmental limits of life, driven by methodological advances that permit agnostic life detection of biology and their respective physical biosignatures and chemical biomarkers. These advances enable mission concepts centered on microbiological processes that facilitate identification of both active life and preserved biosignatures through measurements of metabolism and associated biochemical markers that, on Mars, are more likely to be retained below the surface. Terrestrially, although biological processes can exert a significant influence on Earth’s crust, the presence of habitable conditions does not necessarily imply the existence of cellular life. The Viking missions constituted the first direct life-detection experiments on Mars but produced equivocal outcomes, prompting subsequent exploration strategies to emphasize surface habitability rather than direct biological testing. Leveraging progress in subsurface microbiology and planetary exploration, we contend that Mars missions are now poised to shift toward direct tests for extant microbial activity in the subsurface, with metabolic processes serving as a broadly applicable indicator of life.
Introduction
Mars exploration has largely focused on identifying ancient or extant habitable environments, with particular emphasis on sedimentary materials formed through past aqueous activity. Lacustrine deposits, deltaic systems, and fluvial channels have provided critical insight into the planet’s geologic and climatic evolution. However, despite their importance for reconstructing past environments, these surface and near-surface settings have rarely been interrogated through experiments designed explicitly to detect biological activity. Progressive geological and atmospheric evolution has rendered the martian surface increasingly inhospitable over time, as stable liquid water became scarce and the planet lost its global magnetic field. The resulting exposure to intense ultraviolet and ionizing radiation has created surface conditions that are highly destructive to both living organisms and preserved biosignatures. If life ever arose on Mars and persists today, it is likely to have retreated into the subsurface (Payler et al., 2019), where shielding from radiation and more stable physicochemical conditions may prevail. To date, however, no mission has conducted targeted life-detection experiments within these potentially more favorable subsurface environments, which leaves a critical gap in our understanding of martian habitability.
Although several missions have accessed surface sedimentary deposits, they have not systematically tested these environments for evidence of biological activity. The lack of subsurface exploration has consequently limited our ability to assess the true biological potential of Mars. On Earth, analogous environments, which include subsurface brines, ice-covered systems, and permafrost, are known to host diverse and metabolically active microbial communities (Rothschild and Mancinelli, 2001; Huber et al., 2007; Oren, 2015; Thombre et al., 2020; Shu and Huang, 2022; Mellon et al., 2024; Jo et al., 2022). Comparable environments on Mars remain largely unexplored, specifically the subsurface features of these. The search for extraterrestrial life is further complicated by the possibility that life elsewhere may have originated independently of terrestrial life. With Earth as the only known example, life-detection strategies are inherently shaped by terrestrial biology. To minimize this bias, life-detection experiments must remain as agnostic as possible and target universal features of living systems rather than specific Earth-centric biochemistries. The Viking missions of 1975–1976 represented the first and, to date, the only direct attempts to detect life on Mars. Although several experiments yielded intriguing results, their interpretation was ultimately ambiguous and is now widely attributed to reactive surface chemistry rather than biological processes. In the decades following Viking, planetary exploration strategies largely shifted away from direct life detection and toward the characterization of habitability, effectively separating planetary science objectives from astrobiology and exobiology (Biemann et al., 1977; Klein, 1976, 1978; Levin and Straat, 2016; Mancinelli, 1998). Over the past half-century, however, advances in microbiology, ecology, and technologies for studying extreme environments have substantially expanded our understanding of life’s limits and improved our ability to detect biological activity. These developments enable us to advance the Viking legacy and incorporate lessons learned to reduce false positives and strengthen the validation of biological signatures, whether extant or extinct, in future astrobiology missions.
The objectives of the present study are fourfold. First, we introduce a set of proposed measurements for detecting both extant and extinct life, together with a framework for sample selection and handling. This will include payload instruments that currently have the functionality and whose returned datasets would allow for the scientific meticulousness and resolution for measurements of biological processes and cellular life, respectively. Second, we describe the desired subsurface regolith and evaporite minerals that have the capacity to retain biogenic evidence of extant life along with features that, if preserved under subsurface conditions, would protect the biosignatures of dead or dormant cells. Third, we present the conceptual framework for a 21st-century version of the agnostic Viking life-detection experiments and discuss how these approaches can be adapted for future Mars exploration missions. Finally, we give our recommendations for the international relationships necessary to embark on this type of subsurface mission—that is, allowing for collaborative construction and mission development such that participating space agencies would be able to work together in this next phase of planetary science with a focus on the subsurface.
Motivation: The Need for Subsurface Microbiology Experiments
The martian subsurface represents a substantially more favorable environment for the persistence of extant life and the long-term preservation of biosignatures than the planet’s surface. We further propose that experimental frameworks integrated with subsurface drilling technologies could enable the first definitive detection of either active biological processes or preserved evidence of past life on Mars. In contrast, the martian surface is likely to be among the least hospitable environments for sustaining or preserving life. Chronic exposure to ionizing radiation, extreme thermal variability, highly oxidizing geochemical conditions, limited availability of stable liquid water, impact gardening, and frequent dust storms collectively impose severe constraints on biological survival and the preservation of biosignatures (Dartnell et al., 2007; Pavlov et al., 2012). These processes are also expected to degrade or destroy mineralogical, morphological, and molecular indicators of biological origin. To date, life-detection and habitability investigations on Mars have been largely restricted to surface or near-surface environments, and the absence of unambiguous biosignatures may, therefore, reflect the efficiency of surface processes in erasing biological evidence rather than a definitive absence of life (Hoehler, 2007; Shkolyar et al., 2025; Perl et al., 2026).
For these reasons, the martian subsurface is a more plausible environment in which to search for evidence of life. On Earth, the subsurface hosts an extensive and diverse biosphere, with global microbial cell abundances estimated to exceed 1029 cells Kallmeyer et al., 2012; Magnabosco et al., 2018). These organisms exploit a wide range of redox gradients, nutrients, and water availability within the crust, often occupying chemically and thermally stable environments (Edwards et al., 2012). The resulting diversity of subsurface microbial communities rivals that of surface ecosystems. Notably, the majority of these organisms have not been cultivated in pure culture, which necessitates indirect detection approaches based on metabolic activity, biomolecules, and geochemical signatures (Lloyd et al., 2018; Hoehler et al., 2021, 2025; Seyler et al., 2020). Such agnostic detection strategies are, therefore, well suited for application to Mars. Additionally, the subsurface provides substantial shielding from solar ultraviolet radiation and galactic cosmic rays (Fig. 1). Such protection is particularly critical on Mars, which lacks both a global magnetosphere and a dense atmosphere capable of mitigating radiation exposure. Consequently, the highest probability of detecting either extant life or preserved biosignatures from extinct communities on Mars likely lies beneath the surface. Moreover, the subsurface is expected to host a broad array of potential metabolic energy sources. Reduced gases and minerals, including molecular hydrogen, carbon dioxide, carbon monoxide, iron, manganese, and other transition metals, could sustain chemolithoautotrophic metabolisms within the martian crust (Pierce and Brazelton, 2023; Hoehler et al., 2001; Gary-Bicas et al., 2026). Among these pathways, methanogenesis is particularly well suited to the anticipated redox and thermal conditions of the martian subsurface.
Subsurface habitability gradients and estimated half-life of terrestrial nucleic acids, proteins, and lipids (Zakany et al., 2023). Subsurface sedimentology, closed-system aquifers, buried evaporite deposits (Perl et al., 2025a), and groundwater pockets (Michalski et al., 2013; Pedersen et al., 2014) all have the highest potential to support extant life by allowing water–mineral interactions to yield nutrients for cellular life (Edwards et al., 2012). While different ionic compositions and salinities of groundwater would lead to differences in the eutectic point for brines, all subsurface brine solutions would have a greater chance of protection from the desiccating nature of the martian surface. Moreover, the stability of biochemical structures and metabolites would also be preserved in these crystal habits (Perl et al., 2025b). Image courtesy: Chuck Carter.
Although solar radiation is strongly attenuated with depth, small fluxes of photons may penetrate several centimeters into the martian regolith. On Earth, phototrophic microorganisms adapted to extremely low light levels inhabit shallow subsurface environments, where they are protected from surface stressors while still capable of harvesting solar energy. These organisms, often anoxygenic phototrophs, do not require free oxygen for metabolism. The paucity of atmospheric oxygen on Mars, therefore, does not preclude the existence of analogous microbial strategies in the martian subsurface. Importantly, the martian subsurface is not only the most promising target for life detection but is also likely accessible within the limits of current and near-term drilling technologies (Aadnoy and Looyeh, 2019; Badescu et al., 2019; Zacny et al., 2008, 2013; Bar-Cohen and Zacny, 2021; Golombek et al., 2023; Isambourg et al., 1999; Jahn et al., 2008). While remote geophysical techniques such as ground-penetrating radar, magnetotellurics, and seismic methods provide critical constraints on subsurface structure, direct access to materials remains essential for definitive life-detection experiments. Accessing the martian subsurface is, therefore, a necessary step in the search for life on Mars (Tosi et al., 2026). Finally, it would behoove future payload development to demonstrate technical sensitivity sufficient to detect life at low-biomass thresholds, including single-cell detection limits, as both the instrument sensitivity and the criteria by which biogenicity is assessed must be held to a standard commensurate with the extreme scarcity of biosignatures expected in putative extraterrestrial environments. An understanding of low-biomass ecologies (low relative to terrestrial biomes) would enable a comprehensive assessment of surface-to-subsurface brine environments (Cockell et al., 2019, 2020), where the energetic carrying capacity for biological activity is intrinsically low due to thermodynamic and kinetic constraints rather than a lack of potential cellular diversity. Such energy-limited systems are well documented on Earth, where subsurface brines and low water-activity environments sustain metabolically active, though sparse and slow-growing, microbial communities, providing relevant analogs for similar settings on Mars (Hoehler, 2007; Martínez and Renno, 2013; Nisson et al., 2024, 2023a,b).
The adage of “life detection” implies the ability to measure features that can be reliably distinguished from abiotic processes, such that properties of extant cells or preserved signatures of extinct biological activity can be identified. By design, the search for biosignatures on Mars is focused on the potential remnants of long-dead biological activity (Gibson et al., 2001; Hurowitz et al., 2025) that has survived in the rock record. The linkages of such activity to former biological processes without the source biological record will continue (by definition) to provide a lower burden of proof when compared with repeatable and reactive measurements that are associated with the properties of biology rather than the geological imprints that cells may leave behind. The term “biological validation” allows us to focus on the properties of extant cellular biology in an agnostic methodology. An example of this would have an emphasis on the utility of an information-carrying charged polymer rather than the measurement of that polymer itself (i.e., the sequence on monomers that encode information for replication, expression, mutation, and evolution rather than DNA or RNA alone). This distinction has been categorized into five measurement types (and measurement keys) that have developed into our MOPRH matrix (Fig. 2).
Ordinated sets of independent biological validation experiments. (Top) Performing paired measurements in a defined sequence enables the construction of a constrained parameter space that can be used to support or reject a biogenic interpretation. For extant life, an initial measurement on a parent sample increases the likelihood that a subsequent, independent measurement will yield a result that strengthens the evidentiary burden of the first. A similar logic applies to extinct life, although it depends on the preservation of biological material following cell lysis and typically yields degraded analogs of extant biological signatures. (Bottom) In this sample framework, most observations correspond to two-component experimental tests, whereas higher-confidence detection of extant life may require three or more complementary measurements applied sequentially to the same sample.
A distinguishing feature of active life is metabolic activity that produces kinetic signatures that can deviate measurably from abiotic chemical baselines. Nonbiological reaction rates typically follow an Arrhenius relationship, exhibiting an exponential increase with temperature, whereas biologically mediated reactions often display a temperature optimum, with reaction rates decreasing at temperatures above and below this maximum due to the evolutionary tuning of enzymes to specific functional ranges (Daniel and Danson, 2010). This enzyme-driven kinetic behavior is expected to be largely agnostic to organismal identity and may, therefore, serve as a general indicator of biological activity. One approach to exploiting this distinction is to quantify substrate turnover across a controlled temperature gradient by monitoring the consumption of reactants or the production of products using isotopically labeled compounds. Substrates such as methane or deuterated water can be used to track reaction progress, with stable isotope incorporation rates measured as a function of temperature to derive reaction rate profiles and identify kinetic patterns consistent with metabolism rather than abiotic chemistry (Boschker and Middelburg, 2002; Valentine et al., 2004).
Patterns and collocated structures (O)
Biological systems generate characteristic molecular patterns and structures that can be distinguished from abiotic chemistry, both in composition and spatial organization. Many biomolecules common to terrestrial life, including nucleic acids and aromatic amino acids such as phenylalanine, tyrosine, tryptophan, and, to a lesser extent, histidine, exhibit intrinsic autofluorescence under deep ultraviolet (DUV) excitation. Abiotic synthesis is thermodynamically biased toward smaller, simpler molecules (e.g., glycine, alanine, and valine), whereas biological systems expend metabolic energy to produce and maintain aromatic compounds that play essential functional roles in proteins, including structural stabilization and ion transport (Shock and Schulte, 1990). Consequently, DUV-induced autofluorescence and spectral discrimination provide a useful, agnostic screening approach for identifying organic compounds of interest embedded within abiotic matrices, particularly when combined with spatial co-location analyses using raster detectors and mineralogical context from X-ray fluorescence or optical imaging.
A critical caveat for fluorescence-based life detection is the potential for abiotic false positives. Although targeted fluorescence has been proposed as a life-detection approach on the basis that larger, complex molecules, such as tryptophan, fluoresce while the simpler molecules that dominate abiotic settings typically do not (Creamer et al., 2017; Yoffe et al., 2025), this distinction is not absolute. Tholins, however, complex organic macromolecules produced abiotically by the irradiation of gas- or ice-phase mixtures of cosmically abundant molecules (CH4, N2, NH3, and H2O) by UV photons or energetic particles (Sagan and Khare, 1979; Hodyss et al., 2004), are fluorescent and can produce emission signatures of comparable magnitude to those of kerogen, a biogenic macromolecular organic material derived from the diagenetic transformation of microbial biomass and other biological source material in sedimentary systems (Bailey et al., 2006; Vandenbroucke and Largeau, 2007; Shkolyar et al., 2018; Naraoka et al., 1999). Because tholins are entirely abiotic in origin, yet spectrally overlap with biogenic organics in fluorescence space, their presence in a sample would constitute a false positive in any life-detection assessment relying on fluorescence alone.
Beyond detection, biological systems produce distinctive molecular abundance patterns across chemical classes such as amino acids, lipids, and fatty acids, which differ markedly from abiotic distributions observed in meteorites and laboratory syntheses, where molecular abundance typically decreases exponentially with increasing molecular complexity (McCollom et al., 1999). In contrast, biological systems often exhibit relatively uniform abundances among functionally selected building blocks (Cleaves et al., 2014). Significant deviations from abiotic molecular distributions, therefore, constitute a strong biosignature, offering not only evidence for life but also insight into the underlying biochemistry, including the possibility of non-terrestrial molecular architectures.
Responses to photosynthetic and/or chemical gradients (R)
Direct observation of cellular motility provides a potential means of detecting extant life, as motile behavior reflects active, energy-consuming processes that are distinct from abiotic particle motion. Although many microorganisms rely primarily on Brownian motion, biologically driven motility can be distinguished by deviations from equilibrium diffusion, including non-Brownian dynamics and biased or directional movement in response to external gradients such as chemical cues or photo-stimuli. In thermal equilibrium, Brownian motion obeys detailed balance and is characterized by a mean-squared displacement that scales linearly with time, whereas biologically driven motion exhibits super-diffusive behavior or net drift indicative of energy dissipation (Cates, 2012). In addition, rotational motion constitutes a particularly strong biosignature, as cellular propulsion systems on Earth, including bacterial, archaeal, and eukaryotic flagella, have independently evolved rotary mechanisms that enable propulsion at low Reynolds number (Purcell, 1977; Beeby et al., 2020). The universality of these physical constraints suggests that rotational motility may represent a convergent solution for cellular life elsewhere. Detection of such motion requires high-resolution microscopy capable of resolving near-minimal cell sizes (∼0.1 μm), such as digital holographic (Wallace et al., 2025) or optical microscopy, and necessitates the presence of a liquid medium introduced through controlled wetting of regolith, ice (Zangrando et al., 2020), or subsurface fluids. Each of these mediums can exist in a stable form within the martian subsurface.
Charged polymers and information-carrying molecules (P)
Contemporary understanding of biological information storage is rooted in DNA, a linear, charged polymer whose backbone provides solubility and structural stability while enabling sequence-encoded heredity through interchangeable monomeric units. This framework can be generalized to an agnostic model in which life employs charged (polyanionic or polycationic) polymers capable of maintaining multiple stable sequences and supporting inheritance and evolution (Hutter et al., 2003; Benner, 2017, 2023). Detecting such information-carrying polymers as evidence of extant life, therefore, requires both identification of polymeric structure and characterization of monomer sequence variability. Nanopore sensing offers a powerful, sequence-agnostic approach for this task by measuring characteristic ionic current modulations and dwell-time signatures as individual polymers translocate through nanoscale pores, enabling detection of long-chain charged polymers with unknown subunits (Branton et al., 2008; Deamer et al., 2016; Carr et al., 2020). Recent advances demonstrate that nanopore platforms can discriminate polymer length, charge density, and sequence-dependent features without prior biochemical assumptions, which makes them particularly well suited for life-detection applications beyond Earth-centric biochemistry (Carr, 2022). With appropriate sample preprocessing to remove particulates and a multiplexed array of nanopores spanning a range of diameters, nanopore-based instruments could detect and potentially sequence diverse informational polymers, extending biosignature detection to the fundamental chemical requirements of heredity.
Darwinian evolution results in repeating ionic charges would be the framework for any genetic polymer in any aqueous solution (which, for extant cellular life, would likely contain bioavailable nutrient sources as well). The charge backbone of these biopolymers is able to maintain a near-constant aqueous solubility, which can deter folding and the like. Previous work has noted that all the nonelectrolytes tested gradually lost Watson–Crick base-pairing fidelity and, in turn, developed a sequence-dependent property based on chain length that accounted for a biological function (Hutter et al., 2003). This Polyelectrolyte Theory of the Gene (as noted in Benner, 2023) can potentially be used as a universal sign of extant life since the breaking of this process would not allow for evolutionary behaviors to exist in molecules and, therefore, any microbial communities or even in a single cell, notably stress adaptations.
This represents another agnostic method with which to determine signs of biological activity in lacustrine systems should the observing conditions allow for extant cellular activity (Zwicker et al., 2018) to take place. Benner (2017) noted that, since these repeating charged nucleic acid backbones are a functional property of life within (and requiring) a water/fluid system to exist, this can be done in any aqueous system, whether it is subsurface ice, permafrost, or liquid water (Benner, 2023), as long as the thermodynamic constraints allow. For the analogous ocean worlds settings, this would have clear advantages due to ice crusts on Enceladus and Europa (Garner et al., 2025), as well as plume material ejected from these solar system bodies. It should be noted that potential biomass from the latter can vary greatly compared with the ice surfaces (Tenelanda-Osorio et al., 2021) or martian permafrost and ice.
Morphology and embedded structures (H)
Physical structures indicative of past life are expected to exhibit organized, repeating physical or chemical complexity across multiple spatial scales. At the macroscale, such patterns may manifest as laterally continuous features within sedimentary strata, such as bedding disruptions, lamination breaks, or facies-bound textures, detectable by orbital imagery or rover-scale imaging (Summons et al., 2011; Grotzinger et al., 2014). Meter-scale observations can further constrain candidate features through textural continuity along bedding planes or stratigraphic surfaces, providing geological context for targeted investigation (Lima-Zaloumis et al., 2025). While macroscopic features alone are insufficient to establish biosignatures, they guide focused meso- and microscale analyses, where centimeter-scale textures (e.g., wavy laminae or mat-like fabrics) can be evaluated for repetition and association with larger-scale structures (e.g., Westall et al., 2015). Microscale investigations integrate high-resolution imaging and geochemical measurements to assess the co-location of morphological features, such as cell-like structures or patterned fabrics, with chemical indicators that include isotopic fractionation, organic compounds (Shock and Schulte 1998), and authigenic minerals inconsistent with equilibrium host-rock geochemistry (Drake et al., 2015, 2021; McMahon and Cosmidis, 2022; Buongiorno et al., 2019; Natalicchio et al., 2012). The convergence of physical patterning and geochemical anomalies across spatial scales provides the most compelling evidence for potential biosignatures and reduces ambiguity associated with purely morphological interpretations.
The Regolith Examinations for Validating Extraterrestrial Astrobiological Life Mission Design
The need for a microbiology-driven science payload is at the core of the Regolith Examinations for Validating Extraterrestrial Astrobiological Life (REVEAL) mission design (Fig. 3) with specific instrument types that, it has decided, will all have the capacity to detect independent and dependent lines of evidence for biogenicity with respect to in situ sample analyses by way of our MORPH matrix shown in Figure 2. While this is an example, the science traceability matrix (not shown) meets the minimum spectral and spatial resolutions needed for cellular detections and resolution of organics sourced from biological processes (Cockell, 2026; Nisson et al., 2026; Schopf, 2026). Moreover, our subsurface targets include evaporitic minerals due to the likely presence of fluid inclusions and their high preservation potential of pigments and other biochemical compounds (Vítek et al., 2014; Baqué et al., 2016; Perl et al., 2021, 2025a). Moreover, an updated set of Viking-inspired nutrient seeding experiments can also be conducted as an independent set of observations on surface soils (and subsurface soils, if needed) as our Biomass, Energy, and Nutrients Tracking Observatory (BENTO) experiments (Fig. 4). This would provide a parallel line of observations that would facilitate comparison of subsurface minerals and soils with respect to water activity (aw) and conductivity (dS/m).
Regolith Examination for Validating Extraterrestrial Astrobiological Life (REVEAL) mission design. The framework for our science payload relies on accessing the subsurface of Mars (Tosi et al., 2026) and to assess the sedimentary strata as we exhume regolith in a vertical transect. Biogenicity measurements with respect to any martian ecology (Nisson et al., 2026) would be determined on the aforementioned MORPH framework and as a function of an independent Last Universal Common Ancestor separate from our terrestrial biosphere (Schopf, 2026). Moreover, any positive, negative, or ambiguous biological signal would still be subject to the same design of experiments (Cockell, 2026) such that a collective census was reached based on the payload datasets for geochemical gradients as deeper sediments were analyzed. Image courtesy: Chuck Carter.
Biomass, Energy, and Nutrients Tracking Observatory (BENTO) experiment. The importance of subsurface sampling is in parallel to testing authigenic martian regolith against potential nutrient sources to measure if any potential biochemical reaction takes place. Within the REVEAL mission design, the BENTO experiment emulates what occurred on the Viking mission but within a vertical transect using exhumed martian salt minerals and soils. Martian surface regolith will act as our negative control with later subsurface samples acting as the same for deeper sediments (gray sample dishes). Likely nutrient media would be added to dishes from surface regolith (top purple dishes) and subsurface soils and salt minerals (purple dishes on later rows).
At its highest science mission objective, the REVEAL science payload is designed to address two fundamental questions regarding life detection and biological validation. The first fundamental question concerns the identification of molecular and/or morphological evidence that is indicative of extant or extinct biology. This is whether the in situ experimental evidence and observed dataset reflect an active biological process or preserved cellular structure. The second fundamental question has to do with the reproducible measurement of a molecular signature consistent with a known biological process, which would provide independent confirmation of a putative physical biosignature or chemical biomarker. Should there be an in situ active biosystem in the martian regolith or an exhumed sample of it, the expectation of REVEAL payload observations may not be consistent with the detection of microbial life, as disruption of any ecosystem can temporarily pause metabolic and behavioral processes by introducing stresses. Hence, the “repeatability” of any measurement by the REVEAL payload has a wider range for biological processes and a smaller range for abiotic measurements (e.g., minerals and liquid water). While the BENTO experiment provides a steady-state set of measurements, all REVEAL payload instruments will exercise the main utility of such repeatability requirements, which is that these experiments be repeated at a selected series of regolith depths to expose the instruments to “fresh” sample material. Nisson et al. (2026) provide some insight into what some of these subsurface experiments could be, whereas Tosi et al. (2026) show the pneumatic technology capable of accessing martian subsurface depths from the surface down to at least 50 m (see also Tosi et al., 2024). The expectations for these types of experiments and their potential outcomes are such that regardless of experimental result, the astrobiology and scientific community as a whole will know significantly more regarding the potential ecosystems of ancient and modern Mars (Cockell, 2026).
The BENTO experiment would allow for a measurable chemical and physical representation of the disequilibrium between microbial processes and chemistry. Microbial life (cells) does not generate disequilibria but rather exploits and scatters any already existing chemical gradients as an outcome of metabolism. The BENTO framework will specify at least five gradients that a martian biota could establish (Schopf, 2026). In any redox disequilibria that is present in a martian setting (e.g., photochemical, radiolysis, and serpentinizing), the presence of cellular life and their metabolic processes would likely accelerate the relaxation of those gradients toward equilibrium via uptake of free energy, while maintaining cellular processes that involve repair and replication. Our strategy is more grounded in these processes and their accompanying datasets that show evidence of these states.
As noted in the next section, the manner in which vibrational spectroscopy can also support the measurements of cellular processes is critical to biological validation of extant life. Rather than searching only for chemical byproducts that may be attributed to a biological process in an extinct life paradigm, it is the process itself that can be spectrally measured, state-to-state. Moreover, the binary use of specific instrumentation is not a part of our science traceability matrix, rather it is an ordinated pair of observations (Fig. 2) that yields stronger lines of evidence that support the distinction of biotic, biogenic, abiogenic, and abiotic features (Perl et al., 2020). An example where this would be necessary is in the Kimberley region of Gale crater, Mars, where the Curiosity rover observed elevated manganese oxide concentrations (Lanza et al., 2016) within fracture-filling material that crosscut authigenic sandstones. Carbonate-associated minerals that are common to the martian surface (e.g., calcite) and their subsurface counterparts are capable of producing strong fluorescence emission spectra in the absence of any organic or biological component. This directly implies that positive fluorescence detections, even in aqueous settings where these very same minerals were deposited, cannot be used to uniquely verify authigenic organic material. However, reworking the design of experiments for a biogenicity-driven approach would allow for these measurements to be conducted on already validated biotic features in subsurface regolith to avoid true negative signs of life (McMahon and Cosmidis, 2022).
Raman spectroscopy provides a powerful, nondestructive technique for probing biochemistry and detecting potential biosignatures on Mars by identifying molecular vibrations diagnostic of both mineralogy and organic compounds. In particular, Raman spectroscopy is highly sensitive to conjugated carbon–carbon bonds, which makes it exceptionally well suited for detecting carotenoid pigments produced by halophilic and phototrophic microorganisms. These pigments exhibit strong and characteristic Raman bands that can persist even when embedded within mineral matrices such as sulfates, chlorides, and evaporites, which enhances their potential for long-term preservation in martian environments (Marshall et al., 2007; Jehlička et al., 2014; Perl et al., 2025b). Because carotenoids are energetically costly to synthesize and rare in abiotic systems, their detection, especially when spatially co-located with sedimentary textures or aqueous minerals, constitutes a compelling biosignature candidate (Baqué et al., 2016). Beyond pigments, Raman spectroscopy can identify a wide range of biochemically relevant compounds, including organic functional groups, amino acids, and mineral phases associated with redox gradients, enabling contextual interpretation of habitability (Cockell et al. 2023) and potential biological activity (Westall et al., 2015). The successful deployment of miniaturized Raman instruments on Mars missions such as ExoMars underscores the suitability of the technique for in situ life-detection investigations, where it can serve as both a triage tool and a method for detailed biosignature characterization. While vibrational spectroscopy can aid in biogenicity measurements (and separating the abiotic minerals from the biogenic features), it is paramount that the optical resolution of such measurements is at the cellular level (Wallace et al., 2025) to resolve single cells in subsurface frozen or semi-frozen brine and permafrost systems.
Agnostic measurements for cellular and biogenically sourced organics
Two of our agnostic measurement techniques are vital for biological validation in that their measurements allow for the individual charge of organics and cells as well as testing for metabolic activity as a binary positive or negative signal for life. Using our MORPH matrix format (Fig. 2, top) and having independent measurements on the same parent sample provide a high burden of proof to evaluate biogenicity when deciphering the components of evaporite minerals and entombed fluids (Schopf, 1993; Summons et al., 2014; Neveu et al., 2018; Perl et al., 2021).
Nanopore sensing and polyelectrolyte references
Within salt minerals and brine samples, nanopore sequencing would provide a promising, agnostic approach for detecting extant information-carrying charged polymers by measuring characteristic ionic current disruptions and dwell-time signatures as individual long-chain molecules translocate through nanoscale pores, enabling identification of polymers with unknown monomeric subunits (Branton et al., 2008; Deamer et al., 2016). With appropriate chemical or physical preprocessing to depolymerize macromolecules, nanopore methods may further allow sequence-level characterization of monomer composition, a critical requirement for identifying informational function (Carr et al., 2020; Lin et al., 2022). However, current nanopore technologies face limitations, including reduced sensitivity to branched polymers, dependence on reference libraries for sequence interpretation, and susceptibility to clogging or signal interference from particulate, organic, or inorganic debris. These challenges necessitate upstream sample preparation steps such as filtration, density-based separation, or size exclusion. To broaden detection capability across diverse potential biopolymers, a flight-ready instrument would ideally incorporate multiplexed nanopore, micropore, and millipore arrays spanning pore diameters of approximately 10–104 Å, enabling parallel analysis of polymers across a wide range of sizes and charge densities.
Moreover, the operational use of nanopores in this application has at least two sample-based experimental/technical obstacles that are critical to highlight here with regard to mitigation strategies that can be used for in situ martian subsurface analyses and for brine systems where evaporite minerals (Ehlmann and Edwards, 2014) can enhance the preservation potential for cellular life (Perl et al., 2025a, 2025b; Olson and Lowenstein, 2021). The medium of sample that REVEAL’s payload analyzes will be solid and a mixture of regolith, salt minerals, and potentially ice (Garner et al., 2025) and permafrost, depending upon the abrasion depth at which these features would remain stable in the capped borehole (Fanale et al., 1986; Mellon and Jakosky, 1993). The current state of nanopore sensing relies on an analyte-containing sample that is introduced into a dual chamber with an ionic electrolyte solution (typically KCl and NaCl). The chamber is under an applied voltage that drives the ionic current through the pore. As single-charged molecules inside the sample medium are electrophoretically passed through the pore, the subsequent translocation events processed (Kawabe et al., 2023) are the result of a reduction of ionic current, whose amplitude and duration (e.g., dwell time) are dictated by the chemical and physical states of the molecule (Smith et al., 2024). It is this translocation event frequency, dwell time, and change in current intensity from the initial current that allows for characterization to occur, should there be a reference for comparison of this trio of data.
For martian material in particular, a sampling/analysis architecture that has been proposed for the Agnostic Life Finder instrument allows for solid sample ingestion, where water/liquid/solvent extracted from regolith samples would be the medium for dissolving/liberating any organic and biopolymeric material present in samples (Spacek and Benner, 2022). The risk for analyzing subsurface samples is not having enough extractable polyelectrolyte material. By design, our proposed science mission architecture can mitigate this issue by delivering at a controlled rate into the nanopore a minimum of ∼0.5 mL of extractable material from authigenic regolith, which could produce 104–106 translocation events.
Martian subsurface regolith is likely to contain low biomass when compared with its terrestrial counterparts. Brine environments, particularly in the subsurface, are potentially habitable due to their sustained geochemical flux driven by water–rock interactions and radiolysis. Should the geochemical energy available for metabolic use exceed or be at a given cellular maintenance energy threshold, the resulting volume of cells in a subsurface brine system would not be expected to be “high.” This expectation is fundamental to what typically are low-biomass deep subsurface mineral-brine systems on Earth (Nisson et al., 2024, 2023b). It is, however, universal for cellular life to adapt to stressed conditions, and the magnitude of the stress response is expected to mirror that of lacustrine geobiological settings on Earth. Thomas et al. (2023) have noted that these (or any) microbial adaptations, including representations of those within a series of single-charged polymers, can be measured in a brine system. Taking into account expectations of low biomass and maintenance energy, vast amounts of regolith available for integration, and applying the calculations of (Nisson et al., 2024) to Mars, it stands to reason that a mission architecture to explore Mars’ subsurface can be built with the capability essential to detect life on the red planet.
Radiotracer incubation experiments
The ability to detect metabolic processes independent of phyla identification may also allow for an intermediatory step in life detection. Radiotracer experiments provide an exceptionally sensitive means of detecting metabolic activity in low-biomass environments, particularly where genomic approaches fail or sample extraction is impractical and/or limited in number for remote planetary missions. Such methods have successfully detected active metabolisms, including methanogenesis and sulfate reduction, in Earth’s deep biosphere at cell abundances below 500 cells cm-³ (Beulig et al., 2022). Radiotracer approaches rest on the agnostic premise that any extant life must metabolize to obtain energy, regardless of its biochemical architecture, and that measurable metabolic rate may be extremely low due to substrate limitation. Accordingly, these experiments target plausible martian redox couples that involve substrates and electron acceptors expected to occur on Mars, including CO2/H2-, CO-, acetate-, or formate-driven methanogenesis; iron and sulfate reduction coupled to simple organics or hydrogen; and methane oxidation linked to sulfate or iron reduction. Detection is based not on the magnitude of activity but on its presence, whether occurring over minutes or decades. Radiolabeled nutrient turnover simultaneously probes multiple defining properties of cellular life, energy processing, regulation, growth, and environmental responsiveness, as demonstrated by the Viking lander labeled-release experiments, which measured radiolabeled CO2 evolution following substrate consumption (Klein, 1978; Soffen, 1997; Levin and Straat, 2016). Because radiotracer experiments assess ecosystem-level function rather than that of specific taxa, they remain robust to unknown or non-terrestrial biochemistries and do not rely on Earth-centric molecular libraries, which would be ineffective for identifying truly alien life in the absence of contamination or a measurable abiotic organic background. The Viking life-detection experiments were conducted on surface soils, which we now understand are poor targets for extant life when compared with the subsurface due to the effects of intense UV irradiation, low water activity, and highly oxidizing atmospheric conditions (Dartnell et al., 2007; Pavlov et al., 2012). In contrast, our new generation of radiotracer experiments will target subsurface materials recovered by drilling, where physical shielding and more stable geochemical conditions increase the likelihood of active metabolism. Viking included δ14C-CO2 assimilation experiments to probe autotrophic growth under illuminated and dark conditions; however, growth-based measurements are intrinsically insensitive in energy-limited environments, where metabolic activity may occur without detectable biomass increase (Klein, 1978). By focusing instead on direct metabolic turnover, radiotracer incubations provide substantially greater sensitivity to low-rate biological processes (Beulig et al., 2022). Viking also tested degradation of a limited suite of primarily Earth-centric organic substrates, potentially overlooking metabolisms based on simpler and more universally available compounds. The combination of instruments and proposed experiments therefore emphasizes small organic molecules coupled to multiple plausible martian electron acceptors. Finally, limitations in Viking’s control strategy, particularly the use of combusted soils that may have altered abiotic reactivity, would be addressed through a more comprehensive and representative control framework, including gamma-irradiated samples and parallel experiments types (i.e., killed, medium-only, drill-fluid, post-incubation, others) and background controls (Klein, 1978; Levin and Straat, 2016).
Subsurface regolith examination
All samples acquired, irrespective of lithology or physical form, must undergo a systematic triage process prior to detailed analysis. A lander-based triage system will receive samples directly from the drilling apparatus and provide controlled storage for preliminary characterization and early decision-making. Depending upon drilling productivity, sample processing throughput, and instrument analysis timelines, triage chambers may also function as intermediate holding environments for samples queued for downstream analyses. As such, these chambers must support both short-term assessment and longer-duration storage under tightly regulated environmental conditions. Each triage chamber should incorporate a standardized suite of instruments to enable initial sample assessment, including temperature monitoring (Plesa et al., 2016) and control, pressure regulation (Breyholtz et al., 2011), volatile detection, core logging and orientation, mineralogical characterization, and mass and volume measurements. The system must accommodate variable sample sizes and morphologies, transfer samples to analytical subsystems, discard samples when required, and execute cleaning and sterilization protocols to mitigate cross-contamination. To preserve sample provenance and integrity, individual core segments or cuttings should be maintained in independent, self-contained curation compartments. While Tosi et al. (2026) describe a pneumatic drill design and our regolith exhumation procedures in further detail, a noted feature of this pneumatic process and wellbore design allows for regolith and evaporite minerals to become fluidized with respect to upward flow rate. Since this design is CO2-powered from the martian atmosphere, the rate of sample recovery and ingest to the REVEAL lander would be faster than our payload instruments can process. Therefore, in addition to the sample handing and triage for internal processing, a sterile sample waiting room (Fig. 5) that separates strata is required for regolith, minerals, and icy material to be quantified in sedimentological order without cross-contamination.
Close-up of our proposed subsurface sample cache waiting room. A sterile, contamination-controlled laboratory holding chamber is essential to support rapid subsurface sample retrieval while preserving stratigraphic integrity. Such a system enables samples to be collected, isolated, and curated according to depth and sedimentary context, facilitating robust correlations between measured properties and subsurface stratigraphy. This approach allows systematic comparison of sediments, evaporitic minerals, brine inclusions, permafrost, and ice recovered from increasing depths, thereby preserving spatial context and enhancing interpretation of geochemical and potential biological signals across the subsurface profile. The volume design of soil and mineral tubes will correlate with the rate of martian subsurface regolith that will be exhumed and collected as a function of the ConOps sol planning (Tosi et al., 2026) we have developed. Image courtesy: Chuck Carter.
Thermal regulation within the triage and waiting chambers (Fig. 5) is critical and must maintain samples below temperatures at which eutectic brine formation could occur, as phase transitions may complicate manipulation and bias subsequent analyses (Chevrier and Rivera-Valentín, 2012). Pressure monitoring and control, coupled with continuous volatile sensing, are similarly essential. The system must actively manage the pressure changes associated with sample extraction from the borehole to preserve physical and chemical integrity while enabling controlled capture of evolved gases. Quantitative measurement of released volatiles should be performed within the triage suite. Instruments analogous to the Tunable Laser Spectrometer within the Sample Analysis at Mars payload on the Curiosity rover provide demonstrated capability for high-precision measurements of key gases such as methane, carbon dioxide, and water vapor (Mahaffy et al., 2012; Webster et al., 2015). Expanding the detectable range of volatile species would further enhance biosignature detection potential (Biemann et al. 1976). Accurate logging and orientation of cores or sample fragments are also required to preserve stratigraphic and spatial context (Tosi et al., 2026). Identification of sample top-to-bottom orientation and relative positioning with respect to adjacent samples may be achieved through mechanical indexing within the triage chamber or via metadata transmitted directly from the drilling system. Initial estimates of elemental composition, organic content, and mineralogy can be obtained with the use of a configurable suite of stand-off analytical instruments, enabling informed sample prioritization for detailed downstream analysis (Summons et al., 2011).
Our experimental framework prioritizes the detection of extant life while retaining sensitivity to biosignatures associated with extinct biological systems (Davila et al., 2025; Perl and Baxter, 2020). The overarching objective is to meet the evidentiary burden required to validate life beyond Earth, which necessitates integrating both direct measurements and context-dependent, potentially ambiguous observations within the natural subsurface environment of Mars (Grinspoon et al., 2026). Achieving this goal requires aligning mission design with a contemporary understanding of microbiology, low-biomass ecosystems, and the evolutionary potential of life under energy-limited conditions. The combination of subsurface access and a dedicated microbiology payload represents a substantive departure from previous Mars exploration paradigms and introduces a transformative approach to astrobiology. Unlike prior missions, which primarily targeted surface environments and focused on habitability or ancient biosignatures inferred from organic detections, this mission concept directly links experimental design to testable biological hypotheses. In this respect, Mars has historically been treated differently from ocean worlds such as Europa and Enceladus, which have long been prioritized for life-detection efforts due to the presence of subsurface liquid water (despite comparable challenges in subsurface access). Increasing evidence that the martian subsurface may retain liquid water and chemically favorable conditions for life suggests that it warrants a similarly rigorous, microbiology-driven investigative framework. Accordingly, we advocate an agnostic, subsurface-focused mission strategy that elevates Mars life-detection efforts to the same evidentiary standards applied to ocean worlds.
Moving beyond measurements of habitability
Subsurface environments on both Earth and Mars offer thermally and chemically moderated conditions that are more conducive to life than surface settings. Residual heat from planetary formation and ongoing radiogenic decay elevate subsurface temperatures, promoting the persistence of liquid water and enhancing metabolic reaction rates (Nisson and Perl, 2026). In addition, the subsurface provides effective shielding from ultraviolet radiation and galactic cosmic rays, which are particularly intense on Mars due to the absence of a global magnetosphere and a thick atmosphere (Dartnell et al., 2007). These protective factors significantly increase the likelihood of both biological survival and long-term biosignature preservation (Onstott et al., 2019). Consequently, the martian subsurface represents the most promising environment for detecting either extant life or preserved evidence of extinct biological communities. The integration of subsurface access with a dedicated microbiology payload represents a fundamental shift in Mars exploration strategy, enabling life-detection experiments designed to meet the evidentiary burden required to confirm biological activity. Building on advances in microbiology, instrumentation, and subsurface exploration, this mission concept explicitly targets extant life while retaining sensitivity to preserved biosignatures of extinct communities. Unlike prior Mars missions, which emphasized habitability and ancient surface environments, this approach aligns experimental design directly with hypotheses testable in situ, incorporating both unambiguous and context-dependent measurements within the martian subsurface (Westall et al., 2015). In contrast to Mars, ocean worlds such as Europa and Enceladus have long been prioritized for life detection because of their subsurface liquid oceans, despite significant access challenges. Recent evidence that Mars’ subsurface may retain liquid water and chemically favorable conditions suggests that it warrants a comparable experimental perspective. By adopting an agnostic, microbiology-driven framework and prioritizing subsurface environments, this mission concept aims to elevate astrobiology beyond the detection of potentially biogenic signals toward rigorous validation of life beyond Earth.
International collaboration between space agencies
A subsurface biological validation mission to Mars will require sustained international collaboration to integrate the scientific, technical, and financial capabilities necessary for accessing and interrogating the martian subsurface. The combination of deep drilling, advanced microbiological instrumentation, and planetary protection considerations will likely exceed the scope of any single space agency and benefit from shared expertise developed across international programs, including ESA’s ExoMars drilling and sample-handling systems and NASA’s long-standing leadership in mission design and in situ analysis (NRC, 2007; Goetz et al., 2016; Vago et al., 2017; Steele et al., 2018, 2012; Stromberg et al., 2019). Previous joint efforts, such as the Mars Sample Return campaign, demonstrate the feasibility and scientific value of coordinated mission architectures, shared payload responsibilities, and harmonized planetary protection protocols (Beaty et al., 2019; Carrier et al., 2020). Given the global scientific importance of detecting life beyond Earth, a collaborative framework that includes multiple space agencies, academic institutions, and private partners will be essential to distribute risk, accelerate technological readiness, and ensure that experimental designs meet the highest international standards of evidentiary rigor.
The next generation of experiments: A future biological validation subsurface mission
The Viking lander experiments were rigorously designed and successfully executed within the technological constraints of the 1970s, returning high-quality data from well-controlled analyses of martian soil (Klein, 1978; Levin and Straat, 2016). Subsequent reinterpretation of these results generally suggests that the observed responses were driven primarily by reactive soil chemistry, including oxidizing species, rather than unequivocal biological activity (Biemann et al., 1977; Hecht et al., 2009). These outcomes should not be viewed as a failure but rather as foundational findings that exposed key uncertainties and highlighted the need for follow-on experiments capable of discriminating between chemical and biological processes. Over the ensuing five decades, substantial advances in microbiology, analytical instrumentation, and planetary exploration have refined life-detection strategies and lowered ambiguity, motivating a renewed, biology-centered return to Mars (Wilhelm and Perl, 2026. Nonetheless, no single mission can guarantee the detection of life, and ambiguous results may persist even with modern methods (Cockell, 2026; Calomiris, 2026). Progress toward resolving the question of cellular life on Mars, therefore, requires an iterative, long-term exploration program in which each mission incrementally improves experimental design, expands environmental context, and informs subsequent investigations.
Authors’ Contributions
The formulation, writing, and leadership of this manuscript was done by S.M.P. This manuscript represents the next iteration from the Keck Institute of Space Studies (KISS) workshop “The Biology of Biosignature Detection” led by S.M.P., C.S.C., and W.W.F. This current iteration of work by the REVEAL Science Team includes S.M.P., C.S.C., W.W.F., S.S., K.G.L., M.B.W., F.A.C., T.T., J.L., D.G., C.M.F., K.F., K.L., M.H., S.M.S., L.W.,S.K., H.D., K.B., J.M., A.J.C., P.C., L.G., Y.I., J.B., S.H., P.L.T., D.M.N., A.M., M.G., F.T., M.B., and P.P.
Footnotes
Acknowledgments
The authors thank Sherry Cady, chief editor of Astrobiology, for the privilege of creating this special issue that highlights the historical efforts of the Viking landers and allows us to build bridges toward future astrobiology missions. They also thank the Caltech Keck Institute for Space Studies (KISS) for their support, which motivated this follow-on study and ongoing mission design work, specifically Bethany Ehlmann, Harriet Brettle, Michele Judd, Janet Seid, Antonio Soriano, and the KISS staff. Finally, they thank Chuck Carter who provided the artist interpretations of the martian subsurface habitability gradients (Fig. 1), our REVEAL Mars science lander design (Fig. 3), and the martian subsurface regolith sample waiting room and sample carousel (
).
Author Disclosure Statement
No competing financial interests exist.
Funding Information
No funding was received for this article.
Associate Editor: Michael A. Meyer