Dark microbiome and extremely low organics in Atacama fossil delta unveil Mars life detection limits

The study investigates the detectability limits of biosignatures relevant to life detection on Mars by analyzing Red Stone, an ancient (163–100 Ma) alluvial fan–fan delta in the Atacama Desert that is mineralogically analogous to Martian environments (hematite-rich, clay-bearing mudstones). The research question centers on whether current and near-future rover instruments can detect extremely low levels of organics and microbial biosignatures comparable to those that might exist on Mars. Context is provided by previous Mars missions (Viking, Phoenix, MSL, Mars 2020) that have focused on identifying habitable environments and organics in sites with aqueous histories (e.g., clays), with mixed success and sparse detections of simple organics. The purpose is to evaluate instrument capabilities and strategies (including wet-chemistry derivatization and immunoassays) against a realistic analog where biomass and organics are at or below detection limits, thereby informing the necessity of Mars Sample Return for definitive life detection.

Background includes the emphasis of Mars missions (Viking, Phoenix, MSL Curiosity, Mars 2020 Perseverance, future ExoMars) on detecting habitability and organics, often in clay-rich ancient fluvial-lacustrine environments. Viking and Phoenix did not obtain robust evidence for organics in soils, while SAM on MSL and SHERLOC on Mars 2020 identified simple aliphatic and aromatic molecules (e.g., ~450 ppm organics in Yellowknife Bay mudstone at Gale crater). Hematite, halite, gypsum, and phyllosilicates (smectite, chlorite) are documented on Mars and are present at Red Stone, supporting its analog relevance. The concept of “microbial dark matter” frames the challenge of uncharacterized microbial diversity; the authors adapt this to propose a “dark microbiome” specific to Red Stone’s context. Prior work highlights the Atacama as the driest and oldest desert on Earth and a Mars analog, with fog as a key water source for microbial life. These studies underpin the selection of Red Stone and the focus on instrument detection strategies (Raman, LIBS, DRIFTS, SAM, MOMA, SOLID-LDChip).

Field sampling at the Red Stone outcrop (Caleta Coloso–El Way formation) was conducted in August 2019 with additional campaigns in 2021 (February, May, August, October). Seventeen samples representing stratigraphic horizons (upper weathered conglomerates, cemented conglomerates, sandstones, mudstones, evaporite veins/crusts) were collected with detailed logging, stored appropriately (zip bags, glass tubes with foil lids, Falcon tubes for fragile evaporites), and documented. Dual temperature/relative humidity loggers monitored microclimate in situ. Mineralogy: Bulk and clay-fraction X-ray diffraction (XRD) characterized mineral assemblages (quartz, plagioclase, analcime, calcite, gypsum, halite, chlorite, vermiculite, illite/muscovite, hematite). Additional XRD under controlled relative humidity (1–60% RH) examined interlayer changes (e.g., chlorite swelling). Weathering/diagenetic trends were assessed via A–CN–K, A–C–N, and A–CNK–FM geochemistry. Microbiology: DNA was extracted and 16S rRNA Next-Generation Sequencing (NGS) profiled prokaryotic communities (richness and Shannon diversity indices computed). A culture-dependent approach isolated microorganisms on selective media, with CFU counts and phylogenetic identification via 16S (bacteria) and 18S (fungi) sequencing and phylogenetic analysis. Microscopy included SYBR Green staining and CARD-FISH to detect bacteria and archaea and estimate cell abundances. Organic geochemistry: Total organic carbon (TOC) and bulk δ13C were measured via elemental analysis and IRMS after decarbonation. Lipid biomarkers were extracted (DCM:MeOH), saponified, fractionated into neutral/acidic, further separated (alumina column), and analyzed by GC-MS (HP-5MS column) with internal standards and calibration (C10–C40 alkanes; FAME standards). Ratios of n-fatty acids to n-alkanes were used to infer biomass freshness. Spectroscopy and rover-analog instruments: Raman spectroscopy (532 nm micro-Raman; also 633 nm/1064 nm in complementary setups) targeted mineral and organic signatures; DRIFTS collected NIR and MIR spectra; LIBS (ChemCam lab replica under 7 Torr CO2 at 1.6 m) and time-resolved luminescence (SuperCam lab replica) analyzed elemental/mineralogical composition. SAM-like experiments used flight-analog pyrolysis-GC-MS (Frontier Labs pyrolyzer; Thermo GC-MS) to detect pyrolysis products and MTBSTFA-DMF wet chemistry for derivatization of polar molecules; instrumentation parameters mirrored SAM constraints (heating rates, columns, traps). MOMA testbed (flash pyrolysis at 400/600 °C; derivatization with MTBSTFA after direct or extractive preconcentration) emulated ExoMars analyses. SOLID-LDChip multiplex immunoassay probed molecular biosignatures of microbial taxa in extracts. Data analysis employed standard pipelines for NGS (mothur, dual-index MiSeq strategy) and statistical/community analyses (e.g., richness, Shannon), with geochemical and spectroscopic data compared to reference libraries and standards.

• Red Stone exhibits mineralogy and diagenetic features analogous to ancient Martian fan-deltas: sandstones and mudstones with quartz, plagioclase, analcime, calcite, gypsum, halite, chlorite, vermiculite, illite/muscovite, and hematite; evaporite veins/crusts and halite cementation indicate arid deposition; smectites (saponite, montmorillonite) and chlorite suggest low-temperature diagenesis (~70–90 °C).
• Microclimate: Upper weathered conglomerates experienced highest RH (up to 85.1% overnight/early morning), consistent with fog inputs.
• Microbial biomass and diversity: DNA concentrations were extremely low (≤1 µg DNA per g soil). NGS showed dominance of Alphaproteobacteria and Actinobacteria; richness and Shannon diversity highest in upper conglomerates and slightly elevated near evaporites, correlating with higher RH and suggesting thermotolerant taxa at sun-exposed surfaces.
• Dark microbiome: 8.9% of 16S sequences remained “unclassified,” and 40.8% could not be assigned beyond higher taxonomic ranks, indicating unusually high phylogenetic indeterminacy. Authors define “dark microbiome” as organisms detectable by sequencing whose phylogenetic identity cannot be determined with current databases, potentially novel extant species or relict ancient communities.
• Culture-dependent results: Only 19 unique bacterial and 2 fungal isolates were recovered, with very low CFUs (1.1×10^1–9.0×10^1). Most bacteria were Bacillaceae; isolates reflected mineralogical niches (e.g., Halobacillus in evaporites). Many taxa likely disperse via aeolian dust from coastal origins.
• Organic geochemistry: TOC was very low (max 0.11% dry weight); bulk nitrogen was not detected. δ13C ranged from −19.5 to −26.5‰, with evaporites most 13C-enriched and with higher TOC. Lipids comprised hydrocarbons (n-alkanes) and fatty acids; biomarkers included monounsaturated fatty acids, isoprenoids (pristane, phytane; detected in evaporites), heptadecene, and mid-chain monomethyl alkanes, consistent with phototroph inputs (e.g., cyanobacteria) though these taxa were not directly detected by NGS/culture/microscopy—suggesting relict biosignatures. Mudstones contained hopanes (C27–C31), robust bacterial molecular fossils. n-Fatty acids to n-alkanes ratios were high in upper/evaporite samples (≥16), indicating fresher biomass where water availability is greater.
• Microscopy and cell counts: SYBR Green staining yielded rare cells in a single lower-zone sample; CARD-FISH detected very low bacterial and archaeal abundances across all samples, peaking at ~6×10^3 cells per gram in top conglomerates.
• Spectroscopy and rover-analog detection of organics: Raman (532 nm) verified minerals but did not detect lipid signatures at these concentrations, underscoring the importance of laser parameters and spot size. DRIFTS found organics barely detectable in NIR (one peak at 1.36 µm) but multiple very weak MIR bands attributed to organics.
• SAM-like analyses: Pyrolysis-GC-MS detected aromatics (including oxygenated, chlorinated, and sulfur-bearing), and low-abundance straight-chain alkanes (C12–C35) near instrument detection limits. Wet-chemistry derivatization (MTBSTFA-DMF) revealed derivatized C7–C20 n-carboxylic acids in all samples and proline (only in top conglomerates), indicating active bacterial metabolism there. Detectability on Mars would depend on abundance and instrument operational constraints (temperature limits, analysis time, trap desorption).
• MOMA testbed: Flash pyrolysis detected no organics. After derivatization (direct and extraction-based), only evaporite samples yielded detectable aliphatic carboxylic acids, indicating organics in most samples are below MOMA detection limits.
• SOLID-LDChip: Signals were just above detection threshold but indicated heterotrophic bacteria and cyanobacteria signatures, corroborating the presence of extant and relict biosignatures.
• Overall, while mineralogy is well matched by rover-like instruments (DRIFTS, RLS, LIBS), organic and biosignature levels are at or below detection limits for many instrument modes and techniques.

Findings demonstrate that even in a geologically favorable, Mars-analog delta with clays and hematite, the abundance of organics and cells can be extremely low, challenging in situ detection. The defined “dark microbiome” reflects the limits of current phylogenetic databases and methods in resolving microbial identities in hyperarid, oligotrophic settings; it may represent novel extant taxa or remnants of ancient communities. Correlations between higher humidity/evaporites and increased diversity or fresher lipid signatures underscore the role of transient water availability in sustaining detectable biosignatures. Instrument tests reveal that while mineralogy can be robustly characterized with rover payload analogs, organic detection strongly benefits from wet-chemistry derivatization and may still fail for low-abundance targets, as shown by MOMA testbed outcomes. Immunoassay (SOLID-LDChip) can capture biological evidence beyond basic organics but remains sensitivity-limited at these concentrations. These constraints imply that Martian rocks with similarly low organics may yield non-detections depending on instrument mode, making false negatives likely. Consequently, sample return to Earth, where comprehensive, high-sensitivity multi-technique analyses are possible, is critical for a definitive assessment of past or present life on Mars.

Red Stone provides a unique Mars-analog assemblage combining fan-delta sedimentology, evaporites, hematite, and clay minerals with a sparse, phylogenetically indeterminate microbiome and extremely low organics. The study shows that many rover-relevant techniques can thoroughly characterize mineralogy but struggle to detect organics and biosignatures at such low levels, with wet-chemistry derivatization and immunoassays offering partial improvements. The results underscore the high likelihood of non-detections for similarly low-organic Martian rocks and reinforce the necessity of Mars Sample Return to enable definitive life detection. Future work should refine instrument sensitivities and operational parameters (e.g., laser wavelength/spot size for Raman, enhanced wet-chemistry workflows), target microenvironments with higher water activity (evaporites, clay-rich mudstones), and expand databases and methods to resolve dark microbiome lineages.

Biosignatures and organics in Red Stone are at or near detection limits, challenging even state-of-the-art laboratory instruments and leading to marginal detections by rover testbeds. The detectability of specific compounds depends on instrument parameters and constraints (e.g., temperature limits, analysis time, trap desorption, column characteristics), and flight instruments (e.g., SAM) may have higher detection thresholds than commercial analogs. Wet-chemistry derivatization did not capture all compound classes; MOMA flash pyrolysis returned non-detections without derivatization. Extremely low biomass and site-specific conditions may limit generalizability across all Mars-analog settings.