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

The primary motivation behind past, current, and future Mars missions is the question of whether life ever existed on the planet. Missions like the Mars Exploration Rovers, Phoenix, and the Mars Science Laboratory (MSL) and Mars2020 rovers aimed to identify habitable environments and evidence for life as we know it. Liquid water being a key requirement, many rovers landed in areas with geomorphological or mineralogical evidence of past water, such as clay minerals. These spacecraft carry compositional instruments to identify minerals and search for organic molecules essential for life. Mass spectrometers on various missions can detect organic molecules and building blocks of life. While Viking and Phoenix missions didn't find robust evidence of organics in Martian soils, MSL's SAM and Mars2020's SHERLOC have identified simple organic molecules. Results suggest that organics aren't prevalent on the Martian surface, but this study hypothesizes that instrument limitations and the nature of Martian organics may hinder life detection. To test these limitations, the researchers closely examined Red Stone, a unique site in the Atacama Desert, a well-known Mars analog due to its aridity and age.

The paper draws upon extensive previous research on Mars exploration and the Atacama Desert as a Mars analog. It references studies focusing on the identification of habitable environments on Mars based on evidence of past water, including the findings of past missions like Viking, Phoenix, MSL, and Mars2020. The literature review also incorporates research on the mineralogy of Mars and the presence of clay minerals. Additionally, the paper cites research on organic molecules detected on Mars and the limitations of current instrumentation in detecting low levels of organics. The Atacama Desert's relevance as a Mars analog is established through a review of existing literature demonstrating its geological and climatic similarities to Mars. The paper references studies on the microbial communities found in the Atacama, particularly emphasizing the survival strategies of organisms in hyper-arid conditions.

The study focused on the Red Stone outcrop in the Atacama Desert, a geologically Mars-analog site. Sampling occurred across multiple visits in 2019 and 2021, meticulously documenting the stratigraphic sequence. Samples were analyzed using a range of techniques including X-ray diffraction (XRD) to identify minerals, including a detailed analysis of clay minerals under varying relative humidity (RH). Next-generation sequencing (NGS) determined microbial diversity and identified a "dark microbiome" – microbes detectable but not classifiable. Culture-dependent methods were also employed for microbial isolation and characterization. Biosignature analyses included measurements of total organic carbon (TOC), stable carbon isotopic composition (δ¹³C), and lipid biomarker identification using gas chromatography-mass spectrometry (GC-MS). Microscopic analyses (bright field and chlorophyll autofluorescence), and Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH) were employed to visualize microbial cells. Several testbed instruments intended for Mars missions were used: DRIFTS (Diffuse Reflectance Infrared Fourier Transform Spectroscopy), RLS (Raman laser spectrometer ExoMars simulator), LIBS (Laser Induced Breakdown Spectroscopy), SAM-like pyrolysis, MOMA (Mars Organic Molecule Analyzer) and SOLID-LDChip (Signs of Life Detector). These testbed analyses involved both direct analysis and derivatization techniques (MTBSTFA-DMF) to enhance the detection of polar molecules.

Red Stone's mineralogy closely resembles that of ancient Mars, particularly in its hematite content and clay minerals like smectite and chlorite. The site harbors a microbial community with unexpectedly high phylogenetic indeterminacy ("dark microbiome"), characterized by low DNA yields (1 µg DNA/g soil). Alphaproteobacteria and Actinobacteria dominated the identified phyla. The highest microbial diversity was found in the upper, weathered conglomerates, correlating with higher relative humidity. Organic carbon content (TOC) was extremely low (maximum 0.11% dry weight), with no detectable nitrogen. GC-MS identified hydrocarbons and fatty acids as the main components of the lipid fraction, with stable carbon isotope ratios (δ¹³C) ranging from -19.5‰ to -26.5‰. The presence of certain fatty acids suggested possible sulfate-reducing bacteria. While some biosignatures suggested ancient phototrophs (e.g., cyanobacteria), no extant phototrophic organisms were detected by NGS or microscopy. The high n-fatty acids/n-alkanes ratios in certain samples indicated the presence of relatively fresh biomass. Testbed Mars instruments detected the site's mineralogy but struggled with organic detection. DRIFTS showed limited success in the near-infrared (NIR) but detected some organic bands in the mid-infrared (MIR). SAM-like pyrolysis revealed various organics, but their low abundance suggests they may not be detectable using the flight model. MOMA analysis, even with derivatization, only yielded detectable organics in evaporite samples. SOLID detected microbes, including cyanobacteria, confirming past water availability.

The Red Stone findings highlight the challenges in detecting life on Mars. The extremely low organic content and the presence of a significant "dark microbiome" underscore the limitations of current rover instruments. While the mineralogical similarities between Red Stone and Mars are compelling, the difficulty in detecting even relatively abundant biosignatures using Mars-bound instrumentation suggests that returning samples to Earth is critical for conclusive life detection. The success of wet chemistry derivatization techniques in laboratory settings further stresses the importance of including these techniques in future missions. The study also emphasizes that the "dark microbiome" could represent either truly novel life forms or the fossilized remains of past life, further complicating the search for signs of past life.

Red Stone provides a valuable analog for understanding the challenges of Mars life detection. The study reveals that current and planned Mars missions may miss subtle biosignatures present in low-organic environments. Future research should focus on refining instrument sensitivity, and incorporating improved wet chemistry techniques for sample preparation and analysis. A Mars Sample Return mission is crucial to address definitively whether life ever existed on Mars.

The study's focus on a single, albeit well-characterized, Mars analog site limits the generalizability of its conclusions. The low organic matter concentrations present analytical challenges and may affect the detection limits of various methods. Extrapolating these findings to the entirety of Mars requires careful consideration of the diversity of Martian environments.