Decoding organic compounds in lava tube sulfates to understand potential biomarkers in the Martian subsurface

The search for extraterrestrial life focuses on environments potentially capable of supporting life, including subsurface structures like lava tubes. These tubes, formed during volcanic eruptions, create unique habitable environments and are considered analogous to subsurface structures on the Moon and Mars. Earth's lava tubes are colonized by microorganisms, influencing biogeochemical cycles and creating unique ecosystems sustained by organic matter transported from the surface via seeping water and air currents. This organic matter deposits on cave walls and speleothems, supporting microbial communities and becoming incorporated into speleothems. Biomarkers, distinctive organic molecules indicating specific biosynthetic activities, are used to trace past environmental changes and understand life's history on Earth. In astrobiology, the study expands beyond organic compounds to include biosignatures—a broader range of evidence, including isotopic ratios, mineralogical textures, and molecular distributions—that can reveal both extant and extinct life forms. Lava tubes offer an ideal environment for microbial growth and organic matter preservation due to their protective nature against weathering, sunlight, and extreme temperatures. Sulfate deposits, like gypsum, commonly found in lava tubes, may encapsulate microbial life and organic compounds. While biomarker research in limestone caves is extensive, lava tube studies are limited. This research investigates the organic compound origins in lava tube speleothems to better understand geological processes and biological sources of organic compounds within these unique geological formations, improving the search for biosignatures on other planets.

Numerous studies have explored molecular biomarkers in speleothems from various environments, focusing on karstic caves to understand past vegetation, land use changes, and climatic conditions. Biomarkers, including lipids, are crucial in interpreting past environmental changes and understanding the history of life. Lipids, particularly long-chain *n*-alkanes with an odd carbon number predominance, indicate a vegetation source, while short-chain *n*-alkanes and *n*-alkanoic acids with even carbon number predominance suggest microbial sources. Various lipid indexes, like the carbon preference index (CPI) and average carbon length (ACL), help assess biomass maturity and identify biological sources. In lava tubes, lipid biomarkers provide information on organic matter sources, including capturing natural and human-induced alterations. Analytical pyrolysis (Py-GC/MS) is used to identify organic compounds; however, high-temperature pyrolysis can alter compounds, prompting the use of derivatization techniques like TMAH-Py-GC/MS to improve accuracy. The application of analytical pyrolysis and thermochemolysis (TMAH-Py-GC/MS) is gaining importance in astrobiology and space exploration, particularly for identifying lipid biomarkers and potential biosignatures in Martian samples. Instruments on Mars rovers utilize Py-GC/MS for extraterrestrial organic analysis, highlighting its value in detecting potential signs of past microbial life.

Eleven sulfate-rich mineral samples from six lava tubes on Lanzarote, Canary Islands, were collected. The samples underwent several analyses: 1. **Mineralogy:** Binocular microscope examination, Raman spectroscopy, and X-ray powder diffraction (XRPD) identified minerals. 2. **Thermogravimetric analysis (TGA):** TGA and differential scanning calorimetry (DSC) determined weight loss during decomposition, characterizing sample composition and stability. Weight loss fractions were classified based on temperature ranges: W0 (50–105 °C), W1 (105–200 °C), W2 (200–400 °C), W3 (400–575 °C), and W4 (575–850 °C). 3. **Elemental and stable isotope analysis:** Total organic carbon (TOC) content and carbon isotope ratios (δ¹³C) were measured using an elemental analyzer coupled to a thermal conductivity detector (TCD) and an isotope ratio mass spectrometer (IRMS). Sulfur isotope ratios (δ³⁴S) were determined using an elemental analyzer coupled to an IRMS. 4. **Analytical pyrolysis (Py-GC/MS and TMAH-Py-GC/MS):** Freeze-dried samples were flash-pyrolyzed at 500 °C, and released compounds were separated and analyzed using gas chromatography/mass spectrometry (GC/MS). TMAH-Py-GC/MS was used to protect functional groups during pyrolysis. Van Krevelen diagrams visualized the organic fraction's composition. 5. **Lipid compound screening:** *n*-alkanes and fatty acid methyl esters (FAMEs) were analyzed to identify their relative abundance, average chain length (ACL), and carbon preference index (CPI).

Mineralogical analysis revealed the predominance of calcium (Ca) and sodium (Na) sulfates (gypsum, thenardite, galeite), with minor calcium carbonate (calcite) and sodium chloride (halite). Sample LB02 also contained iron and magnesium silicates. Sulfur isotope (δ³⁴S) values indicated two origins: volcanic (low δ³⁴S values, e.g., Montaña Rajada) and oceanic (higher δ³⁴S values, e.g., Paso Esqueleto). Total organic carbon (TOC) content ranged from 0.08% to 0.77%, varying significantly among lava tubes (highest in Las Breñas, lowest in Montaña Rajada). Carbon isotope (δ¹³C) values showed variations among lava tubes, with higher values (e.g., Los Naturalistas and Las Breñas) suggesting microbial alteration of organic matter, and lower values (e.g., Montaña Rajada, Monte Corona Puerta Falsa, and Paso Esqueleto) suggesting input of fresh, unaltered organic material. Py-GC/MS analysis revealed diverse organic compounds, including furans, carbohydrates, *n*-alkanes/alkenes, and aromatics. Specific compounds varied among samples, with some suggesting microbial activity (e.g., furans in Paso Esqueleto) and others suggesting vegetation influence (e.g., lignin-derived compounds in Paso Esqueleto). Lipid analysis revealed *n*-alkanes and FAMEs. *n*-alkane distributions varied among lava tubes, with some indicating microbial activity (short-chain *n*-alkanes) and others suggesting vegetation input (long-chain *n*-alkanes). FAMEs were dominated by palmitic acid (C₁₆₀) and stearic acid (C₁₈₀), with variations suggesting contributions from microbial activity and vegetation. Branched alkanes and FAMEs, commonly associated with bacterial biosynthesis, further supported the microbial influence. The presence of 10-methylhexadecanoic acid methyl ester (10-Me C₁₆ FAME) in some samples may indicate sulfate-reducing microbial species.

The findings indicate a complex interplay of geological processes (volcanic and oceanic inputs) and biological activity (microbial and vegetation) shaping the organic composition of Lanzarote lava tube speleothems. The variations in TOC, δ¹³C, and lipid profiles reflect the influence of microbial communities and surface-derived organic matter. The presence of microbial biomarkers, such as branched alkanes and FAMEs, strongly suggests microbial activity within these environments. The identification of lignin-derived compounds and long-chain *n*-alkanes indicates a contribution from above-ground vegetation, although the extent varies among samples. The observed differences among lava tubes likely reflect variations in age, environmental conditions, and the extent of microbial activity and/or alteration of original organic material. The use of both Py-GC/MS and TMAH-Py-GC/MS provided a more complete picture of the organic compounds, reducing the potential for pyrolysis artifacts and enhancing biomarker identification. The study highlights the potential of sulfate speleothems as repositories of organic matter, including potential biosignatures. These results have significant implications for understanding potential biosignatures in analogous Martian environments and the search for life beyond Earth.

This multi-analytical study of Lanzarote lava tube sulfate speleothems provides crucial insights into the complex interplay of geological and biological factors in these subsurface environments. The identification of various organic compounds, particularly microbial biomarkers, indicates the potential for preserving evidence of past or present life in similar settings on other planets. Future studies should focus on analyzing larger datasets, improving techniques for distinguishing between ancient and recent organic matter, and exploring the preservation potential of various minerals in extreme environments. This research enhances our understanding of potential biosignatures and refines strategies for detecting life beyond Earth.

The highly porous and powdery nature of the speleothems hindered the differentiation between surface and interior compositions, potentially affecting the interpretation of organic matter preservation. The study did not directly determine the age of the organic compounds, making it challenging to definitively assign specific temporal periods to microbial activity or organic influx. Further research is needed to clarify the specific types of microorganisms responsible for observed biomarker distributions and to understand the processes governing organic matter preservation within the speleothems.