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

Lava tubes are promising astrobiological targets because they form protective subsurface habitats that shield organic matter and microbial communities from weathering, UV radiation, and temperature extremes. These environments on Earth are analogs to subsurface lava tubes identified on the Moon and Mars. Organic matter enters lava tubes via seeping water and air currents, depositing onto walls and speleothems and potentially preserving molecular biomarkers that record past vegetation, land use, climate, and microbial activity. While biomarker studies in carbonate cave speleothems are common, few have focused on volcanic lava tubes. The research question addressed here is whether sulfate-rich speleothems from Lanzarote lava tubes host preservable organic compounds and diagnostic lipid biomarkers, and how mineralogy and stable isotopes constrain the geological and biological origins of those organics. The study has implications for detecting biosignatures in analogous Martian subsurface settings where sulfates are known to occur.

Previous work has established that cave speleothems can archive environmental and biological signals, including lipid distributions (e.g., n-alkanes, n-alkanoic acids), which can differentiate between plant (long-chain, odd-over-even) and microbial (short-chain, even-over-odd; branched) sources. Analytical pyrolysis (Py-GC/MS) and thermochemolysis using TMAH (TMAH-Py-GC/MS) enable detection of diverse organic families with minimal sample preparation and are increasingly applied in astrobiology, including in Mars-analog studies and flight-like protocols (e.g., MOMA on ExoMars and SAM on Curiosity). Sulfate minerals on Mars (e.g., at Jezero crater) have been highlighted for their preservation potential of environmental conditions and possible biosignatures. On Earth, lava-tube sulfates like gypsum can encapsulate organic matter and microorganisms. However, high-temperature pyrolysis can generate artifacts (e.g., decarboxylation of fatty acids to alkanes), so derivatization approaches are needed to stabilize labile functional groups for more reliable biomarker interpretation.

Study area and sampling: Eleven sulfate-rich samples were collected from six Lanzarote lava tubes in May 2021 (Paso Esqueleto—PE; Montaña Rajada—MR; Maguez/El Ermitaño—LE; Las Breñas—LB; Monte Corona Puerta Falsa—MCPF; Los Naturalistas—CN). Sites span historical to Pleistocene ages and varying surface uses. Samples were collected with sterilized tools into sterile containers, stored at 4 °C, and freeze-dried for pyrolysis. Permits: RES-AUT 11/2021 and 4443/2021. Mineralogy: Initial binocular microscopy (×20–×60). Raman spectroscopy (Horiba XploraPlus, 532 nm) with database comparison (Crystal Sleuth, RRUFF). When needed, bulk X-ray powder diffraction (Philips X’Pert Pro MPD; Co anode 40 kV/40 mA; 3–85° 2θ; 0.017° step; 100 s/step; spinning; back-loading) with identification via PANalytical High Score Plus v4.9. Thermogravimetric analysis (TGA/DSC): Discovery SDT 650 (DSC/TGA), N2 atmosphere (50 mL/min). About 5 mg dry sample, 50–900 °C at 20 °C/min. Weight-loss fractions: W0 (50–105 °C), W1 (105–200 °C; moisture and very labile OM), W2 (200–400 °C; intermediate OM), W3 (400–575 °C; recalcitrant OM), W4 (575–850 °C; mineral fraction). Replicate error ≤0.2%. Elemental and stable isotope analysis: TOC quantified with Flash 2000 HT elemental analyzer (TCD) after decarbonization (1 M HCl), triplicate (2–5 mg). δ13C measured by EA-IRMS (Thermo Fisher Delta V Advantage). Standards: IAEA-CH-3, IAEA-CH-6, IAEA-600; precision ±0.1‰. δ34S of one sulfate per tube via Elemental Analyzer Costech ECS 4100 and Temperature Conversion EA coupled to Thermo Fisher Delta V IRMS; normalized to CDT using IAEA S-2 and S-3; reproducibility ±0.1‰. Analytical pyrolysis: Double-shot pyrolyzer (Frontier 3030D). Flash pyrolysis at 500 °C for 1 min based on TG-optimized release of organics. GC-MS: Shimadzu GC-2010 + QP2010 Plus (70 eV); Zebron ZB-5HT column (30 m × 0.25 mm × 0.10 μm), He 1.2 mL/min. Thermochemolysis (TMAH-Py-GC/MS) applied to preserve and detect carboxylic acids as FAMEs and minimize pyrolytic artifacts. Molecular families interpreted via van Krevelen-type diagrams and lipid indices (CPI, ACL). Statistical analysis: one-way ANOVA with Tukey for TOC and δ13C differences among caves.

- Mineralogy: Predominant Ca and Na sulfates (gypsum, thenardite, galeite); minor calcite (high-Mg in MCPF03) and halite. LB02 gypsum crust overlies basaltic host rock (iron- and magnesium-silicates detected). MR01 lacks gypsum, hosting thenardite and galeite, indicative of a young, minimally weathered system. - Thermogravimetry: Most samples (except LB) lost 69–97% of total mass in W1 (105–200 °C), reflecting dehydration of hydrated minerals (e.g., gypsum) and concurrent decomposition of very labile OM. LB samples show substantial W4 (>550 °C) losses (mineral decomposition) and a notable W3 contribution (recalcitrant OM: 13.9% and 9.9% relative loss for LB01 and LB02). MR01 exhibits minimal total weight loss (0.5% vs. 15.0–32.3% others), consistent with absence of gypsum. - Sulfur isotopes (δ34S ‰ VCDT; one per tube): Two genetic groups: volcanic (low δ34S), and marine (high δ34S). Values: PE01 22.12; PE04 −0.83; MR01 −0.83; LE01 6.29; LE05 20.17; LB01 (not reported); LB02 (not reported); MCPF01 (not reported); MCPF03 21.29; MCPF05 (not reported); CN03 4.54. Young/little-weathered tubes (MR, CN) show volcanic/low δ34S; others (PE, LB, parts of MCPF, LE05) show marine sea-spray signatures (~20–22‰). - Carbon and TOC: TOC ranges 0.08–0.77%. MR lowest (0.08%), LB highest (0.77%). δ13C varies across caves: LB reaches the lowest value (−32‰), LE the highest (−23‰). Patterns suggest dominant C3 vegetation inputs with variable microbial processing; higher δ13C in CN and LB attributed to microbial alteration; lower δ13C in MR, MCPF, PE linked to fresher plant-derived inputs. - Molecular composition (Py-GC/MS): van Krevelen patterns show mixtures of furans, carbohydrates, n-alkane/alkene pairs (C9–C31/32), phenols, aromatics, hydroaromatics, PAHs, and lignin-derived methoxyphenols. PE shows EPS-related compounds (furans, carbohydrates) and mixed alkyl sources; LB shows low-functionalized aromatics/PAHs; MR resembles PE04/LB02 with lignin-derived compounds; CN dominated by phenols and branched alkanes; LE dominated by aromatics and methoxyphenols; MCPF samples show n-alkane/alkene pairs, aromatics, carbohydrates, PAHs. - Lipid biomarkers: FAMEs detected after TMAH derivatization, dominated by C16:0 (palmitic) and C18:0 (stearic) across caves, with short even-over-odd chains indicative of microbial input. Branched FAMEs (bacterial) present variably: highest in MCPF03 (21.4%), then MCPF01 (8.8%), MCPF05 (2.2%). CN sample: lipids constitute 49.5% of pyrolysable OM; n-alkanes show even-over-odd pattern (CPI 0.9) with dominant C12 and 74.8% branched alkanes—consistent with microbial sources. LB01/02 dominated by short-chain n-alkanes (ACL 12.9–15.4) with bimodal distributions (C15 and C27 in LB02), indicating combined microbial and plant wax inputs (C27–C31). LE lacks >C23 n-alkanes, supporting microbial dominance (ACL ~14.5). MCPF shows a sequence of alteration: MCPF01 (long-chain plant wax dominance; ACL 25.1), MCPF03 (more short-chain, microbial influence; ACL 20.4), MCPF05 (short-chain dominance; ACL 17.7). PE01 displays bimodal distribution with strong long-chain vegetation signal (CPI 2.7; ACL 20.9), while PE04 shows microbial-dominated unimodal C15 with abundant branched alkanes (49.7%). - Astrobiology relevance: Hydrated sulfates contain structurally bound water; gypsum is known on Mars. Sulfate speleothems can trap organics and microorganisms, potentially preserving ancient biosignatures; combined mineralogical, isotopic, and molecular data identify candidate biosignatures (branched alkanes, branched FAMEs, short-chain FAMEs) and constrain sources.

The multi-proxy approach (mineralogy, TG/DSC, δ34S/δ13C, Py-GC/MS/TMAH-Py-GC/MS) disentangles geological versus biological inputs to sulfate speleothems. δ34S distinguishes volcanic degassing-derived sulfates in younger/pristine tubes (low δ34S) from marine sea-spray sulfates in older or more open systems (high δ34S), linking mineral formation pathways to environmental context. TOC and δ13C show that organic inputs are largely C3-derived but variably processed by subsurface microbial communities; higher δ13C and abundant branched alkanes in CN and LB reflect microbial alteration, whereas lower δ13C in MR, MCPF, PE align with fresher plant inputs. Molecular profiles further resolve sources: long-chain n-alkanes (C27–C31, high CPI/ACL) indicate plant wax contributions; short-chain n-alkanes, even-over-odd short-chain FAMEs, and branched alkanes/FAMEs implicate microbial membranes and activity. TG evidence that most organic decomposition coincides with gypsum dehydration suggests at least a portion of OM is incorporated within hydrated sulfate structures, consistent with potential longer-term preservation. These results validate that sulfate speleothems can archive diagnostic lipid biomarkers and environmental signals, supporting their use as terrestrial analogs for detecting and interpreting biosignatures in Martian subsurface sulfates.

Sulfate speleothems from Lanzarote lava tubes are dominated by gypsum, thenardite, and galeite, with minor calcite and halite; mineral assemblages and δ34S values reveal both volcanic and marine sulfate origins. TG indicates major mass loss between 105–200 °C due to dehydration of hydrated minerals concomitant with decomposition of labile OM, implying some OM is structurally or texturally incorporated. δ13C patterns reflect C3 vegetation inputs variably modified by microbial processing. Molecular and lipid screening show widespread microbial biomarkers (short-chain and branched alkanes; branched FAMEs; dominance of C16:0 and C18:0) alongside vegetation markers (long-chain n-alkanes). MCPF samples illustrate a gradient from fresh plant-derived OM to microbially altered OM. The integration of Py-GC/MS and TMAH-Py-GC/MS provides rapid, sensitive detection of lipid biomarkers that can discriminate OM sources. Overall, sulfate speleothems are promising archives of biosignatures with direct implications for life-detection strategies in Martian lava tubes and sulfate deposits.

- The porous, thin crust or powdery nature of sulfate speleothems prevented separate characterization of surface versus interior fractions, limiting assessment of OM age and preservation state. - High-temperature pyrolysis can generate artifacts (e.g., decarboxylation of fatty acids to alkanes); although TMAH thermochemolysis was applied to stabilize acids, residual ambiguity in alkane sources remains. - δ34S was measured on one sample per lava tube, which may not capture intra-cave variability. - Low TOC contents and small sample sizes constrain detection of low-abundance biomarkers. - Environmental heterogeneity (age, ventilation, proximity to entrances) and potential recent inputs complicate temporal attribution of OM (ancient vs. recent).