From bio to geo: Assessing lipid biomarker integrity and preservation through an environmental lithification gradient
Authors: L. Finkel Pablo , Carrizo Daniel , Parro Victor , Vignale Federico , Farías María Eugenia , Sánchez-García Laura 
Published: July 2024

Lipids, characterized as organic compounds insoluble in water, play a vital role in the composition of biological cell membranes. Their widespread presence in terrestrial life renders them significant as universal indicators of life. Unlike many other biomolecules, lipids exhibit notable geostability, demonstrating greater resistance to thermal and diagenetic processes within suitable protective matrices. Consequently, they possess a unique ability to effectively preserve information about their origin and metabolic processes for extensive periods, potentially spanning billions of years (Vinnichenko et al., 2020). This longevity is particularly crucial in astrobiology, given the vast geological timescales of planetary bodies. The biogenic nature and chemical resilience of lipid compounds make them exceptional targets for investigating extraterrestrial life.The scope of our work was to investigate the preservation of lipids across a gradient of lithification. We scrutinized lipid biomarkers extracted from three distinct stages of microbial mat development: a fresh microbial mat actively undergoing growth, a lithifying microbial mat in the transition phase towards petrification, and a fully lithified microbialite, an organo-sedimentary deposit representing the geo-endmember in the high-altitude lacustrine ecosystem. Lipids extracted from the samples were fractionated into three organic fractions comprising defunctionalized hydrocarbons (apolar fraction), alkanoic acids (acidic fraction), and alcohols (polar fraction), each subjected to analysis using gas chromatography coupled with mass spectrometry for the identification of individual lipid compounds. Additionally, compound-specific isotopic analysis facilitated the elucidation of their metabolic pathways of synthesis, aiding in the attribution of lipid origin based on the 13C/12C ratio (&#948;13C), a key feature to strengthen biological source allocation.The fresh microbial mat, composed of three coloured layers, exhibited the highest diversity and abundance of lipid biomarkers. Its upper orange layer displayed a predominance of long-chain n-alkanes, indicative of plant-derived inputs from the surrounding environment, while the intermediate green layer and the lower pink layer showed elevated levels of phytol, phytene, and squalene, suggesting the presence of photosynthetic prokaryotes. All layers were rich in saturated and polyunsaturated fatty acids, primarily associated with active prokaryotic life and microalgae.As the samples transitioned towards lithification, the abundance of complex lipid features such as methyl branches (isoprenoids), cyclic structures (hopanoids and sterols), functional groups (hydroxy and carboxy), or double bonds (unsaturated alkanes or fatty acids) progressively decreased. Consequently, the lithifying mat displayed lower concentrations of linear, saturated (n-alkanes), and methyl-branched compounds compared to the fresh mat, although still higher than those found in the microbialite. The latter exhibited minimal presence of lipids, among which pure hydrocarbon skeletons (n-alkanes) were relatively abundant, representing the most resilient lipid residues, which could be called geolipids. Notably, the microbialite concentrations of n-alkanes approached to a higher extent those of the lithifying mat and fresh microbial mat compared to other lipid families (Figure 1), thus illustrating how linear hydrocarbons can show a selective recalcitrance over other lipid structures. Still, it was interesting to note some preservation of unsaturations, methyl branching (e.g., isoprenoids &#8211; Figure 1C) and functional groups (hydroxyl or carboxyl terminal groups) in the microbialite.Overall, we report a loss of complexity as the samples transitioned toward the geo end-member, losing most of their labile features and retaining mostly linear and simple hydrocarbon chains. While these &#8220;simpler&#8221; biomarkers retain a higher potential for preservation in the geological record, the deciphering of their biogenicity becomes highly challenging, as it is the case for the short hydrocarbon chains detected at Gale Crater on Mars (C10 &#8211; C12n-alkanes, SAM instrument; Freissinet et al., 2019). Determining their carbon isotopic composition helps discriminating their biogenic versus abiotic origin (i.e., 13C depleted signature) and assigning a potential biosource. Finally, the detection of minimal amounts of functional groups and double bonds in the microbialite &#8211; the most lithified sample &#8211; indicates that despite their low concentrations, there seem to be mechanisms preventing labile features from being obliterated early in carbonate-rich matrices. Such mechanisms should be studied in depth to elucidate the conditions involved in a potential long-term preservation in regard to Mars exploration.This work was funded by the Spanish Ministry of Science and Innovation/State Agency of Research MCIN/AEI/10.13039/501100011033 and by &#8220;ERDF A way of making Europe&#8221;, through grants PRE2020-093795 (P.L.F.), ESP2017-87690-C3-3-R (D.C.), PID2021-126746NB-I00 (V.P.), and RYC2018-023943-I (L.S.-G.)Figure 1. Differences in hydrocarbon concentrations across sample types. A) n-alkanes arranged by increasing carbon number, B) alkenes (unsaturated alkanes) arranged by increasing carbon number, C) isoprenoid compounds and D) polycyclic (mainly pentacyclic) triterpenoids, which were only detected in the fresh microbial mats. Mean concentration of all compounds per sample is depicted as a straight line to facilitate comparison between samples.&#160;ReferencesFreissinet C, Glavin DP, Buch A, et al. Detection of long-chain hydrocarbons on Mars with the Sample Analysis at Mars(SAM) instrument. In: Ninth International Conference on Mars 2019, Pasadena, CA, 2019; p. 2.Vinnichenko G, Jarrett AJM, Hope JM, et al. Discovery of the Oldest Known Biomarkers Provides Evidence for Phototrophic Bacteria in the 1.73 Ga Wollogorang Formation, Australia. Geobiology 2020;18(5):544&#8211;559; doi: 10.1111/gbi.12390.