Simulating Mars Drilling Mission for Searching for Life: Ground-Truthing Lipids and Other Complex Microbial Biomarkers in the Iron-Sulfur Rich Río Tinto Analog

Abstract

Sulfate and iron oxide deposits in Río Tinto (Southwestern Spain) are a terrestrial analog of early martian hematite-rich regions. Understanding the distribution and drivers of microbial life in iron-rich environments can give critical clues on how to search for biosignatures on Mars. We simulated a robotic drilling mission searching for signs of life in the martian subsurface, by using a 1m-class planetary prototype drill mounted on a full-scale mockup of NASA's Phoenix and InSight lander platforms. We demonstrated fully automated and aseptic drilling on iron and sulfur rich sediments at the Río Tinto riverbanks, and sample transfer and delivery to sterile containers and analytical instruments. As a ground-truth study, samples were analyzed in the field with the life detector chip immunoassay for searching microbial markers, and then in the laboratory with X-ray diffraction to determine mineralogy, gas chromatography/mass spectrometry for lipid composition, isotope-ratio mass spectrometry for isotopic ratios, and 16S/18S rRNA genes sequencing for biodiversity. A ubiquitous presence of microbial biomarkers distributed along the 1m-depth subsurface was influenced by the local mineralogy and geochemistry. The spatial heterogeneity of abiotic variables at local scale highlights the importance of considering drill replicates in future martian drilling missions. The multi-analytical approach provided proof of concept that molecular biomarkers varying in compositional nature, preservation potential, and taxonomic specificity can be recovered from shallow drilling on iron-rich Mars analogues by using an automated life-detection lander prototype, such as the one proposed for NASA's IceBreaker mission proposal.

Fingerprinting molecular and isotopic biosignatures on different hydrothermal scenarios of Iceland, an acidic and sulfur-rich Mars analog

“…www.nature.com/scientificreports/ Molecular and isotopic lipid analysis. The lyophilized samples were extracted with a mixture of DCM:MeOH (3:1, v/v) to obtain a total lipid extract (TLE) by (i) ultrasonic bath (3x; further details in 54 ) the MAT samples (1-3 g dw), or (ii) Soxhlet extraction (24 h; details in 55 ) the rest of samples (~ 30 g dw). In all cases, internal standards (tetracosane-D50, myristic acid-D27, 2-hexadecanol) were added prior to extraction.…”

“…The remaining lipidic extract was then acidified with HCl (37%) to remove K + from the solution by precipitation of KCl, then recovering the liberated carboxylic groups by liquid-liquid extraction with n-hexane (acidic fraction). Further separation of the neutral fraction into non-polar (hydrocarbons) and polar (alkanols and sterols) was done according to a method extensively described elsewhere 55 . Aliquots of the acidic fraction were heated with 1 ml of 10% BF 3 in MeOH at 80 °C for 30 min to methylate the fatty acids.…”

Geomicrobiological Heterogeneity of Lithic Habitats in the Extreme Environment of Antarctic Nunataks: A Potential Early Mars Analog

“…Raw sequences were processed in MOTHUR software v.1.43.0 (Schloss et al, 2009), using a custom script based upon MiSeq SOP (Kozich et al, 2013), as it has been employed before by our group in Sanchez-Garcia et al, 2018, Azua-Bustos et al, 2019, Lezcano et al, 2019, Sanchez-Garcia et al, 2020b further explained in Supporting Information Figure S3. To identify putative contaminant sequences remaining within the samples that were not detected with the negative control approach, 'decontam' R package (Davis et al, 2018) was used by employing the frequency-based identification method (i.e., odd high abundance of specific taxa on low DNA-concentration samples) with a threshold of 0.1.…”

“…In this study, the deeper characterization allowed to complete the partial taxonomic adscription of the archaeal community coming from the lower quality of the sequences and the impossibility to find appropriate counterparts in the databases (similarly to Pessi et al, 2015;Zhang et al, 2020). Together, they also point to a high suitability of both open (i.e., sequencing) and close (i.e., immunoassays) techniques to describe microbial communities from extreme environments, despite the small divergences that could inherently appear between these approaches (Fernandez-Martinez et al, 2019;Lezcano et al, 2019;Sanchez-Garcia et al, 2020a). However, both techniques lack of an explicit identification of active microorganisms, so future studies would benefit from also including metatranscriptomics or metaproteomics analysis for a better discussion of these results.…”

The Complex Molecules Detector (CMOLD): A Fluidic-Based Instrument Suite to Search for (Bio)chemical Complexity on Mars and Icy Moons

“…We have been using FSI for many years, both in the laboratory and in multiple field campaigns, and the sensitivities of detection obtained range from 0.1 to 10 ppm for biochemical compounds and 10 3 to 10 4 cells or spores per milliliter (Fernandez-Calvo et al , 2006 ; Rivas et al , 2008 ; Parro et al , 2011a ; Blanco et al , 2013 ; Lezcano et al , 2019 ; García-Descalzo et al , 2019 ; Sánchez-García et al , 2020 ).…”