Multi-Technique Characterization of 3.45 Ga Microfossils on Earth: A Key Approach to Detect Possible Traces of Life in Returned Samples from Mars

The NASA Mars 2020 Perseverance rover is actively exploring Jezero crater to conduct analyses on igneous and sedimentary rock targets from outcrops located on the crater floor (Maaz and Seitah formations) and from the delta deposits, respectively. The rock samples collected during this mission will be recovered during the Mars Sample Return mission, which plans to bring samples back to Earth in the 2030s to conduct in-depth studies using sophisticated laboratory instrumentation. Some of these samples may contain traces of ancient martian life that may be particularly difficult to detect and characterize because of their morphological simplicity and subtle biogeochemical expressions. Using the volcanic sediments of the 3.45 Ga Kitty's Gap Chert (Pilbara, Australia), containing putative early life forms (chemolithotrophs) and considered as astrobiological analogues for potential early Mars organisms, we document the steps required to demonstrate the syngenicity and biogenicity of such biosignatures using multiple complementary analytical techniques to provide information at different scales of observation. These include sedimentological, petrological, mineralogical, and geochemical analyses to demonstrate macro- to microscale habitability. New approaches, some unavailable at the time of the original description of these features, are used to verify the syngenicity and biogenicity of the purported fossil chemolithotrophs. The combination of elemental (proton-induced X-ray emission spectrometry) and molecular (deep-ultraviolet and Fourier transform infrared) analyses of rock slabs, thin sections, and focused ion beam sections reveals that the carbonaceous matter present in the samples is enriched in trace metals (e.g., V, Cr, Fe, Co) and is associated with aromatic and aliphatic molecules, which strongly support its biological origin. Transmission electron microscopy observations of the carbonaceous matter documented an amorphous nanostructure interpreted to correspond to the degraded remains of microorganisms and their by-products (extracellular polymeric substances, filaments horizontal ellipsis ). Nevertheless, a small fraction of carbonaceous particles has signatures that are more metamorphosed. They probably represent either reworked detrital biological or abiotic fragments of mantle origin. This study serves as an example of the analytical protocol that would be needed to optimize the detection of fossil traces of life in martian rocks.