The presence of perchlorate in the Martian soil may limit in-situ resource utilization (ISRU) technologies to support human outposts. In order to exploit the desiccation, radiation-tolerant cyanobacterium Chroococcidopsis in Biological Life Support Systems based on ISRU, we investigated the perchlorate tolerance of Chroococcidopsis sp. CCMEE 029 and its derivative CCMEE 029 P-MRS. This strain was obtained from dried cells mixed with Martian regolith simulant and exposed to Mars-like conditions during the BIOMEX space experiment. After a 55-day exposure of up to 200 mM perchlorate ions, a tolerance threshold value of 100 mM perchlorate ions was identified for both Chroococcidopsis strains. After 40-day incubation, a Mars-relevant perchlorate concentration of 2.4 mM perchlorate ions, provided as a 60 and 40% mixture of Mg- and Ca-perchlorate, had no negative effect on the growth rate of the two strains. A proof-of-concept experiment was conducted using Chroococcidopsis lysate in ISRU technologies to feed a heterotrophic bacterium, i.e. an Escherichia coli strain capable of metabolizing sucrose. The sucrose content was fivefold increased in Chroococcidopsis cells through air-drying and the yielded lysate successfully supported the bacterial growth. This suggested that Chroococcidopsis is a suitable candidate for ISRU technologies to support heterotrophic BLSS components in a Mars-relevant perchlorate environment that would prove challenging to many other cyanobacteria, allowing a ‘live off the land’ approach on Mars.

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<p class="western" align="justify"><span lang="en-US">Martian regolithic soil is considered an inhospitable </span><span lang="en-US">environment</span><span lang="en-US"> to life as we know it with low availability of nutrients and the presence </span><span lang="en-US">of powerful oxidants, namely perchlorate </span><span lang="en-US">salts</span><span lang="en-US">. Extreme microorganisms such as cyanobacteria of the genus </span><span lang="en-US"><em>Chroococcidiopsis</em></span><span lang="en-US"> dominate rock-dwelling communities in extreme deserts resembling the actual Martian environment. The strain </span><span lang="en-US"><em>Chroococcidiopsis</em></span><span lang="en-US"> 029, extremely tolerant to desiccation, ionizing, and UV radiation, can thrive in Mars-like conditions in a dried state. In the present work, we investigated the response of </span><span lang="en-US"><em>Chroococcidiopsis</em></span><span lang="en-US"> 029 when grown for a 3-week period using Martian regolith simulant containing 2.4 mM </span><span lang="en-US">perchlorate anions</span><span lang="en-US">. The growth either in the planktonic cells or biofilm life style was monitored following the in chlorophyll </span><span lang="en-US"><em>a</em></span><span lang="en-US"> content. The cellular and molecular responses to 2.4 mM </span><span lang="en-US">perchlorate anions</span><span lang="en-US"> was studied following cell viability according to: i) PCR-PMA assay, ii) changes in gene expression of three SOD-coding genes (</span><span lang="en-US"><em>soda 2.1, soda.2, and sodC</em></span><span lang="en-US">), and iii) production of intracellular ROS as rev</span><span lang="en-US">e</span><span lang="en-US">aled by CLSM. Results suggested that perchlorate did not compromise cell viability and that a significant over-expression of three SOD isoforms occurred after the one-week exposure with a greater expression of the membrane-bound MnSOD (</span><span lang="en-US"><em>sodA 2.1</em></span><span lang="en-US">) in comparison to the cytoplasmic isoforms MnSOD (</span><span lang="en-US"><em>sodA 2.2</em></span><span lang="en-US">) and Cu/ZnSOD (</span><span lang="en-US"><em>sodC</em></span><span lang="en-US">). The accumulation of ROS within the cells was observed after 1-day exposure to perchlorate. Future investigations on the effect of Mars-like conditions in hydrated biofilms with 2.4 mM ClO4- and Martian regolith simulant will be carried out supported by the Europlanet scholarship 202</span><span lang="en-US">4</span><span lang="en-US">. These results are relevant for the habitability of Mars and the development of In-situ Resource Utilization.</span></p>