Perchlorate stress responses of Haloferax volcanii and implications on the habitability of Mars
Authors: Gries Anne , Heinz Jacob , Schulze-Makuch Dirk 
Published: September 2022

&lt;p&gt;&lt;strong&gt;Introduction&lt;/strong&gt;&lt;/p&gt;
&lt;p&gt;All known terrestrial life forms require liquid water to survive and grow. Therefore, under the conditions present on Mars today, sub-zero brines might be one of few environments allowing for microbial life to persist. These brines likely contain perchlorates which have been found in martian regolith (Hecht et al. 2009). Whilst their hygroscopicity as well as their potential to lower the freezing point of water support the presence of liquid water, ion-specific toxic characteristics such as chaotropicity may be detrimental for many organisms.&lt;/p&gt;
&lt;p&gt;An organism thriving under very high salt concentrations is the halophilic archaeon &lt;em&gt;Haloferax volcanii&lt;/em&gt; isolated from the Dead Sea (Mullakhanbhai and Larsen, 1975). Whilst halophilic archaea require salt, most importantly sodium chloride (NaCl), to survive, they only tolerate limited amounts of sodium perchlorate (Oren et al. 2014).&lt;/p&gt;
&lt;p&gt;Here we present the concept and preliminary results of experiments investigating the stress responses of &lt;em&gt;H. volcanii&lt;/em&gt; growing in medium containing sodium perchlorate (NaClO&lt;sub&gt;4&lt;/sub&gt;). To determine stress caused specifically by NaClO&lt;sub&gt;4&lt;/sub&gt;, and not by general ionic or oxidative stress or low water activity, results are compared to stress responses caused by NaCl and glycerol.&lt;/p&gt;
&lt;p&gt;&lt;strong&gt;Methods&lt;/strong&gt;&lt;/p&gt;
&lt;p&gt;&lt;em&gt;H. volcanii&lt;/em&gt; was grown at 40&amp;#176;C in altered DSMZ medium #97, where MgSO&lt;sub&gt;4&lt;/sub&gt; was replaced by MgCl&lt;sub&gt;2&lt;/sub&gt; and the concentration of NaCl was lowered from 4 to 1.7&amp;#160;mol/kg, in order to allow the addition of different amounts of NaClO&lt;sub&gt;4&lt;/sub&gt;, glycerol, or additional NaCl (up to NaCl saturation). Growth was tracked by measuring the optical density at 600 nm and regularly verified by colony forming unit (CFU) counts. Morphology was observed by light microscopy. Additional methods such as proteomics will be applied in upcoming experiments for analysing the stress responses in cells grown under the highest possible stress conditions.&lt;/p&gt;
&lt;p&gt;&lt;strong&gt;Preliminary Results&lt;/strong&gt;&lt;/p&gt;
&lt;p&gt;&lt;em&gt;H. volcanii&lt;/em&gt; is able to grow in medium containing 1.7&amp;#160;mol/kg of NaCl up to saturating concentrations, as was expected based on the very high tolerated NaCl range already described by Mullakhanbhai and Larsen (1975). Although visibly reduced, growth occured also in medium containing up to at least 0.6&amp;#160;mol/kg NaClO&lt;sub&gt;4&lt;/sub&gt;, which was achieved by letting the cells adapt to incrementally increasing NaClO&lt;sub&gt;4&lt;/sub&gt; concentrations.&lt;/p&gt;
&lt;p&gt;Fully substituting NaCl by NaClO&lt;sub&gt;4&lt;/sub&gt; did not support any growth and resulted in complete death of the cell culture, as suggested by lack of CFUs. Hence, it is likely that the ClO&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt; anion cannot provide the necessary conditions for cell metabolism accomplished by Cl&lt;sup&gt;-&lt;/sup&gt; and possibly exhibits additional stress factors like chaotropic destabilization of biomacromolecules, as observed recently in the halotolerant yeast &lt;em&gt;Debaryomces hansenii&lt;/em&gt; (Heinz et al. 2022). Although the NaCl tolerance of &lt;em&gt;H. volcanii &lt;/em&gt;is higher, the tolerance for NaClO&lt;sub&gt;4&lt;/sub&gt; is much lower than that of &lt;em&gt;D. hansenii&lt;/em&gt;. Unlike the yeast, &lt;em&gt;H. volcanii&lt;/em&gt; has no cell wall and, as a prokaryote, no cell compartmentalisation in general, which might cause increased susceptibility towards the destabilizing properties of perchlorate. Various cell morphologies, ranging from coccoid to rod shaped as well as varied sizes were observed under the different stress conditions, calling for additional research.&lt;/p&gt;
&lt;p&gt;&lt;strong&gt;Outlook&lt;/strong&gt;&lt;/p&gt;
&lt;p&gt;Further experimentation is needed to confirm the abovementioned results and to determine the maximum solute concentrations at which &lt;em&gt;H. volcanii&lt;/em&gt; can grow. Stress responses of cells grown under these conditions will be determined thereafter. By generating this data, we aim to better understand microbial responses to perchlorate stress and thereby further elucidate the habitability of martian brines and possibly can propose potential biomarkers for upcoming life detection missions on Mars.&lt;/p&gt;
&lt;p&gt;&amp;#160;&lt;/p&gt;
&lt;p&gt;&lt;strong&gt;References&lt;/strong&gt;&lt;/p&gt;
&lt;p&gt;Hecht MH, Kounaves SP, Quinn RC, West SJ, Young SMM, Ming DW, Catling DC, Clark BC, Boynton W V., Hoffman J, DeFlores LP, Gospodinova K, Kapit J, Smith PH (2009) Detection of perchlorate and the soluble chemistry of martian soil at the phoenix lander site. Science 325:64&amp;#8211;67 . doi: 10.1126/science.1172466&lt;/p&gt;
&lt;p&gt;Heinz J, Doellinger J, Maus D, Schneider A, Lasch P (2022) Perchlorate-Specific Proteomic Stress Responses of Debaryomyces hansenii Could Enable Microbial Survival in Martian Brines. 1&amp;#8211;25, preprint available at bioRxiv, doi: 10.1101/2022.05.02.490276.&lt;/p&gt;
&lt;p&gt;Mullakhanbhai MF, Larsen H (1975) Halobacterium volcanii spec. nov., a Dead Sea halobacterium with a moderate salt requirement. Archives of Microbiology 104:207&amp;#8211;214 . doi: 10.1007/BF00447326&lt;/p&gt;
&lt;p&gt;Oren A, Elevi Bardavid R, Mana L (2014) Perchlorate and halophilic prokaryotes: Implications for possible halophilic life on Mars. Extremophiles 18:75&amp;#8211;80 . doi: 10.1007/s00792-013-0594-9&lt;/p&gt;