title: Aliens Among Us: Microbial Life of Analogous Martian Environments
Creators: name: Lee, Cameron J
Publisher: name: Griffith University
Year: 2024
Subjects: subject: astrobiology
subject: extremophiles
subject: halophiles
subject: microbiology

description: Extremophiles are a classification of microorganisms that are capable of growth in environments too extreme for human conditions. Isolation and identification of novel organisms from extreme environments have vastly increased understanding of how these organisms adapt, survive, and thrive in extreme conditions, extending the limits of what we define as "life". Astrobiology utilises the advancements and knowledge gained from studying extremophiles to search for life on other planets beyond Earth. It's thought that if life were to exist elsewhere in the universe it would be simple single celled organisms. Halophiles are a subclassification of microorganisms that have adapted to saline/hypersaline environments. A hypersaline environment is defined as an extreme environment with salinity &gt;3.5% upward to saturation (35%). Halophilic and halotolerant microorganisms are diverse across all three domains of life, the majority of which are from prokaryotic classes Gammaproteobacteria and Haloarchaea. Adaptive strategies for halophilic organisms make sure they don't lose water through osmosis by generating a hyperosmotic cytoplasm, these strategies are "high salt-in", which accumulates K+ and Cl- ions intracellularly to balance concentration, and "low-salt, organic solute-in", which uses organic solutes (osmolytes) to create a more flexible osmotic adaptation for organisms. Present day Mars is known as a "cold and dry" planet with little surface water activity. Evidence has pointed to pockets of surface water containing various salts in extreme concentrations. These salts include chlorides and perchlorates, the latter of which has been noted as ubiquitous on the planet's surface. It's expected that any life that survives on the surface in these water pockets would be adapted to extreme salinities and perchlorate levels. Further in situ evidence has identified significant concentrations of manganese oxides coating rocks in the Gale Crater of Mars. This thesis sought to identify novel manganese oxidising and perchlorate-reducing halophiles that may be capable of surviving present day Martian conditions. Twelve hypersaline environments domestic and international were sampled and enriched for manganese oxidising and perchlorate tolerant/reducing halophiles. Culture dependent studies revealed 25 isolates capable of manganese oxidation and sustained growth in elevated perchlorate concentrations. These strains belonged Pseudomonadota (synonym Proteobacteria) and Bacillota (synonym Firmicutes) phyla, though predominantly from the Halomonas genus. Four strains, LES-1, PAR-7, PAR-8, and SSL-5 were selected for further analysis. The substrate utilisation requirements varied among isolates in terms of growth density. The rates of oxidation among the halophiles varied between the strains, PAR-7 had the most rapid rate of oxidation followed by PAR-8 and LES-1. Strain SSL-5 was inconclusive on time course studies despite recorded manganese oxidation activity. Perchlorate tolerance also varied among the strains, PAR-7 and SSL-5 had tolerance &gt;5.0% while LES-1 and PAR-8 had tolerance up to 2.0%. Molecular analysis revealed strains LES-1, PAR-7, and PAR-8 were closest related to Halomonas halodenitrificans DSM 735 at 86.06%, 86.44% and 86.15%, respectively. The strains were ~99% similar between themselves, though phenotypically different. Isolate SSL-5 was 87.45% similar to Halomonas shengliensis CGMCC 1.6444. The genome analysis of isolates revealed genes associated with manganese oxidation called multicopper oxidases, these genes were encoded in each genome except for SSL-5. Another manganese oxidation molecular pathway is the cytochrome c-type biogenesis operon, Ccm. The Ccm operon was identified in all four isolates. No known direct perchlorate reduction pathways were identified in the four isolates, indicating the reduction activity could be due to novel mechanisms. Genome similarity for the four isolates indicates they're novel Halomonas species. Metagenomic analysis of two hypersaline environments revealed that the microbial composition of Kati Thanda-Lake Eyre, South Australia and Lake Parachalmic, Victoria was significantly different. Kati Thanda-Lake Eyre was predominantly Haloarchaea in distribution, ~68%, and Proteobacteria comprised ~13%. However, Lake Parachalmic was dominantly Bacteria at ~95% and Proteobacteria comprised ~59%. Various multicopper oxidase like genes were identified within the environments in small distributions, &lt;0.0015% and 0.43% of the total function in Kati Thanda-Lake Eyre and Lake Parachalmic, respectively. No known perchlorate reduction mechanisms were noted in the hypersaline environments. The results of this current study have built upon the microbial diversity in Australian hypersaline environments, particularly with insights into their astrobiology related significance. Bacteria that can oxidise manganese and tolerate increased perchlorate in hypersaline conditions were isolated for the first time and have significant potential for Martian related viability studies, thus detecting life on Mars.