Abstract EANA2025-187

Extreme environments present a range of hostile conditions for microbial life, including high temperatures, extreme pH, and fluctuating salinity. Microbial communities surviving under such stress have evolved unique biochemical strategies, often displaying distinct pigmentation and odor. These extremophiles are key candidates for astrobiological research due to their adaptability and potential applications in bioremediation, bioleaching, and space mission support. In Saudi Arabia, geothermal activity has led to the formation of several hot springs, particularly in the Al Lith region. “Ain al Harrah” stands out with a temperature of 85°C. The region also features a hypersaline pink lake with salinity exceeding 20%. These sites offer promising habitats for extremophiles and provide valuable insights into microbial survival strategies and early life evolution.

This study aims to isolate and characterize culturable microorganisms from three hot springs including Ain al Harrah and the pink lake. Genomic and metagenomic analyses were conducted to investigate genes and pathways involved in survival under extreme conditions, toxic compound degradation, and metal transformation processes.

Water and sediment samples were collected for microbial cultivation. Sediments (0.5g) were sprinkled on agar plates, and 1L of water was filtered through 0.22µm membranes. Serial dilutions were plated on high- and low-nutrient agar prepared with 50% native water. Incubation was performed at 35°C and 60°C for two weeks. Thirty axenic isolates were selected based on morphology and thermophilic traits for whole genome sequencing on the PromethION platform. Raw sequencing data were assessed using LongQC and FastQC, assembled with Flye, Canu, and Unicycler, and polished using Medaka. Genome quality was evaluated using QUAST, CheckM2, and BUSCO. Taxonomic classification and plasmid detection were performed with GTDB-Tk and Mob-suite. Prokka was used for functional annotation. For metagenomic data, the nf-core/mag and MetaWRAP pipelines were used to reconstruct and quantify the Metagenome Assembled Genomes (MAGs).

The selected isolates displayed genes for biofilm formation, heat/cold/oxidative stress resistance, DNA repair, motility, sporulation, and energy conservation. Three genomes—Luteimonas terrae THG-MD21, Massilia litorea LPB0304, and Bacillus infantis DSM 19098—showed ANI <95%, suggesting potential novelty. Metagenomic analysis revealed complete iron and sulfur metabolic pathways and methanotrophy potential. Genes for chlorite dismutation (cld) and perchlorate reduction (pcrABCD) were identified, especially in Chloroflexota, Desulfobacterota, Actinomycetota, and Acidobacteriota.  Genomes in the Abyssobacteria phylum encoded 3 of 4 essential perchlorate reductase subunits. Additional metal metabolism genes—citA, citB, soxA, dsrAB, mtrAB, mtoA, phoA, sitAs—were widespread across the MAGs.

These findings highlight the microbial communities’ potential for biotechnological applications in extreme environments. Future work will evaluate their capacity to survive and function under simulated Martian conditions, further informing their relevance to astrobiology and in-situ resource utilization.