The Life of a Halophile: Hypersaline Environments and their Microbial Residents

Kurzfassung

Introduction: Extreme saline environments serve as valuable analogues for astrobiological research. Despite their seemingly hostile and harsh conditions, these habitats support the growth of a wide variety of extremophilic organisms, particularly halophilic archaea. These microorganisms exhibit remarkable resistance to various stress factors, including desiccation, radiation, and osmotic pressure. Hypersaline ecosystems are present in both modern and ancient marine environments, such as salt caverns, salt flats, and evaporating salt lakes, where water gradually evaporates, leaving behind concentrated salt deposits. Methods: In this study, samples were collected from diverse hypersaline environments, including a saltsaturated brine from ancient Zechstein Sea deposits in Lüneburg, Germany, as well as salt flats and lakes in South America, including the Salar de Uyuni in Bolivia (Fig. 1) and nearby lakes. Environmental parameters were recorded, and microbial diversity was analysed through 16S rRNA (V3/V4) amplicon sequencing. Additionally, culturable microorganisms were enriched and isolated. Selected archaeal isolates were tested for their resistance to astrobiologically relevant stressors, followed by whole-genome sequencing. Results: The analysed saline environments exhibited salt concentrations ranging from 10% (w/v) to full saturation. In the salt brine of Lüneburg, 16S rRNA sequencing revealed the presence of halophilic organisms with a microbial community dominated by Halobacterales, alongside halophilic bacteria such as Desulfohalobiaceae and Thiohalorhabdaceae. In addition, a high abundance of the order Nanosalinales (phylum Nanohaloarchaeota) was detected - an archaeal lineage that has yet to be cultured independently and typically found in symbiosis with other haloarchaea, making them promising candidates for pure culture isolation. Similarly, in the Salar de Uyuni and nearby lakes, Halobacterales and Nanosalinales were prevalent, while the genus Salinibacter emerged as one of the dominant bacterial taxa. Microscopic analysis indicated notable morphological diversity, with isolates displaying pleomorphism. Pure cultures from the genera Halorubrum, Haloarcula, Natronomonas, Natrinema and Halolamina from both habitats were successfully cultivated and isolated. Stress tests on selected isolates displayed tolerance to gamma irradiation (>1000 Gy), heavy ions (>500 Gy) and UV-radiation (up to 300 J/m²) as well as the ability to thrive in NaCl concentrations up to 30% (w/v). Additionally, isolates exhibited enhanced growth in typically toxic perchlorates concentrations of > 0.5 – 1%, similar to levels detected on Mars. Whole-genome sequencing (WGS) of twoarchaeal isolates revealed multiple copies of the 16S rRNA operon with sequence divergences of up to 5%, a phenomenon previously observed in halophilic archaea. This genetic variability may contribute to the remarkable stress resistance of haloarchaea. Conclusion: Hypersaline environments serve as promising analogue sites for extraterrestrial habitats. They host a diverse range of halophilic microorganisms capable of withstanding high radiation and extreme salt concentrations. This study describes the microbial community of diverse hypersaline environments and highlights the potential of these accessible ecosystems for astrobiological research.