A Simple and Effective Method for Solid Medium Cultivation of Strictly Hydrogen- and Sulfur-oxidizing Chemolithoautotrophs Predominant in Deep-‍sea Hydrothermal Fields.

Strictly hydrogen- and sulfur-oxidizing chemolithoautotrophic bacteria, particularly members of the phyla Campylobacterota and Aquificota, have a cosmopolitan distribution in deep-sea hydrothermal fields. The successful cultivation of these microorganisms in liquid media has provided insights into their physiological, evolutionary, and ecological characteristics. Notably, recent population genetic studies on Sulfurimonas (Campylobacterota) and Persephonella (Aquificota) revealed geographic separation in their populations. Advances in this field of research are largely dependent on the availability of pure cultures, which demand labor-intensive liquid cultivation procedures, such as dilution-to-extinction, given the longstanding assumption that many strictly or facultatively anaerobic chemolithoautotrophs cannot easily form colonies on solid media. We herein describe a simple and cost-effective approach for cultivating these chemolithoautotrophs on solid media. The results obtained suggest that not only the choice of gelling agent, but also the gas phase composition significantly affect the colony-forming ratio of diverse laboratory strains. The use of gellan gum as a gelling agent combined with high concentrations of H2 and CO2 in a pouch bag promoted the formation of colonies. This contrasted with the absence of colony formation on an agar-solidified medium, in which thiosulfate served as an electron donor, nitrate as an electron acceptor, and bicarbonate as a carbon source, placed in anaerobic jars under an N2 atmosphere. Our method efficiently isolated chemolithoautotrophs from a deep-sea vent sample, underscoring its potential value in research requiring pure cultures of hydrogen- and sulfur-oxidizing chemolithoautotrophs.