Isolation of two species of Caldatribacterium (Atribacterota) and the importance of folate for their culturability

The phylum Atribacterota is ubiquitous in anoxic environments where it plays important roles in syntrophic carbon and hydrogen metabolism; however, only two species have been isolated. Here, we report the isolation of two additional species representing a new family, Caldatribacterium saccharofermentans from a hot spring and Caldatribacterium inferamans from a deep fractured-rock aquifer. Both were co-enriched on carbohydrates with sulfate-reducing bacteria (SRB). Despite unsuccessful attempts to isolate them on the defined enrichment medium, we finally isolated both species by adding yeast extract to the medium. We show that folate was the key factor provided by the SRBs and the yeast extract and that folate provided in vitamin solutions was inadvertently removed by filter sterilization according to standard media preparation protocols. We further confirm the absence of folate biosynthesis pathways across the phylum and suggest that folate precipitation during media preparation limits cultivation of Atribacterota in toto. The two Caldatribacterium species share unusual features with other Atribacterota , including three lipid membrane-like layers (LMLs), with the inner LML that appears to surround a nucleoid, and a high percentage of transmembrane proteins. This study expands the culturability of Atribacterota and identifies conserved features, including sugar fermentation, unusual cell ultrastructure, and vitamin dependencies necessary for their cultivation.