Isolation of genome-predicted Caldatribacterium (Atribacterota) reveals pervasive microbial cultivation problem due to folate precipitation

Most bacterial phyla have few or no pure cultures, including Atribacterota, comprised of ubiquitous anaerobes. Here, we report genome-guided enrichment and isolation of two Atribacterota species representing a new family, Caldatribacterium saccharofermentans from a hot spring, and Caldatribacterium inferamans from a deep aquifer. Both were co-enriched with sulfate-reducing bacteria and initially resisted isolation, which we link to inadvertent removal of precipitated folic acid by filter-sterilization of unbuffered Wolin’s vitamin solution. We then predict folate auxotrophy across the Atribacterota and ~29% of all bacteria, with extensive auxotrophy in 27% of phyla. Since ≥604 of 791 ( ≥ 76%) media with folic acid additions in the MediaDive database use unbuffered vitamin solutions in which folic acid is likely removed during filter-sterilization, we propose that folate auxotrophy limits culturability in defined media en masse. We also uncover unusual features of Caldatribacterium, including three lipid membrane-like layers (LMLs), with the inner LML surrounding the nucleoid, and a high percentage of secreted proteins, supporting a unique cell biology of Atribacterota. Most bacterial phyla have few or no representatives in pure culture. Here, Hedlund et al. isolate two new species within the phylum Atribacterota with unique cell biological features. In addition, they reveal a common mistake with media preparation leading to folate removal, which might explain some cultivation challenges.