Cultivation and Enrichment of “Caldatribacterium,” a Member of the Candidate phlyum Atribacteria (OP9)

Event Center BC

Atribacteria (OP9) is candidate phylum with no representatives in pure culture that is found in various anaerobic environments worldwide. Although recent metagenomic and single-cell genomic data have opened a window into the physiological potential of the Atribacteria, cultivation of members of this candidate phylum would greatly enhance the ability to understand their metabolic capacity and ecology. Members of Caldatribacterium, a lineage of the Atribacteria present in some geothermal springs, were previously found to be abundant in thermophilic in situ enrichments on lignocellulosic substrates (corn stover) in Great Boiling Spring, NV. In an attempt to cultivate Caldatribacterium in the laboratory, these enrichments were used as inoculum in media with xyloglucan as a sole carbon source. This yielded stable laboratory cultures of Caldatribacterium, present at ~2-5% of the total population as assessed by fluorescence in situ hybridization (FISH) and quantitative polymerase chain reaction (qPCR). Additional enrichments were performed using different individual sugars as substrates. Of these, fucose gave the highest abundance of Caldatribacterium (~50%), and the dilution-toextinction technique was performed to attempt to isolate Caldatribacterium. The results from (q) PCR of the dilution to extinction process indicated that the samples of bacteria could be high in abundance and possibly a pure sample. Further experimentation with FISH verified that in fact the samples were not pure, but still had significantly high percentages (>90%) of Caldatribacterium. These highly enriched will be useful in determining the metabolic capabilities of this lineage of Atribacteria.