The coexistence of uncultured heterotrophic bacteria belonging to the phylum Chloroflexi has often been observed in anaerobic ammonium oxidation (anammox) reactors fed with synthetic nutrient medium without organic carbon compounds. To determine if coexisting Chloroflexi in anammox reactors scavenge organic matter derived from anammox bacterial cells, the present study was conducted to investigate the substrate uptake pattern of the uncultured Chloroflexi present in an anammox reactor and to clarify if they take up microbial products derived from anammox bacterial cells. To accomplish this, combined microautoradiography and fluorescence in situ hybridization (MAR–FISH) was conducted. Phylogenetic analysis revealed that 36% of the clones analyzed in this study were affiliated with Chloroflexi. The sequence similarities to Anaerolinea thermophila and Caldilinea aerophila within the phylum Chloroflexi were only 81.0–88.7% and 80.3–83.8%, respectively. The uncultured Chloroflexi were found to incorporate sucrose, glucose, and N-acetyl-glucosamine. The 14C-tracing experiment revealed that the uncultured Chloroflexi were clearly MAR-positive, indicating the utilization of decaying anammox bacterial cell materials. Taken together, these results indicate that coexisting uncultured Chloroflexi in anammox reactors scavenge organic compounds derived from anammox bacterial cells.

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