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.

Candidate phylum Saccharibacteria (former TM7) are abundant and widespread in nature, but little is known about their ecophysiology and detailed phylogeny. In this study phylogeny, morphology, and ecophysiology of Saccharibacteria were investigated in activated sludge from nine wastewater treatment plants (WWTPs) from Japan and Denmark using the full-cycle 16S rRNA approach and microautoradiography (MAR) - fluorescence in situ hybridization (FISH). Phylogenetic analysis showed that Saccharibacteria from all WWTPs were evenly distributed within subdivision 1 and 3 and in a distinct phylogenetic clade. Three probes were designed for distinct saccharibacterial groups, and revealed morphotypes representing thin filaments, thick filaments, and rods/cocci. MAR-FISH showed that most probe-defined Saccharibacteria utilized glucose under aerobic-, nitrate reducing-, and anaerobic conditions. Some Saccharibacteria also utilized N-acetylglucosamine, oleic acid, amino acids, and butyrate, which are not predicted from available genomes so far. In addition, some filamentous Saccharibacteria exhibited β-galactosidase and lipase activities determined using a combination of enzyme-labeled fluorescence and FISH (ELF-FISH). No uptake of acetate, propionate, pyruvate, glycerol, and ethanol was observed. These results indicate that Saccharibacteria is a phylogenetically diverse group and play a role in the degradation of various organic compounds as well as sugar compounds under aerobic-, nitrate reducing-, and anaerobic conditions.

We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that “ Brocadia ”-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 μm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas -, Nitrosospira -, and Nitrosococcus -like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira -like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH ₄ ⁺ and NO ₂ ⁻ consumption rates decreased from 0.68 and 0.64 μmol cm ⁻² h ⁻¹ at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 μmol cm ⁻² h ⁻¹ at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH ₄ ⁺ and NO ₂ ⁻ and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira -like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O ₂ or organic compounds, which consequently established suitable microenvironments for anammox bacteria.

Although the anaerobic ammonium oxidation (anammox) process has attracted attention regarding its application in ammonia wastewater treatment based on its efficiency, the physiological characteristics of anammox bacteria remain unclear because of the lack of pure-culture representatives. The coexistence of heterotrophic bacteria has often been observed in anammox reactors, even in those fed with synthetic inorganic nutrient medium. In this study, we recovered 37 draft genome bins from a long-term-operated anammox column reactor and predicted the metabolic pathway of coexisting bacteria, especially Patescibacteria (also known as Candidate phyla radiation). Genes related to the nitrogen cycle were not detected in Patescibacterial bins, whereas nitrite, nitrate, and nitrous oxide-related genes were identified in most of the other bacteria. The pathway predicted for Patescibacteria suggests the lack of nitrogen marker genes and its ability to utilize poly-N-acetylglucosamine produced by dominant anammox bacteria. Coexisting Patescibacteria may play an ecological role in providing lactate and formate to other coexisting bacteria, supporting growth in the anammox reactor. Patescibacteria-centric coexisting bacteria, which produce anammox substrates and scavenge organic compounds produced within the anammox reactor, might be essential for the anammox ecosystem.