Highly enriched anammox within anoxic biofilms by reducing suspended sludge biomass in a real-sewage A²/O process

This study proposes a novel strategy of stably enriching anammox in mainstream, based on the competitive difference to NO₂^- between anoxic biofilms and suspended sludge. A modified anaerobic-anoxic-oxic (A²/O) process run for 500 days with actual municipal wastewater. Microbial analysis revealed that anoxic-carrier biofilms had a significantly higher abundance of anammox (qPCR: 0.74% - 4.34%) than suspended sludge (P< 0.001). Batch tests showed that anammox within anoxic-carrier biofilms contributed to significant nitrogen removal, coupled with partial-denitrification (NO₃^- → NO₂^-). The anammox genus, Ca. Brocadia, was highly enriched when suspended sludge was accidentally lost. Further batch tests found that reducing suspended biomass helped anammox enrichment in anoxic-carrier biofilms, because the suspended sludge had strong NO₂^- competition (NO₂^- → N₂) with anammox (increased nirK). Metagenomic sequencing revealed that Ca. Brocadia dominates in the anoxic-carrier biofilms, and is the most important narG contributor to NO₃^- → NO₂^-, which could have promoted the competition of NO₂^- with heterotrophic bacteria. For this A²/O process, the low effluent total nitrogen (8.9 mg ± 1.0 mg N/L) was attributed to partial-denitrification coupling with anammox, demonstrating that this process is applicable to the general influent N-concentration range (30 mg - 50 mg NH₄⁺-N/L) of municipal wastewater treatment plants (WWTPs). Based on the special competitive preference of anammox for NO₂^-, this study provides a promising and practical alternative for enriching anammox bacteria in municipal WWTPs.