Competitive enrichment of comammox Nitrospira in floccular sludge

The discovery of complete ammonium oxidation (comammox) has subverted the traditional perception of two-step nitrification, which plays a key role in achieving biological nitrogen removal from wastewater. Floccular sludge-based treatment technologies are being applied at the majority of wastewater treatment plants in service where detection of various abundances and activities of comammox bacteria have been reported. However, limited efforts have been made to enrich and subsequently characterize comammox bacteria in floccular sludge. To this end, a lab-scale sequencing batch reactor (SBR) in the step-feeding mode was applied in this work to enrich comammox bacteria through controlling appropriate operational conditions (dissolved oxygen of 0.5 ± 0.1 g-O₂/m³, influent ammonium of 40 g-N/m³ and uncontrolled longer sludge retention time). After 215-d operation, comammox bacteria gradually gained competitive advantages over counterparts in the SBR with a stable nitrification efficiency of 92.2 ± 2.2 %: the relative abundance of Nitrospira reached 42.9 ± 1.3 %, which was 13 times higher than that of Nitrosomonas, and the amoA gene level of comammox bacteria increased to 7.7 ± 2.1 × 10⁶ copies/g-biomass, nearly 50 times higher than that of conventional ammonium-oxidizing bacteria. The enrichment of comammox bacteria, especially Clade A Candidatus Nitrospira nitrosa, in the floccular sludge led to (i) apparent affinity constants for ammonium and oxygen of 3.296 ± 0.989 g-N/m³ and 0.110 ± 0.004 g-O₂/m³, respectively, and (ii) significantly low N₂O and NO production, with emission factors being 0.136 ± 0.026 % and 0.023 ± 0.013 %, respectively.