Anaerobic ammonium oxidation (anammox) process is a highly effective and economic technology for nitrogen removal from wastewater. However, the slow growth of anammox bacteria and sludge flotation often hinder its field application. Ion adsorption and crystal precipitation can potentially promote the sludge granulation and hence address the above issues. This study investigated two approaches to support anammox granulation through Mg2+ adsorption and magnesium ammonium phosphate (MAP) precipitation. Mg2+ addition improved the specific anammox activity (SAA) by 4.09 to 4.75-fold compared to MAP-mediated ones, which could be explained by the upregulations of nitrogen and inorganic carbon metabolisms. The active extracellular polymeric substances generation at metabolites level may also favor the granulation in Mg2+-mediated anammox. However, sludge loss halted the continuous size increase of sludge. Differently, MAP promoted granulation by physically increasing the granular density, which allowed for a greater retention of sludge within the reactor. However, the co-growth of MAP precipitates with anammox may lead to mass transfer limitations, resulting in down-regulated gene expressions and metabolites in inorganic carbon metabolism, which negatively impacted the SAA. Overall, both strategies achieved comparable nitrogen removal capacities. Nevertheless, the co-growth of MAP and anammox was promising for effectively mitigating sludge flotation. Our study provided strategies and omics-based evidences for anammox granulation and activity variations, benefiting anammox practical applications.@en

In the completely autotrophic nitrogen removal over nitrite (CANON) process, the conflicting oxygen requirements of anammox and ammonium-oxidizing bacteria often lead to retardation in anammox activity. However, our study achieved stable nitrogen removal with a maximum capacity of 1096 g-N/m3/d in a 20 m3 CANON reactor under long-term intensive aeration. The anammox bacteria unusually distributed in the outer layer of the biofilm and demonstrated remarkable oxygen tolerance. Their activity only declined by 18.5 % under 2.0 mg/L of dissolved oxygen. When anammox bacteria encountered oxygen exposure, they adopted some strategies. Metatranscriptomics revealed that Candidatus Kuenenia, the dominant anammox species in our system, downregulated its gene expressions involved in carbon metabolism and oxidative phosphorylation. This may reduce electron leakage that combines with O2, thereby minimizing the generation of reactive oxygen species (ROS). By contrast, the secretion of extracellular proteins and conversion of O2·− were upregulated to eliminate ROS promptly. This behavior endowed Ca. Kuenenia with a unique oxygen detoxification pathway: O2·− were initially converted to H2O2 by superoxide dismutase SOD2 and superoxide reductase dfx (major role), followed by reduction to H2O via non-heme chloroperoxidase cpo (a newly recognized mechanism in the oxygen detoxification of anammox) and catalase katE. These results expanded the current knowledge of anammox alleviating oxidative stress.@en