Elucidating how calcium inhibits anammox systems and a novel recovery strategy using cation exchange resin:...

The application of anaerobic ammonium oxidation (anammox) process is hindered by the susceptibility of anaerobic ammonium-oxidizing bacteria (AnAOB) to inhibitors including excessive Ca²⁺ concentrations. In this study, the mechanisms of Ca²⁺ inhibition and subsequent recovery using cation exchange resin (CER) were systematically investigated by analyzing the characteristics of anammox granular sludge combined with metagenomic analysis. Under 400 mg/L Ca²⁺ stress, the nitrogen removal rate (NRR) remained stable despite a sharp decrease in AnAOB abundance. This resilience was attributed to increased extracellular polymeric substance secretion for Ca²⁺ sequestration and functional compensation owing to the activation of the nitrification-denitrification process. When Ca²⁺ concentration raised to 800 mg/L, a dense CaCO₃ precipitate layer formed on the granules, protecting the internal AnAOB but severely limiting substrate mass transfer, leading to a reduction in the NRR from 2.0 to 1.5 kg N/m³/d. Under this dense shield, metabolic pathways shifted toward biosynthesis, and carbon fixation and nucleotide metabolism were activated to support cellular proliferation. CER treatment removed the precipitate layer, restored mass transfer, and fully restored nitrogen removal performance. This recovery was accompanied by peak abundances of auxiliary bacteria (Fimbriimonas and Chthonomonas) and metabolic signatures, indicating a transition from stress defense to growth-oriented homeostasis. This study elucidated the response behavior of the anammox system under graded Ca²⁺ stress and during the recovery process, and proposed a novel recovery method suitable for industrial application.