Roles of EDTA washing and Ca²⁺ regulation on the restoration of anammox granules inhibited by copper(II)

Highlights: • 80.5% of the Cu in anammox granules was introduced via adsorption. • Cu(II) internalized on/into AnAOB cells plays a crucial role in toxicity. • EDTA washing contributes to the detoxification of anammox granules. • Ca²⁺ can stimulate the re-growth of damaged anammox consortium. - Abstract: We investigated the feasibility of using ethylene diamine tetraacetic acid (EDTA) washing followed by Ca²⁺ enhancement for the recovery of anammox reactors inhibited by Cu(II). Kinetic experiments and batch activity assays were employed to determine the optimal concentration of EDTA and washing time; and the performance and physiological dynamics were tracked by continuous-flow monitoring to evaluate the long-term effects. The two-step desorption process revealed that the Cu in anammox granules was primarily introduced via adsorption (approximately, 80.5%), and the portion of Cu in the dispersible layer was predominant (accounting for 71.1%). Afterwards, the Cu internalized in the cells (approximately, 14.7%) could diffuse out of the cells and be gradually washed out of the reactor over the next 20 days. The Ca²⁺ addition that followed led to an accelerated nitrogen removal rate recovery slope (0.1491 kgN m⁻³ d⁻²) and a normal biomass growth rate (0.054 d⁻¹). The nitrogen removal rate returned to normal levels within 90 days and gradual improvements in granular characteristics were also achieved. Therefore, this study provides a new insight that externally removing the adsorbed heavy metals followed by internally repairing the metabolic system may represent an optimal restoration strategy for anammox consortium damaged by heavy metals.