Resetting substrate loading enables recovery of inhibited anammox granular reactors through energy-associated...

Substrate inhibition remains a critical bottleneck for the stable operation and recovery of anaerobic ammonium oxidation (anammox) reactors. Although reducing influent substrate concentrations is the most commonly adopted recovery strategy, the appropriate reset point for restarting an inhibited reactor remains poorly understood. Here, using a continuous substrate-inhibited anammox granular reactor as a model system, we showed that severe inhibition occurred at influent ammonium and nitrite concentrations of 400 and 480 mg·L^-1, respectively. Notably, reducing the influent concentrations to 300 and 360 mg·L^-1, which had previously sustained stable high-rate nitrogen removal, was insufficient to restore reactor performance. Successful recovery was only achieved after further decreasing the influent concentrations to 150 and 180 mg·L^-1, respectively. Metatranscriptomic and genome-resolved analyses showed that the dominant anammox bacterium, Candidatus Kuenenia stuttgartiensis (AMX1), remained in a state of energy-metabolism dysregulation under substrate inhibition, as evidenced by significant upregulation of hydrazine dehydrogenase (hdh; log₂FC = 1.78) and a markedly increased hydrazine dehydrogenase-to-hydrazine synthase subunit A (hdh/hzsA) ratio. Altered expression of NADH-ubiquinone oxidoreductase chain genes, together with adenylate energy charge measurements, further demonstrated that substrate inhibition trapped anammox bacteria in a low-energy state. By integrating adenosine triphosphate (ATP) levels, specific anammox activity, and substrate concentrations, we found that substrate inhibition caused a decoupling between substrate concentration and anammox activity. Overall, this study establishes a practical and reliable strategy for recovering substrate-inhibited continuous-flow anammox granular reactors dominated by Ca. Kuenenia stuttgartiensis and provides mechanistic insight into the energy-dependent basis of the reset point.