Strategy-dependent anammox tolerance to sulfadiazine: A cascade from EPS defense to microbial network cooperation

Anammox is highly sensitive to antibiotic stress. However, the dynamics of electron transfer, the response patterns of key metabolic enzymes, and their molecular interaction mechanisms under different exposure strategies remain unclear. This study compared two sulfadiazine (SDZ) exposure strategies, repeated-exposure (R0-R3) and elevating-concentration (R4), to evaluate their impacts on system stability and resilience. Under R0-R3, intermittent antibiotic shocks induced cumulative inhibition, leading to specific anammox activity (SAA) and total nitrogen removal efficiency (TNRE) declining continuously. In contrast, R4 maintained TNRE above 85 % even at 50 mg/L SDZ. Although SAA peaked at 1 mg/L and declined at higher concentrations, biomass accumulation largely compensated for this loss. Mechanistic analysis showed that tightly bound proteins (TB-PN) in extracellular polymeric substances (EPS) were markedly depleted from R0 to R3, disrupting electron flow and weakening key enzymatic functions. Molecular docking revealed that SDZ could bind spontaneously to key enzymes, particularly nitrite reductase (NIR). In R4, the TB-PN-dominated EPS matrix reduced SDZ binding through physical shielding and preserved catalytic activity. It also supported a coordinated defense involving EPS restructuring, electron transfer, enzyme protection, and microbial cooperation. These findings identify TB-PN as a central element of multiscale defense and provide a basis for optimizing anammox stability under antibiotic stress.