Rapid granulation versus functional imbalance of aerobic granular sludge under F-53B and OBS stress: A ROS-mediated...

F-53B and OBS, two major alternatives to perfluorooctane sulfonate (PFOS), are frequently detected in wastewater treatment systems; however, their effects on aerobic granular sludge (AGS) formation and functional stability remain poorly understood. Therefore, this study investigated the impacts of long-term F-53B and OBS exposure on sludge performance and microbial community dynamics during AGS granulation. Compared with the control reactor, both F-53B and OBS significantly stimulated extracellular polymeric substance (EPS) secretion (R1: 103.09 ± 1.73 mg/g VSS; R2: 132.63 ± 4.05 mg/g VSS; R3: 115.23 ± 1.53 mg/g VSS), thereby accelerating granulation (R1: 48 d; R2: 42 d; R3: 46 d). However, the enhanced granulation rate came at the expense of treatment performance, as total nitrogen removal decreased from 74.86 ± 1.78% in R1-66.78 ± 2.28% and 67.69 ± 2.23% in R2 and R3, respectively. Further analyses revealed that F-53B and OBS increased intracellular reactive oxygen species (ROS) levels to 133.84 ± 0.46% and 121.67 ± 0.55% of the control, respectively, accompanied by increases of 20.0% and 12.8% in the proportion of membrane-damaged cells. Multi-omics analyses demonstrated that excessive ROS activated the RpoS/c-di-GMP signaling pathway and upregulated key genes involved in glycolysis and the tricarboxylic acid cycle, thereby promoting EPS-producing bacteria such as Candidatus_Competibacter (R1: 10.69 ± 0.68%; R2: 15.52 ± 1.67%; R3: 11.32 ± 0.91%) to synthesize more EPS as a protective barrier and accelerate granule aggregation. During this process, microbial carbon and energy allocation shifted from nutrient removal toward EPS production, resulting in competitive suppression of key functional microorganisms involved in nitrogen and phosphorus removal, such as Candidatus_Accumulibacter and Nitrosomonas, ultimately reducing treatment efficiency. Enzyme activity assays and molecular docking further confirmed that both compounds interfered with antioxidant enzymes and weakened ROS-scavenging capacity. Overall, F-53B and OBS induced a ROS-mediated metabolic trade-off in AGS, promoting rapid granulation through excessive EPS production while compromising the stability of nitrogen and phosphorus removal. These findings provide new insights into the ecological risks of PFOS alternatives and offer a scientific basis for improving the stability of AGS-based wastewater treatment processes.