Mechanisms of combined tetracycline and sulfadiazine stress inhibiting nitrogen transformation in aerobic granular sludge: Insights from oxidative stress, energy metabolism and functional genes.

From the perspective of linking structural damage, oxidative stress and molecular regulation, this study clarified how antibiotic inhibited nitrogen metabolism in aerobic granular sludge (AGS). Long-term antibiotic stress severely damaged AGS structure, causing blurred boundaries, surface cracks and collapse zone. As a primary response, AGS continuedly increased extracellular polymeric substances (EPS) secretion, which partially mitigated antibiotic toxicity through adsorption and shielding effects. However, persistent toxicity triggered 160.2% higher reactive oxygen species (ROS) level than the control. Excessive ROS disrupted cellular redox homeostasis and suppressed the activities of nitrogen-transforming key enzymes (e.g., AMO, NOR, NR, NIR) with an inhibition range of 22.4∼31.9%, and energy metabolism enzymes responsible for ATP synthesis with a 40.2% activity reduction. Furthermore, this oxidative stress cascade extended to the central carbon metabolism, as nearly all genes encoding tricarboxylic acid (TCA) cycle enzymes were remarkably downregulated. This TCA cycle inhibition further cut off the supply of ATP and some electron donors (e.g., NADH and FADH2) essential for nitrification and denitrification, forming a self-reinforcing inhibitory loop. Overall, this study demonstrated that antibiotics inhibit AGS nitrogen transformation through a well-defined "oxidative stress-energy metabolism-functional gene" cascade mechanism.