Multivariate analysis study on mechanism of 3-oxo-C12-HSL enhancing low temperature stress resistance of activated sludge.

Low-temperature stress markedly suppresses the metabolic activity of activated sludge microorganisms, thereby compromising the operational stability of biological wastewater treatment systems. This study investigated associations between 3-oxo-C12-HSL-related quorum-sensing (QS) responses and physiological, transcriptional, and metabolic changes in activated sludge following rapid temperature decline. At 15°C and 8°C, lasIg was upregulated with log2FC values of 1.02 and 3.01, respectively, accompanied by 27.79- and 32.87-fold increases in endogenous N-(3-oxo-dodecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) levels, reaching maximum concentrations of 25.05 and 32.90 μg g⁻1 VSS. With 3-oxo-C12-HSL accumulation, QS-related genes, including lasRg, luxRg and sdiAg, were significantly upregulated, with log2FC values ranging from 1.13 to 4.84. These changes coincided with the upregulation of genes involved in extracellular polysaccharide biosynthesis, energy metabolism, oxidative phosphorylation, fatty acid metabolism, and antioxidant defense. Meanwhile, differential metabolites associated with the pentose phosphate pathway (PPP), glycolysis (EMP), and the tricarboxylic acid cycle (TCA) increased by 1-104%. These molecular changes were accompanied by pronounced physiological responses, including a 73.37-508.91% increase in extracellular polysaccharides (PS), an increase in ATP from 3278.82 to 7016.70 nmol L⁻1, a 24% decrease in reactive oxygen species (ROS), and a 16% increase in total antioxidant capacity (T-AOC). Exogenous 3-oxo-C12-HSL supplementation further enhanced these responses, supporting a potential functional role potential functional association in the low-temperature response of activated sludge. Overall, 3-oxo-C12-HSL was associated with coordinated changes in polysaccharide formation, energy metabolism, antioxidant defense, and fatty acid-related responses, suggesting its potential as a candidate QS signal for low-temperature biological wastewater treatment.