Polymyxin B improved the nitrogen removal capacity of mainstream anammox process under low temperature through regulating electron transfer and virulence factor

Anaerobic ammonium oxidation (anammox) represents a low-carbon, cost-effective approach for biological nitrogen removal, but its widespread application in mainstream municipal wastewater treatment has long been restricted by the high sensitivity of anaerobic ammonium-oxidizing bacteria (AnAOB) to low ambient temperatures. This study explored whether polymyxin B supplementation could recover AnAOB metabolic activity under 15 °C stress, with a focus on its regulatory effects on biofilm assembly, electron transport efficiency and virulence factor expression. Gradient doses of polymyxin B (0–0.4 mg L −1 ) were continuously added to mainstream anammox reactors receiving influent with 70 mg L −1 total nitrogen. Results showed that polymyxin B addition gradually raised nitrogen removal efficiency (NRE) from 75.0% to 82.3%. Compared with the control reactor, the experimental group achieved 1.98, 1.40 and 1.48-fold elevations in specific anammox activity (SAA), extracellular polymeric substance (EPS) content and heme c level, respectively. Metagenomic annotation indicated that AnAOB relative abundance in P3 was 36.3%, equivalent to a 2.14-fold increase relative to the control. Further analysis revealed the anammox system facilitated dense biofilm formation by upregulating genes encoding lipopolysaccharides, alginate and polar flagella. Polymyxin B modulated energy, amino acid and carbohydrate metabolism to boost electron transfer in the anammox system. This study offers technical references for enhancing AnAOB adaptability under mainstream wastewater conditions, and support exogenous additives as a feasible tool for full-scale anammox engineering.