A new strategy for maintaining the cold-resistant activity of activated sludge: The low-temperature protective properties of biofilms and the study of microbial diversity

• Additives enriched biofilm-formers Pseudomonas & Comamonas. • Putrescine boosted microbial activity at all temperatures. • Key biofilm genera supported community stability under cold stress. • Additives enabled resilient, stable cold-stress communities. Activated sludge is a key technical step for wastewater treatment. However, low temperatures adversely affect the pollutant removal efficiency of activated sludge, primarily due to a substantial drop in microbial activity. In general, the activity of activated sludge is markedly suppressed at temperatures below 15°C. The precise and rapid modulation of microbial activity and abundance in activated sludge under low-temperature conditions is pivotal for addressing this challenge. In this study, we systematically examined the individual effects of betaine, alginic acid, and putrescine on biofilm development within a low-temperature range (8-16 °C) in activated sludge systems. Despite the established protective functions of betaine, alginic acid, and putrescine for sludge under stress conditions, their synergistic potential for constructing cold-adapted biofilm communities has not been explored. The results show that the betaine-alginic acid combination enhanced microbial diversity by 4.7-fold at 12°C compared to conventional sludge. The dominant bacterial species in the low-temperature activated sludge were identified as Pseudomonas and Comamonas , which were found to be capable of maintaining stable biofilm biomass (14-18 mg/L). Conversely, putrescine treatment enhanced the abundance of the cryotolerant Rhodooccus at the expense of overall microbial diversity in the sludge. Alginic acid was shown to contribute to enhanced biofilm structural stability by promoting sustained EPS production. Betaine enabled microorganisms to sustain their metabolic activity under low-temperature stress. While, the superior protective effect of putrescine on low-temperature biofilms was attributed to its role in modulating microbial putrescine function. This study provides a novel strategy for bioaugmentation using biocompatible compounds to enhance low-temperature biofilm performance.