Alginate and Glycine Betaine Enhance Biofilm Formation in Psychrophilic Activated Sludge Systems.

Activated sludge systems at sub-optimal temperatures (8°C-16°C) suffer from inhibited biofilm development, diminished extracellular polymeric substances (EPS) secretion and reduced carbon/nitrogen removal. To counteract this, we investigated the synergistic effect of alginic acid (AA, a structural polysaccharide) and betaine (BET, a compatible solute) in sequencing-batch reactors (SBRs) at 8°C-16°C The combined AA + BET amendment elicited a rapid, pronounced response, doubling attached biofilm biomass compared to the control, a significantly greater enhancement than with single amendments. This suggests a synergistic effect in which BET mitigates osmotic stress, thereby improving microbial capacity to utilise AA for structural development. Microbial community analysis revealed that the combined addition of AA and BET reshaped the microbial community, enabling the consortium to acquire a high capacity for EPS production, which conferred cold tolerance, while simultaneously suppressing the growth of filamentous Acidovorax. Principal component analysis (PCA) segregated the AA + BET community, and increased diversity indices indicated greater functional richness. The control group contained 11.43% of microbial taxa that were distinct from those in the experimental group; these taxa, likely lacking targeted regulation, exhibited a lower contribution to the activated sludge process (ASP) system. This dual-stress-alleviation strategy effectively orchestrates a resilient, EPS-rich biofilm with a psychrotolerant core microbiome, offering a practical approach to maintaining robust activated-sludge performance during cold weather However, further pilot-scale validation and techno-economic assessment are needed before practical implementation in full-scale wastewater treatment systems.