Exogenous trimethylglycine alleviates low-temperature stress in anammox consortia: Insights from 15N tracing, physiology, and multiomics.

The application of anammox technology in municipal wastewater treatment plants with low water temperatures has been severely hindered because of the temperature sensitivity of anammox bacteria (AnAOB). Glycine betaine (trimethylglycine, TMG), a key cryoprotectant, is rarely synthesized endogenously by AnAOB, and its regulatory mechanism remains unclear. This study investigated how exogenous TMG alleviates low-temperature inhibition of anammox activity by comparing TMG-amended (R3) and unamended groups (R2) at 15°C, with a 30°C group (R1) used as an optimal-temperature reference. The results demonstrated that the nitrogen removal efficiency (NRE) decreased from 81.7% at 30°C to 46.7% at 15°C. Nevertheless, at 15°C, NRE was significantly restored to 70.2% with TMG addition. 15N labeling experiments revealed that TMG increased anammox-associated N2 production by 2.5-fold without enhancing heterotrophic denitrification. From a physiological perspective, TMG stabilized cellular structure, restored Na+/K+ ATPase activity and ATP levels, and reduced reactive species accumulation. Multiomics analysis further indicated that TMG addition was associated with enhanced transcription of genes involved in anammox metabolism, reactive species detoxification, membrane lipid repair, and choline/TMG-related metabolic potential. The detection of homocysteine, L-methionine, and N,N-dimethylglycine suggested possible involvement of TMG-associated methyl metabolism. Moreover, exogenous TMG was cost-competitive and did not substantially promote heterotrophic denitrification, representing a feasible strategy for stabilizing anammox performance in cold-region wastewater treatment.