Enhanced denitrification under saline Conditions: Glycine betaine as a key osmoprotectant

Biological denitrification is significantly inhibited by salinity, which adversely affects microbial activity and reduces efficiency. This study aimed to evaluate the impact of salinity on denitrification performance and assess the potential of glycine betaine (GB) as an osmotic pressure regulator and protective agent. Results indicated that under the optimal conditions, including an influent nitrate concentration of 51.03 mg L^-1, C/N ratio of 5.42, pH value of 8.95, and salinity of 1.05 %, the nitrate removal efficiency was predicted to reach 100 %. However, a sharp decline (56.09 ± 4.52 %) in nitrate removal efficiency occurred when salinity increased from 0 % to 3 % within the initial 6 h. This inhibition was mitigated by adding 25 mg L^-1 GB, which enhanced nitrate removal efficiency by 2.19 times. GB promoted the secretion of extracellular polymeric substances (EPS), especially polymeric protein, a critical contributor to salinity resistance. Metagenomics analysis revealed that GB improved denitrification process by upregulating key genes involved in nitrogen and carbon metabolism. Furthermore, the relative abundance of Na⁺ transporter genes, K⁺ transporter genes, and GB absorption and synthesis genes rose with GB addition, underscoring the indispensable role of GB in alleviating osmotic stress and accelerating microbial metabolism. These findings emphasize the detrimental effects of salinity on denitrification and demonstrate the potential of GB as an osmoprotectant, enabling efficient nitrogen removal under saline conditions.