Research on the Performance and Metabolic Mechanisms of Nitrogen Removal by Heterotrophic Ammonia-Assimilating Microorganisms under Salinity Stress
Conventional biological nitrogen removal processes, such as nitrification-denitrification (AO) and anaerobic ammonium oxidation (Anammox), have inherent limitations under high-salinity conditions. Salinity stress readily inhibits denitrification, leading to reduced nitrogen removal efficiency, accompanied by emissions of the greenhouse gas N2O and loss of nitrogen resources. Heterotrophic ammonia assimilation has emerged as a promising alternative for nitrogen removal from high-salinity wastewater; however, its salinity tolerance threshold and the underlying mechanisms by which salinity affects nitrogen removal performance remain insufficiently understood. In this study, an internal-bag aeration reactor was developed to promote ammonia assimilation as the dominant nitrogen removal pathway. Under optimized conditions (sodium acetate as carbon source, C/N ratio of 9.0, pH 8.0, initial MLSS of 3000 mg/L), the effects of increasing salinity (0 - 90 g/L NaCl) on nitrogen and Chemical Oxygen Demand (COD) removal were systematically investigated in both batch and continuous-flow modes. Batch experiments identified 60 g/L NaCl as the critical salinity threshold for ammonia assimilation-based nitrogen removal. Total nitrogen (TN) removal efficiencies were 75.17% and 60.21% at 0 and 60 g/L NaCl, respectively. When salinity increased to 70 g/L, TN removal efficiency sharply decreased to 45.82%, further declining to only 22.48% at 90 g/L. Within the 0 - 60 g/L salinity range, ammonia-assimilating microorganisms actively enhanced biomass, extracellular polymeric substances (EPS), proteins, and polysaccharides to establish effective osmotic protection barriers. Microbial community analysis revealed salinity-driven succession, with salt-tolerant phyla (Pseudomonadota and Thermodesulfobacteriota) and genera (Marinobacterium and Halomonas) becoming dominant. Metatranscriptomic analysis further demonstrated that salinity stress upregulated the transcriptional expression of genes involved in ion transport, compatible solute synthesis, and protein repair, while concurrently downregulating nitrogen and energy metabolism genes. Continuous-flow experiments operated stably for 220 days under varying salinity conditions. Within the 060 g/L salinity range, the system exhibited stable performance. Notably, at 20 g/L NaCl, the top six phyla (Pseudomonadota, Bacillota, Bacteroidota, Chloroflexota, Actinomycetota, and Thermodesulfobacteriota) formed an evenly distributed community with maximum total abundance. After salinity was reduced from 90 g/L to 10 g/L, nitrogen removal performance rapidly recovered, demonstrating robust functional resilience.