Heterotrophic nitrification and aerobic denitrification (HN-AD) represents an innovative biological nitrogen removal strategy for saline wastewater treatment. However, how HN-AD microbes could be applied to environmental microbiomes and conduct nitrogen metabolic performance remains ambiguous. Here we established synthetic heterotrophic microbiomes using halophilic HN-AD strains - biofilm-forming Pseudomonas kunmingens 8-C and Acinetobacter johnsonii 2–1-H, and characterized nitrogen metabolism in pure-cultured strains and microbiomes. The pure-cultured HN-AD strains removed ammonium primarily via ammonium assimilation (> 46 % contribution) and heterotrophic nitrification, comprehensively validated by nitrogen balance, ¹⁵N stable isotopic labeling tests, enzyme activity assays and functional gene identification. Four synthetic halophilic microbiomes constructed by biofilm-forming and HN-AD strains achieved ammonium and total nitrogen removal efficiencies of 76-92 % and 72–86 %. Biofilm-forming strains facilitated heterotrophic microbiome assembly by shaping microbial communities through the deterministic assembly process. Notably, initial functional strains selectively recruited environmental microbes with efficient ammonium-assimilating capacity, manifested as a stable and relatively high abundance of glnA gene in microbiomes. But the invasion of microbes consequently led to the overwhelming dominance of ammonium assimilation over nitrification in microbiomes. Our results provided a framework for constructing environmental microbiomes using functional microbes and highlighted the distinct nitrogen metabolism shifting from HN-AD pure-cultured bacteria to microbial consortia.

Oxygen is essential for heterotrophic ammonia assimilation (HAA), driving the microbial degradation of organic compounds and ammonia assimilation in aerobic wastewater treatment. However, the mechanisms underlying carbon-nitrogen transformation and microbial community assembly by heterotrophs under aerobic conditions remained elusive. This study investigated the impact of aeration rates (0.1, 0.5, 1, and 3 L/min·L) on the pollutant removal and microbial dynamics of HAA bioreactors over a 120-day operation period to improve the performance of the system by regulating the microbial oxygen affinity. The NH₄⁺ -N and COD removal efficiencies of the highest aeration rate (3 L/min·L) were as high as 94.8 % and 96.8 %. Batch tests on nitrogen balance verified that nitrogen removal was attributed to assimilation rather than nitrification. The kinetic and mass balances analysis highlighted enhanced microbial activity and substrate utilization at increased aeration rates. Halomonas, emerged as dominant taxa, correlating with improved ammonia assimilation under higher aeration rates. Increased aeration enhanced the microbial robustness and reduced the modularity of microbial interactions, and stochastic processes emerged as the primary drivers of community assembly. The aeration rates of 1-3 L/min·L were considered as the parameter range of optimal pollutant removal, and the aeration rate parameter close to 1 L/min·L was considered as the optimal efficiency combined with the cost. This study provides valuable insights for optimizing biotechnological applications and engineering microbial systems for enhanced environmental performance.

Food waste leachate is a high–strength wastewater characterized by refractory organics, high–salinity and elevated ammonium concentrations, posing challenges for effective treatment and nitrogen resource recovery. In this study, a novel strategy integrating UV/PMS advanced oxidation pretreatment with aerobic heterotrophic ammonium assimilation (HAA) was employed to enhance microbial nitrogen assimilation and carbon removal. Long–term monitoring revealed that the UV/PMS–HAA system achieved superior NH₄⁺–N and COD removal efficiencies of 84.04 % and 90.74 % compared to the control. EPS analysis indicated higher protein content and tighter sludge structure, supporting improved microbial aggregation. Microbial diversity was significantly enhanced in the UV/PMS–HAA system, with enrichment of functional genera such as Halomonas, Pseudomonas and Thauera. Network and robustness analysis revealed intensified microbial cooperation and reduced disturbance sensitivity. Substantial upregulation of ammonium assimilation genes (gdhA, glnA, gltB), while nitrification–related genes (amoA, hao) were nearly absent, confirming a heterotrophic assimilation–dominated pathway. Enzyme activity analysis further supported this trend, with elevated GS, GOGAT activity and higher intracellular Glu, Gln, and TAA levels in the UV/PMS–HAA. UV/PMS pretreatment effectively reshaped microbial structure and function, promoting nitrogen recovery through assimilation rather than loss via nitrification, and provides a promising solution for treating complex nitrogen–rich wastewaters.