Heterotrophic ammonium assimilation (HAA) represents an emerging biological nitrogen removal strategy for saline wastewater treatment. Developing halophilic HAA microbiome into granular form enables simultaneously improve the sludge settleability and robustness. However, the regulatory mechanism of hydraulic shear force (HSF) on granule characteristics and microbial community ecology of the HAA microbiome remain unclear. This study investigated the effects of HSF, controlled by reactor height-to-diameter ratios (H/D = 1, 2.5, 5, and 10), on the nitrogen removal performance, granule morphology, and microbial community assembly. The constructed aerobic HAA granules were uniformly micro-sized (230–280 μm) yet exhibited high density (1030–1115 kg/m3) and excellent settleability. Notably, under moderate HSF condition (H/D = 5), the abundance of key HAA-related genes (glnA, gltB, and gdhA) and the enzyme activities of GS, GOGAT, and GDH were maximized, corresponding to highest ammonium removal efficiency. Across all four systems, the constructed aerobic HAA granules strictly performed assimilation function, with no detectable ammonia-oxidizing genes (AMO), nitrifying bacteria, or nitrogen loss. Increasing HSF imposed strong selective pressure on the aerobic HAA granules, resulting in a linear increase in deterministic community assembly while reducing microbial diversity. In system with an H/D of 5, the dominant genera Ponticoccus and Marinobacillus acted as network hubs, maintaining microbial community stability. Overall, this study successfully established micro-sized aerobic HAA granules, and revealed the regulatory effects of HSF on their granule characteristics, microbial community assembly, and nitrogen metabolism. This study provides valuable insights for the design and optimization of stable HAA-based systems for saline wastewater treatment.

Drawing inspiration from the self-regulating carbon–nitrogen cycling of saline ecosystems, this study investigates how substrate flux and biomass density co-regulate the structure and function of marine sediment-derived halophilic heterotrophic ammonia assimilation (HAA) microbiome cultivated in saline ammonia-containing wastewater with a COD/N radio of 20 under volumetric exchange ratios (VER) of 75 %, 50 %, and 25 % and mixed liquor suspended solids (MLSS) increasing from 5 to 15 g/L. The combined variation in VER and MLSS generated a gradient in food-to-microorganism radio (F/M). With increasing in biomass, COD removal efficiencies peaked at 94.4–99.3 % at 15 g MLSS/L, whereas ammonia removal efficiencies reached at 90.3–96.8 % at 12.5 g MLSS/L before declining. A VER of 25 % reduced sludge activity, while a VER of 75 % impaired floc settleability. The directed HAA community shifted in substrate flux and biomass density, centering on dominant genera such as Halomonas and Marinobacter, ultimately forming a stable microbiome.

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.

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.

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.

Microbially induced carbonate precipitation (MICP) presents a promising strategy for the softening and purification of produced water. However, produced water from heavy oil reservoirs exhibits high salinity, refractory organics, particularly with hardness ions such as Ca²⁺ and Mg²⁺, all of which substantially inhibit microbial mineralization activity. Industrially viable continuous-flow operational strategies remain underdeveloped, and the underlying biomineralization mechanisms are not yet fully elucidated. Here, we report the successful construction of an engineering microbiome through substrate gradient acclimation, achieving continuous and stable precipitation of Ca²⁺ (87.28 %) and Mg²⁺ (84.16 %). The process also revealed the sequential transformation of organic functional groups under high salinity perturbation. Hydroxyl groups (−OH) in extracellular polymeric substances preferentially bound divalent cations under neutral conditions, whereas carboxyl groups (−COO⁻) served as nucleation sites for carbonate formation under alkaline conditions. Extracellular carbonate precipitation predominated, while a minor fraction of amorphous magnesium carbonate was accumulated intracellularly. The engineering microbiome, dominated by urease-positive and hydrocarbon-degrading taxa, tolerated extreme salinity and hardness through metabolic complementarity and coordinated gene regulation. These findings demonstrate a robust, continuous-flow MICP process for HPW treatment, offering a foundation for industrial-scale integration with improved stability, efficiency, and microbiome resilience in complex environments.

Marine microorganisms have an inherent advantage in the treatment of saline wastewater due to their halophilic properties. Ammonium assimilation is the most important and common nitrogen conversion pathway in the ocean, which means that it may be a suitable nitrogen removal strategy under high salinity conditions. However, the targeted construction of engineering microbiomes with ammonium assimilation function for nitrogen recovery has not been realized. Here, we constructed four halophilic ammonium assimilation microbiomes from marine microbial community under varying chemical oxygen demand (COD) to nitrogen (COD/N) ratios. The regulation of COD/N ratio on microbial self-assembly was explored at the phenotypic, genetic, and microbial levels. The results of nitrogen balance tests, functional genes abundance and microbial community structure confirmed that the microbiomes regulated by different COD/N ratios all performed obligate ammonium assimilation functions. >93% of ammonium, 90% of TN, 98% of COD, and 82% of phosphorus were simultaneously removed by microbial assimilation under the COD/N ratio of 20. COD/N ratios significantly affected the self-assembly of microbiomes by selectively enriching heterotrophic microorganisms with different preference for organic carbon load. Additionally, the increase of COD/N ratio intensified the competition among species within the microbiome (the proportion of negative connections of microbial network increased from 5.0% to 24.4%), which may enhance the stability of community structure. Taken together, these findings can provide theoretical guidance for the construction and optimization of engineering microbiomes for synergistic nitrogen removal and recovery.

The contradiction between theoretical metabolism of ammonium assimilation and experiential understanding of conventional biosystems makes the rational optimization of the ammonium-assimilating microbiome through carbon to nitrogen (C/N) ratios perplexing. The effect of different C/N ratios on ammonium-assimilating biosystems was investigated in saline wastewater treatment. C/N ratios significantly hindered the nutrient removal efficiency, but ammonium-assimilating biosystems maintained functional stability in nitrogen conversions and microbial communities. With sufficient biomass, higher than 86% ammonium and 73% phosphorus were removed when C/N ratios were higher than 25. Ammonium assimilation dominated the nitrogen metabolism in all biosystems even under relatively low C/N ratios, evidenced by the extremely low abundances of nitrification functional genes. Different C/N ratios did not significantly change the bacterial community structure of ammonium-assimilating biosystems. It is anticipated that the ammonium-assimilating biosystem with advantages of clear metabolic pathway and easy optimization can be applied to nutrient removal and recovery in saline environments.