Development of a psychrotolerant composite microbial agent for nitrogen removal and its nitrogen metabolism pathways

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Although NR characteristics of single psychrotolerant bacteria have been extensively studied, synergistic interactions between functionally distinct psychrotolerant nitrogen-removing consortia remain unexplored. In this study, a composite microbial agent, designated NDC-6, was generated by coculturing a psychrotolerant nitrifying consortium NC1 ( Pseudomonas veronii HN1, P. poae HN2, and P. peli HN3) and an aerobic denitrifying consortium DC1 ( Aeromonas sp. AD1, P. extremaustralis AD2, and Serratia liquefaciens AD3) at a 1:1 inoculation ratio, and its NR performance was systematically evaluated. After 3 days of incubation at 10°C, NDC-6 achieved removal efficiencies of 89.3%, 88.1%, 85.5%, and 95.3% for NH 4 + -N, NO 3 − -N, total nitrogen (TN), and chemical oxygen demand (COD), which were significantly higher than those of individual strains or single-function consortia. Sodium succinate was identified as the optimal carbon source, which simultaneously improved biomass growth and NR efficacy of NDC-6. Optimal culture conditions determined using response surface methodology were as follows: C/N ratio, 6; temperature, 10.2°C; pH, 7.2; and shaking speed, 156 rpm. Under these conditions, the maximum TN removal efficiency reached 89.8%. Nitrogen balance and functional gene expression ( hao , nap A, nir S, nir K, cnor B, and nos Z) analyses revealed that NDC-6 achieved complete NR through both assimilatory and dissimilatory pathways. The dissimilatory mechanism relied on synergistic metabolism and functional complementation between NC1 and DC1, mediated by their respective functional genes. This study provides mechanistic insights into the biological treatment of nitrogen-containing wastewater, particularly under low-temperature conditions, and offers a novel strategy for such treatment. Psychrotolerant bacteria Composite microbial agent Biological nitrogen removal Nitrification Denitrification Metabolic pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Substantial amounts of nitrogen-containing pollutants have been persistently entering the aquatic systems over the recent decades, owing to extensive application of fertilizers in farmlands, direct discharge of untreated domestic sewage, and inadequate treatment of industrial effluents from sectors, such as chemical manufacturing, pharmaceuticals, and food processing [ 1 ]. Excessive nitrogen causes eutrophication of natural water bodies and seriously threatens ecosystem functioning and human health [ 2 ]. Therefore, efficient treatment of nitrogen-contaminated wastewater is currently a critical environmental issue that needs to be solved urgently. Biological nitrogen removal (BNR) has emerged as a prominent research focus worldwide in the field of wastewater treatment in view of the powerful functions of microbial metabolism and transformation, including ammoniation, nitrification, and denitrification. Compared with traditional physical or chemical methods, BNR exhibits remarkable advantages, such as higher efficiency, lower energy consumption, and superior ecological benefits [ 3 ]. However, environmental temperature is a key factor that limits the actual application of BNR in wastewater purification [ 4 ]. Currently, most known functional microorganisms carry out nitrogen removal (NR) under mesophilic conditions, and the optimal temperature for microbial growth and physiological metabolism is 25–37°C, with the best metabolic activity occurring at 20–25°C [ 5 ]. Low-temperature environment (< 15°C) can damage the cell membrane of microorganisms, seriously inhibit the metabolic activity of enzymes and the protein synthesis rate, and slow down the growth rate of functional microbes, ultimately resulting in unsatisfactory NR performance [ 6 ]. For example, reducing the temperature from 35 to 10°C drastically decreased the aerobic denitrification rate of ammonia-oxidizing archaea from 20.64 to 7.75 mg N/(L·h), with a concomitant decrease in total nitrogen (TN) removal efficiency from 98.5–60.9% [ 7 ]. Similar effects have been reported for Glutamicibacter arilaitensis EM-H8 [ 8 ] and Raoultella ornithinolytica strain YX-4 [ 9 ]. Compared with mesophilic nitrogen-removing bacteria, psychrotolerant bacteria can proliferate extensively over broad low-temperature ranges while maintaining high NR activity. Therefore, developing psychrotolerant bacteria with enhanced NR capabilities could be a potential strategy for improving wastewater purification efficiency under low-temperature conditions. In recent years, several researchers have isolated psychrotolerant functional microorganisms from low-temperature environment and used them for treating nitrogen-containing wastewater, achieving certain removal effects. Yang et al. [ 10 ] obtained Bacillus simplex H-b, a psychrotrophic heterotrophic nitrification and aerobic denitrification (HNAD) bacterium, from the frozen soil in Northeast China. B. simplex H-b exhibited excellent NR capabilities, with 82.2% ammonium nitrogen (NH 4 + -N, 60 mg/L), 67.3% nitrate nitrogen (NO 3 − -N, 63 mg/L), and 78.7% nitrite nitrogen (NO 2 − -N, 10 mg/L) removal efficiencies at 10°C. Pseudomonas fragi EH-H1 exhibited high-efficiency HNAD performance at 15°C with NH 4 + -N, NO 3 − -N, and TN removal efficiencies of 100%, 99.1%, and 97.7%, respectively [ 11 ]. Ma et al. [ 12 ] found that psychrotolerant bacteria employed diverse strategies, including maintenance of membrane fluidity, expression of cold shock proteins, structural regulation of enzymes, and accumulation of compatible solutes, to counteract cold-induced stress. Despite promising application prospects in wastewater purification, single microbial species poses inherent limitations in degrading complex pollutants because of metabolic constraints and stringent cultivation requirements. Consequently, single microbial strains achieve much lower NR efficiency when treating actual wastewater with complex and unstable composition [ 13 ]. Fluctuating wastewater treatment efficiency and substandard effluent quality constrains practical implementation of psychrotolerant functional bacteria in full-scale wastewater treatment plants [ 14 ]. To improve the treatment efficacy of nitrogen-containing wastewater, some researchers have focused on developing composite microbial agent (CMA) by coculturing two or more functionally distinct but compatible (i.e., non-antagonistic) microorganisms [ 15 ]. CMA has been shown to exhibit excellent synergistic effects in wastewater treatment, which can improve the pollutant-removal efficiencies and broaden metabolic pathways by leveraging the metabolic complementarity and interspecies cooperation among different strains [ 16 ]. Compared with single strains, CMA exhibits superior operational stability and ecological adaptability, especially for actual wastewater treatment. The composite consortium B-Cl, comprising B. marisflavi B1 and B. marisflavi B5 in a volume ratio of 3:2, achieved a removal efficiency of 86.9% for 2,4-dichlorophenol, higher than that of B1 (69.9%) and B5 (75.5%) alone [ 17 ]. Shi et al. [ 18 ] used acclimated CMA, consisting of indigenous sediment microorganisms, functional bacteria ( B. natto ADT), and supplemental bacteria ( Lactobacillus bulgaricus , Enterococcus , Lactobacillus , etc.) in a volume ratio of 10:1:1, for in-situ remediation of polluted black-odorous streams. After treatment, the concentrations of NH 4 + -N decreased by 74.0% and 76.3%, underscoring the effectiveness of the CMA. While CMA has demonstrated broad applicability in bioremediation of polluted environment, including petroleum hydrocarbon degradation in oil-contaminated soil [ 19 ], organic matter decomposition of kitchen waste [ 20 ], and immobilization of heavy metal [ 21 ], its potential for treating nitrogen-contaminated wastewater under low-temperature conditions remains underexplored. Thus, it is imperative to screen psychrotolerant NR-functional bacteria, construct high-efficiency microbial consortia with optimized functions, and further explore the synergistic metabolic mechanisms among strains to enhance bioremediation performance in cold climates. In this study, a psychrotolerant CMA with efficient NR performance was developed to improve the NR efficacy at low-temperature conditions. The specific objectives of this study were to (i) isolate and screen a series of psychrotolerant bacteria with NR capacities from river sediment, frozen soil, and activated sludge in cold regions during winter; (ii) construct a psychrotolerant CMA for efficient removal of nitrogen based on principles of ecological niche separation and synergistic metabolisms; (iii) optimize the key factors affecting NR by CMA using response surface methodology (RSM) based on the Box–Behnken design; (iv) elucidate the NR pathways employed by CMA via polymerase chain reaction (PCR) amplification of key genes encoding nitrifying and denitrifying enzymes, together with nitrogen mass balance analysis. The development and characterization of psychrotolerant CMA provides mechanistic insights into the treatment of nitrogen-contaminated wastewater in regions with cold climate and offers novel solutions for it. Materials and methods Materials Samples for isolating and screening functional strains with NR capability were obtained from river sediment, frozen soil, and activated sludge from a sewage treatment plant in Shenyang, Liaoning Province, China during winters with temperatures below 5°C. The collected samples were stored in a refrigerator at 4°C until use. Culture medium A mineral salt medium (MSM) containing 0.2 g/L MgSO 4 ·7H 2 O, 0.01 g/L CaCl 2 ·2H 2 O, 0.001 g/L FeSO 4 ·7H 2 O, and 2 mL/L of trace element solution, pH 7–7.2, was used. The trace element solution was as described by Yang et al. [ 22 ]. An enrichment medium (EM) was used to enrich psychrotolerant nitrifying and denitrifying bacteria from samples under low-temperature conditions. The EM consisted of an MSM base supplemented with 2.5 g/L C 6 H 5 Na 3 O 7 ·2H 2 O, 0.32 g/L NaNO 3 , 0.25 g/L (NH 4 ) 2 SO 4 , and 0.044 g/L KH 2 PO 4 . The Luria broth (LB) medium, used for isolating and purifying specific functional strains, contained 10 g/L peptone, 5 g/L yeast extract, and 10 g/L NaCl and had a pH of 7–7.2. The psychrotolerant nitrifying bacteria were screened using a nitrification medium (NM), which consisted of an MSM base supplemented with 2.5 g/L C 6 H 5 Na 3 O 7 ·2H 2 O, 0.5 g/L (NH 4 ) 2 ·SO 4 (equivalent to 0.105 g/L NH 4 + -N), and 0.044 g/L KH 2 PO 4 . The psychrotolerant denitrifying bacteria were screened using a denitrification medium (DM), which consisted of an MSM base supplemented with 2.5 g/L C 6 H 5 Na 3 O 7 ·2H 2 O, 0.64 g/L NaNO 3 (equivalent to 0.105 g/L NO 3 − -N), 0.044 g/L KH 2 PO 4 , and 1 mL/L bromothymol blue (BTB, 1% w/v). A composite bacterial medium (CBM), serving as the basal medium for cultivating the CMA and optimizing key influencing factors, was prepared by supplementing MSM with the following components: 2.5 g/L C 6 H 5 Na 3 O 7 ·2H 2 O, 0.25 g/L (NH 4 ) 2 ·SO 4 (equivalent to 0.0525 g/L NH 4 + -N), 0.32 g/L NaNO 3 (equivalent to 0.0525 g/L NO 3 − -N), and 0.044 g/L KH 2 PO 4 . All the above-mentioned chemical reagents were purchased from the Sinopharm Chemical Reagent Co., Ltd. (China). Unless otherwise specified, these chemicals were of analytical or higher purity grade. The agar plate medium was prepared by adding 20 g agar per liter of liquid medium. All culture media used in this study were sterilized at 121°C for 30 min before use. Isolation and screening of psychrotolerant functional bacteria Enrichment and isolation of psychrotolerant bacteria Fifty milliliters of activated sludge or 5 g of river sediment/frozen soil samples was added to 100 mL EM in 250 mL sterilized conical flasks. Several sterilized glass beads were put in the flasks, followed by incubation at 10°C with shaking at 150 rpm for 3 h. After complete suspension and dispersion of the sludge/sediment, 10 mL aliquots were transferred to 100 mL fresh EM and cultivated at 10°C for 5 days with shaking at 150 rpm to facilitate bacterial proliferation and enrichment. The same process was repeated several times until significant NR effects were detected in the EM. Subsequently, the enriched cultures were serially diluted with sterile deionized water in concentration gradients of 10 − 1 , 10 − 2 , 10 − 3 , 10 − 4 , 10 − 5 , and 10 − 6 . Thereafter, 1 mL of diluted bacterial suspensions were coated onto the LB agar plates and incubated in an inverted position in a constant-temperature incubator at 10°C for 7 days. Colony morphology and bacterial growth were regularly monitored. Distinct individual colonies were selected for streak purification on fresh LB agar plates. The purification process was repeated through multiple streaking cycles until pure cultures were confirmed under an optical microscope (Leica DMi1, GER). Screening of psychrotolerant nitrification bacteria Purified isolates were prepared as cell suspensions in sterile deionized water (optical density at 600 nm (OD 600 ) = 1.0). A 10% (v/v) inoculum of cell suspensions was added to 100 mL NM and cultured at 10°C with shaking at 150 rpm for 3 days. The NH 4 + -N-removal performance of the isolates was evaluated by calculating the removal efficiency. The strains with superior NH 4 + -N-removal efficiency (> 85% at 10°C)) were screened as candidates for subsequent investigation. Screening of psychrotolerant aerobic denitrification bacteria A 100 µL cell suspension (OD 600 = 1.0) of purified isolates was coated onto the DM agar plates and incubated in an inverted position in a constant-temperature incubator at 10°C for 7 days. Distinct individual colonies exhibiting intrinsic blue coloration or blue halo were selectively transferred to 100 mL liquid DM. The cultures were incubated at 10°C for 3 days with shaking at 150 rpm. The strains demonstrating exceptional NO 3 − -N removal capabilities (> 85% efficiency at 10°C) were prioritized for subsequent investigation. Identification of psychrotolerant functional bacteria Colony characteristics of the isolated strains were observed after 4 days of cultivation on respective agar plates. Gram staining for all pure screened strains was performed using the Gram Strain Kit (Bestbio, China), and the cellular morphology was analyzed via scanning electron microscopy (SEM, Zeiss Geminis, GER). Genomic DNA was extracted from each strain using an Ezup Column Bacterial Genomic DNA Extraction Kit (SK8255, Sangon Biotech, Shanghai, China), followed by PCR amplification of the 16S rRNA gene with the universal primers 27F and 1492R (Table S1 ). The amplification product was sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). The isolated strains were taxonomically identified by comparing the 16S rRNA gene sequence homology with other registered strains in the GenBank of the National Centre for Biotechnology Information using the BLAST algorithm. The neighbor-joining algorithm (1000 replicates) was employed to construct a phylogenetic tree of the isolates with the MEGA 11.0 software. Construction of psychrotolerant CMA The Oxford cup agar diffusion assay was performed to investigate the antagonistic interactions among the isolated bacterial strains on LB agar plates [ 23 ]. Non-antagonistic functional strains were cultivated at 10°C for 2 days with shaking at 150 rpm. Following centrifugation at 8000 rpm for 10 min, the bacterial cells were washed with sterile deionized water and resuspended to obtain bacterial suspensions (OD 600 = 1.0) of each functional strain. The suspensions of strains sharing an identical metabolic function (nitrification or denitrification) were combined in equal volume ratios to form the nitrifying consortium NC1 and denitrifying consortium DC1. Subsequently, these functional consortia were pooled in equal volumes to form the CMA. The cell suspensions of each individual strain, single-functional consortium, and the CMA were inoculated at an inoculum volume of 10% (v/v) into 100 mL CBM and cultivated at 10°C for 3 days with shaking at 150 rpm. By comparing the biomass growth (OD 600 ) and the NR performance of different inoculated strain combinations, the optimal culture strategy was determined. The nitrifying (NC1) and denitrifying (DC1) consortiums were inoculated at varying ratios (4:1, 3:1, 2:1, 1:1, 1:2, 1:3, and 1:4) with a total inoculum volume of 10% (v/v) into 100 mL CBM. Based on the comparison of the biomass growth and NR performance of CMA after 3-day cultivation, the optimal inoculation ratio was determined to construct the CMA. The CMA under the optimal inoculation ratio was named NDC-6. Influence of carbon source Sodium citrate (SC; 2.5 g/L), sodium acetate (SA; 2.09 g/L), sucrose (Suc; 1.45 g/L), glucose (Glu; 1.53 g/L), and sodium succinate (SS; 2.07 g/L) were individually supplemented into CBM as sole carbon sources. Each carbon source was adjusted to provide an equivalent carbon content of 612 mg/L in the CBM to evaluate its effect on the NR performance of NDC-6 at low temperature. Cell suspensions (OD 600 = 1.0) of the NC1 and DC1 were inoculated at optimal ratios with a total inoculum volume of 10% (v/v) into the 100 mL CBM containing different carbon sources, followed by aerobic cultivation at 10°C for 3 days with shaking at 150 rpm. Comparative analyses of biomass growth and NR efficiencies of NDC-6 were used to determine the optimal carbon source. Optimization of culture condition for NR by NDC-6 The key cultivation factors influencing NR by NDC-6 were optimized using RSM based on a Box–Behnken design. The C/N ratio (A), temperature (B), pH value (C), and shaking speed (D) were selected as independent variables and were coded low (− 1) or high (+ 1) by keeping 0 as the mid-point. The coded and actual levels of these process variables are represented in Table S2. The initial TN concentration in the CBM medium was maintained at 0.105 g/L, with C/N ratios adjusted by varying the concentration of the optimal carbon source. The shaking speed was modulated to regulate the dissolved oxygen (DO) content in the culture medium. The TN removal efficiency was considered a response variable. A total of 29 experimental runs with a four-factor, three-level Box–Behnken design were conducted in 250 mL conical flasks containing 100 mL CBM and 10 mL mixing inoculum of consortia NC1 and DC1 to optimize variable ranges and levels. After 3 days of cultivation, TN- removal efficiency in each experimental run was calculated. A quadratic polynomial regression model was implemented to characterize the experimental response data, facilitating identification of optimal conditions and assessment of the significance of variable interactions. Nitrogen metabolism genes Genomic DNA was exacted from the isolated strains using a DNA extraction kit (B518255, Sangon Biotech, Shanghai, China). PCR amplification was performed targeting functional genes, namely hao , nap A, nir S, nir K, cnor B, and nosZ , which encoded key enzymes involved in nitrogen metabolism. The specific target primers used in PCR are described in Table S1 and PCR reaction system and process are detailed in Table S3. Analytical methods Biomass growth was characterized by measuring the OD 600 of culture broth using a spectrophotometer (Agilent UV-Vis, USA) [ 24 ]. After centrifuging the culture broth at 8000 rpm for 10 min, the concentrations of COD, NH 4 + -N, NO 3 − -N, NO 2 − -N, and TN in the supernatant were quantified using standard spectrophotometric methods [ 25 ]. Gas chromatography (Agilent 8850, USA) was used to detect the concentration of N 2 O in gas samples. The organic nitrogen (organic-N) in the culture broth primarily comprised extracellular proteins synthesized during bacterial growth and intracellular proteins released upon cell lysis. The concentration of organic-N was calculated by subtracting the concentrations of inorganic nitrogen (including NH 4 + -N, NO 3 − -N, and NO 2 − -N) from the TN concentration in the supernatant after centrifugation. The concentration of intracellular nitrogen (intracell-N) was calculated by subtracting the TN concentration in the centrifuged supernatant from that in the culture broth [ 26 ]. Gaseous nitrogen (gaseous-N) loss was determined by subtracting the TN concentration in the culture broth after 3-day incubation from the initial TN concentration. The pollutant removal efficiency ( RE ) was determined as Eq. (1): (1) The pollutant removal rate ( RR ) was calculated as Eq. (2): (2) where, c 0 and c t (mg/L) represent the pollutant (NH 4 + -N, NO 3 − -N, TN, and COD) concentrations in the initial culture medium and centrifugal culture broth after t hours of cultivation, respectively. Statistical analysis Triplicate measurements were performed for all experiments, and results are presented as mean value ± standard deviation (SD). Experimental data were plotted using the Origin software (Pro 2021, Origin Lab Corporation, USA). The Design-Expert software (v 13.1.0, Stat-Ease Inc., USA) was employed to implement the Box–Behnken response surface experimental design, analysis of the acquired regression and graphical presentation of experimental data, and to generate three-dimensional (3D) response surface plots. Statistical analysis was performed using one-way analysis of variance (ANOVA) in IBM SPSS Statistics (v. 27.0, Chicago, USA) to evaluate the interaction between response surface variables and the response value and to determine the intergroup difference (significance threshold: p < 0.05). Results and discussion Isolation, screening, and identification of functional bacteria Eighteen psychrotolerant nitrogen-removing bacterial strains were isolated and preliminarily screened from activated sludge, river sediments, and frozen soil in northern China during winter when temperatures were consistently below 5°C. These strains exhibited capabilities for removing NH 4 + -N, NO 3 − -N, and TN. After re-screening, three strains each of nitrifying and aerobic denitrifying bacteria were obtained. The colony morphology and cellular characteristics of these six strains are detailed in Table 1 and Fig. S1 . Table 1 Colonial and cellular morphological characteristics of six isolated psychrotolerant bacteria Function Strain Colony morphology Gram staining Cell morphology GenBank Accession No. Nitrification HN1 Light yellow and transparent, circular morphology with irregular edges, smooth and moist surface with a center protrusion, and 2–3 mm in diameter. G– Straight long rod, (1.5–3.0) µm × (0.6–0.8) µm PP913934 HN2 Slightly yellow and transparent, circular morphology with regular edges, smooth and moist surface with a center protrusion, and 3–4 mm in diameter. G– Straight rod, (1.0–2.5) µm × (0.6–0.8) µm PP911455 HN3 Light yellow and semi-transparent, irregular in morphology, smooth, moist and glossy surface with a center protrusion, and 1–2 mm in diameter. G– Curved rod, (1.0–2.0) µm × (0.8–1.0) µm PP911456 Denitrification AD1 White and transparent, circular morphology with regular edges, smooth and moist surface with a center high protrusion, and 2–4 mm in diameter. G– Short rod, (0.6–0.8) µm × (0.6–0.8) µm PP913935 AD2 Light yellow and transparent, irregular in morphology, smooth and moist surface with a center protrusion, and 2–3 mm in diameter. G– Straight long rod, (2.0–3.0) µm × (0.6–0.8) µm PP911450 AD3 White and opaque, circular morphology with regular edges, smooth and moist surface with a center high protrusion, and 2–3 mm in diameter. G– Curved rod, (1.5–2.5) µm × (0.8–1.0) µm PP913933 Three screened nitrifying strains were taxonomically identified and designated as Pseudomonas veronii HN1, P. poae HN2, and P. peli HN3 based on the results of 16S rRNA gene sequence alignment against the GenBank database and phylogenetic tree analysis (Fig. S2A-C). After culture in NM at 10°C for 3 days with shaking at 150 rpm, these isolates exhibited significant removal of NH 4 + -N, achieving REs of 86.8 ± 1.7%, 85.7 ± 1.8%, and 86.4 ± 2.1%, with average removal rate of 1.27 ± 0.15, 1.25 ± 0.13, and 1.26 ± 0.16 mg/L/h, respectively, higher than that of B. simplex H-b (0.74 mg/L/h) at 10°C [ 10 ]. The Pseudomonas species have been extensively documented in previous studies for their significant nitrification capabilities under diverse environmental settings. Yang et al. [ 27 ] isolated a novel acid-resistant bacterium, P. citronellolis YN-21, which exhibited exceptional heterotrophic nitrification ability under acidic conditions with NH 4 + -N RR of 7.84 mg/L/h at pH 5.0. P. aeruginosa WS-03 could effectively remove NH 4 + -N at 30°C with an RR of 8.96 mg/L/h [ 28 ]. According to several reports, Pseudomonas species, characterized by exceptional growth rates, remarkable environmental adaptability, and superior NR efficiency, exhibit significant competitive advantages over conventional nitrifying bacteria [ 29 ]. Therefore, we selected these three isolated Pseudomonas species as candidates for CMA construction. Isolated aerobic denitrifying strains exhibited the highest sequence identity (≥ 99%) with Aeromonas sp. strain J223, P. extremaustralis 14 − 3, and Serratia liquefaciens strain ATCC 27592 in the GenBank database (Fig. S2D-F). Thus, the three strains were identified and designated as Aeromonas sp. AD1, P. extremaustralis AD2, and Serratia liquefaciens AD3, which exhibited remarkable NO 3 − -N removal performance during 3-day cultivation in DM at 10°C and shaking at 150 rpm, with corresponding REs of 85.7 ± 1.6%, 86.3 ± 1.8%, and 85.8 ± 1.8%. Furthermore, the average RR of NO 3 − -N by these three strains were 1.25 ± 0.21, 1.26 ± 0.17, and 1.25 ± 0.15 mg/L/h, respectively, surpassing that of Acinetobacter tandoii MZ-5 (1.04 mg/L/h at 25°C) [ 30 ] and Arthrobacter arilaitensis Y-10 (0.35 mg/L/h at 15°C) [ 31 ]. During cultivation, all three bacterial strains accumulated detectable levels of NO 2 − -N, with concentrations of 2.49 ± 0.37 mg/L ( Aeromonas sp. AD1), 2.25 ± 0.41 mg/L ( P . extremaustralis AD2), and 2.73 ± 0.46 mg/L ( S . liquefaciens AD3). Chen et al. [ 32 ] demonstrated that Aeromonas sp. HN-02, an HNAD bacterium, could maintain activity at 5°C, with RRs of 0.9 and 22.3 mg/L/h for ammonia and COD, respectively. P. extremaustralis , a psychrophilic bacterium native to the extreme environments in Antarctica, has been reported as an antibacterial agent [ 33 ] and a phenacetin-degrading strain [ 34 ]; however, its potential application in NR, especially under low-temperature conditions, has received limited attention. In addition, this study potentially represents the first documented evidence of the denitrification capability of S. liquefaciens , revealing a previously uncharacterized metabolic trait of this species. Construction of psychrotolerant CMA Pairwise antagonism assays conducted on the six screened bacterial isolates did not reveal any detectable antagonistic interactions, which indicated that these strains did not inhibit the growth of each other and can be cocultured for the preparation of CMA. Therefore, the CMA developed in this study comprised a psychrotolerant nitrifying consortium NC1 (including P. veronii HN1, P. poae HN2, and P. peli HN3) and an aerobic denitrifying consortium DC1 (including Aeromonas sp. AD1, P. extremaustralis AD2, and S. liquefaciens AD3). The results of comparative experiment illustrated in Fig. 1 indicate that the CMA achieved REs of 89.3 ± 1.8%, 88.3 ± 2.2%, 85.8 ± 1.3%, and 95.5 ± 2.7% for NH 4 + -N, NO 3 − -N, TN, and COD, respectively, in CBM, which were obviously improved compared with those of the individual strains and single-functional consortia cultivated under identical conditions. Furthermore, the CMA exhibited the highest OD 600 of 1.99 ± 0.03 among different strain combinations. These results indicate that when phylogenetically distinct microorganisms coexist in the same environment but occupy different ecological niches, they can establish mutualistic, symbiotic relationships and leverage the synergistic metabolic interactions to improve microbial proliferation and contaminant removal efficacy [ 35 ]. Jing et al. [ 36 ] reported similar findings, demonstrating that the composite microbial consortium ECT, formed by combining three screened petroleum-degrading strains, Ralstonia sp. CP, Rhizobium sp. BX, and Acinetobacter sp. FL, achieved superior petroleum-degrading efficiency compared with individual strains. During CMA-mediated NR, the lowest concentration of accumulated NO 2 − -N was 0.63 ± 0.37 mg/L, indicating complete denitrification. Overall, the CMA exhibited excellent NR performance under low-temperature conditions, which indicated that the two functional bacterial consortia could be cocultured for synergistic elimination of nitrogen. Consequently, the cocultivation-derived CMA, integrating both nitrifying and denitrifying consortia, was selected for subsequent studies. The biomass growth of CMA and its NR performance at varying inoculation ratios of nitrifying and denitrifying consortia after 3 days of cultivation at 10°C with shaking at 150 rpm are depicted in Fig. 2 . At 1:1 inoculation ratio of NC1 and DC1 consortia, the CMA achieved the highest REs of 89.3 ± 1.5% for NH 4 + -N, 88.1 ± 1.8% for NO 3 − -N, 85.5 ± 2.1% for TN, and 95.3 ± 2.3% for COD. Concurrently, it exhibited maximal biomass growth (OD 600 , 1.98 ± 0.03) and minimal NO 2 − -N accumulation (0.68 ± 0.12 mg/L). The inoculation ratio significantly affected the growth of CMA and its NR efficiency. As reported by Guo et al. [ 37 ], the inoculation ratio determined the initial relative abundance of functionally different microorganisms, thereby influencing synergistic metabolic interactions within the cocultured microorganisms. Ebadi et al. [ 38 ] proposed that the decontamination performance of composite bacterial consortium is regulated by a dynamic equilibrium involving competitive relationships for nutrients (e.g., carbon, nitrogen, and phosphorus) and synergistic metabolic interactions among functionally distinct microorganisms. Notably, interspecies synergistic metabolism played a decisive role in the overall decontamination capacity of the system. Interspecies synergistic metabolism can be fully exploited only when functionally diverse bacterial strains are combined at their optimal inoculation ratios, thereby maximizing the RE of pollutants [ 36 ]. Therefore, the CMA cultured under the optimal inoculation ratio was designated NDC-6. Effects of carbon sources on NR The biomass growth and NR performance of NDC-6 using different carbon sources were evaluated, as shown in Fig. 3 . When sodium succinate was used as the sole carbon source, with incubation at 10°C for 3 days and shaking at 150 rpm, NDC-6 achieved peak values for both biomass growth (OD 600 , 1.99 ± 0.01) and TN-removal efficiency (87.8 ± 1.1%). When cultured with sodium acetate or sodium citrate as sole carbon sources, NDC-6 maintained secondary biomass amount and NR efficacy relative to that achieved with the optimal carbon source, exhibiting OD 600 values of 1.93 ± 0.01 and 1.97 ± 0.01, and TN-removal efficiencies of 79.0 ± 1.6% and 83.2 ± 1.4%, respectively. Relative to other carbon sources, NDC-6 exhibited the poorest growth (OD 600 : sucrose, 0.39 ± 0.01; glucose, 0.49 ± 0.01) and TN removal (sucrose 37.7 ± 1.3%; glucose 52.0 ± 2.1%) when cultured with sucrose or glucose as a carbon source. Numerous studies have indicated that sodium succinate and sodium citrate—intermediate metabolites in the tricarboxylic acid cycle during bacterial respiration—were more readily utilized by nitrifying or denitrifying bacteria and enhanced the activity of nitrate reductase [ 39 ]. Moreover, Wei et al. [ 40 ] demonstrated that sodium acetate, a small molecule organic compound with a simple metabolic pathway, induced higher efficiency of electron generation, transfer, and competition, thereby promoting rapid bacterial proliferation and complete denitrification. Collectively, sodium succinate, sodium citrate, and sodium acetate have been identified as optimal carbon sources for various nitrifying and denitrifying bacteria. For instance, the use of sodium succinate as the carbon source yielded the highest NH 4 + -N-removal efficiency in HNAD mixed bacteria HY-1 [ 41 ]. Sodium citrate served as an optimal carbon source for Acinetobacter calcoaceticus TY1 [ 42 ]. Wang et al. [ 43 ] reported significant enhancement of the denitrification efficiency of Paracoccus versutus JUST-3 by sodium acetate. In contrast, when macromolecular organic compounds, such as glucose or sucrose, were used as sole carbon source, they underwent hydrolysis into small organic acids prior to microbial utilization. This process reduced the carbon assimilation efficiency, inhibited bacterial growth and proliferation, and consequently compromised the NR performance. Similar findings were reported in studies on Rhizobium sp. WS7 [ 44 ] and Thauera linaloolentis [ 40 ] wherein sucrose was more unfavorable for denitrification. Therefore, based on its maximal promotion of NDC-6 growth and NR synthesis, sodium succinate was identified as the preferred carbon source and employed in subsequent experiments. Optimization of culture conditions for NR by NDC-6 Twenty-nine experimental Box–Behnken design matrices for independent variables (C/N ratio, temperature, pH, and shaking speed) and corresponding response variable ( Y ) for TN-removal efficiency are presented in Table S4. Following second-order polynomial regression fitting, the quadratic polynomial regression equation in terms of coded independent variables was obtained (Eq. ( 3 )). $$\:\text{Y}\text{}\text{=}\text{}\text{84.4}\text{-1.59}\text{A}\text{+1.31}\text{B}\text{-5.05}\text{C}\text{-1.64}\text{D}\text{+0.72}\text{AB}\text{-0.32}\text{AC}\text{-0.18}\text{AD}\text{+0.18}\text{BC}\text{+0.83}\text{BD}$$ $$\:\text{-1.01CD-2.21}{\text{A}}^{\text{2}}\text{-7.28}{\text{B}}^{\text{2}}\text{-8.26}{\text{C}}^{\text{2}}\text{-3.65}{\text{D}}^{\text{2}}$$ 3 As presented in Table 2 , the significance test value ( F- value, 208.26) and low probability value ( p < 0.0001) for the established quadratic regression model in ANOVA indicated that the model could effectively describe the impact of various factors on the TN-removal efficiency. The determination coefficient R ² and adjusted R 2 were 0.987 and 0.983, respectively, which were close to the predicted R ² of 0.983. The model exhibited nonsignificant lack of fit ( p > 0.05), indicating negligible pure error and good agreement between the model-predicted and experimental values [ 45 ]. Collectively, these findings indicated that the fitted quadratic polynomial model for TN removal by NDC-6 had high accuracy and reliability in predicting the optimal values for the independent variables and analyzing the interactive effects of these variables on the TN-removal efficiency. Table 2 ANOVA of the fitted quadratic polynomial model for TN removal by NDC-6 Source Sum of squares Mean square F value P value (prob > F ) Significance Model 995.1 71.1 73.7 < 0.0001 ** A-C/N 30.8 30.8 31.9 < 0.0001 ** B-Temperature 26.3 26.3 23.3 < 0.0001 ** C-pH 258 258 267 < 0.0001 ** D-Shaking speed 23.0 23.0 27.9 0.0002 ** AB 2.06 2.06 2.13 0.166 AC 0.41 0.41 0.42 0.528 AD 0.13 0.13 0.13 0723 BC 0.12 0.12 0.13 0.727 BD 7.08 7.08 7.33 0.017 * CD 0.25 0.25 0.25 0.622 A 2 33.9 33.9 35.2 < 0.0001 ** B 2 376 376 390 < 0.0001 ** C 2 386. 386 400 < 0.0001 ** D 2 73.2 73.2 75.9 < 0.0001 ** Residual 13.5 0.96 Lack of Fit 10.7 1.07 1.55 0.358 not significant Pure Error 2.77 0.69 Cor Total 1008.5 Note: ** very significant, P value<0.01; * significant, P value<0.05 The ANOVA results revealed that all linear terms (A, B, C, and D) in the established model exhibited p -values less than 0.01, signifying that each of the four factors significantly affected the TN removal by NDC-6. This result was consistent with previous reports that C/N ratio, temperature, pH, and DO are critical environmental factors for BNR [ 46 ]. Using the F -value analysis, the factors influencing TN removal were ranked according to their influence degree as follows: pH > shaking speed > C/N ratio > temperature. pH had the most significant impact on TN removal. Similar findings were reported for the optimization of conditions for coculture of B. cereus G2 and B. pumilus G5 by Peng et al. [ 47 ], who demonstrated that excessively acidic or alkaline environments remarkably compromised the NR performance of CMA. Notably, temperature had a relatively weaker influence on TN removal compared with the other three factors. The reason might be that NDC-6 was composed of multiple psychrotolerant strains, which had a certain tolerance to low-temperature environments and a broad temperature-adaptation range. In addition, temperature and shaking speed significant influenced TN removal ( p < 0.05), signifying that the NR performance of NDC-6 was susceptible to the interactions of these two environmental parameters. The 3D response surface plots illustrating the interactive effects of the four independent variables (C/N ratio, temperature, pH, and shaking speed) on the removal of TN by NDC-6 are presented in Fig. 4 . With increasing C/N ratio and temperature, the TN-removal efficiency by NDC-6 initially increased and subsequently decreased (Fig. 4 A). The highest TN-removal efficiencies were achieved at a C/N ratio of approximately 5–6 and a temperature around 10°C. Carbon sources serve as electron donors essential for denitrification [ 48 ]. Carbon deficiency triggers competitive inhibition between heterotrophic nitrifying bacteria and aerobic denitrifying bacteria for limited carbon resources, causing concurrent decline in NH 4 + -N- and NO 3 − -N-removal efficiencies [ 49 ]. In contrast, excessive carbon availability stimulates the overproliferation of heterotrophic bacteria, which consumes substantial amounts of DO, resulting in the deterioration of water quality and carbon wastage. Therefore, effective enhancement of NR can only be achieved when the C/N ratio is maintained within an optimal range. Temperature stress induces microbial oxidative stress and cellular damage, thereby impairing the nutrient-removal capacity [ 43 ]. At lower temperatures, enzymatic activity is inhibited, reducing metabolic rates and substantially diminishing the NR performance [ 50 ]. On the contrary, excessively high temperatures induce enzyme denaturation [ 51 ]. Optimal metabolic rates can only be maintained within a defined temperature range to enhance NR. As illustrated in Fig. 4 F with the increase in initial pH of CBM and shaking speed of the oscillating incubator, the efficiency of TN removal by NDC-6 exhibited parabolic variation trends, characterized by an initial increase followed by a decrease. The highest efficiency of TN removal was achieved at initial pH in the range of 7.0–7.5 and a shaking speed of 150–160 rpm. Similar to other NR-functional strains, such as Paracoccus versutus JUST-3 [ 43 ] and Glutamicibacter halophytocola MD1 [ 52 ], NDC-6 preferred a neutral or weakly alkaline environment (pH 6–9). Exposure to strongly acidic or alkaline conditions induces aggregation of surface charge on bacterial cells, disrupting the integrity of cell structure and inhibiting microbial proliferation capacity and enzymatic activity, thereby impairing nitrogen transformation [ 53 ]. Consequently, precise pH regulation is a critical determinant in optimizing microbial NR. In this study, DO level in the culture medium was adjusted by regulating the shaking speed of the oscillating incubator. Jin et al. [ 54 ] found that the DO content increased linearly with shaking speed within a certain range. Preliminary experiments in this study indicated that the DO content in the culture medium in 250 mL conical flask was approximately 3.3 mg/L at a shaking speed of 100 rpm. Each 10 rpm increment in shaking speed increased the DO content by approximately 0.4 mg/L. The highest TN-removal efficiency was observed for the shaking speed of 150–160 rpm, corresponding to a DO content of 5.3–5.7 mg/L. Li et al. [ 55 ] reported that excessively low DO content inhibited the activity of ammonia monooxygenase in nitrifying bacteria, reducing the nitrification performance. Moreover, low DO concentrations suppress the catalytic efficiency of denitrifying enzymes, thereby slowing down mass transfer and substrate utilization [ 56 ]. Conversely, an excessive high DO content not only inhibits the activity of nitrite reductase in the denitrification process but also induces competition for electrons between O 2 and NO 3 − -N, leading to incomplete nitrate reduction [ 57 ]. This highlights the importance of optimizing the shaking speed to ensure efficient removal of nitrogen by NDC-6. The cultivation conditions optimized for maximizing TN removal were as follows: A = 0, B = 0.075, C = − 0.3, and D = − 0.2. Accordingly, the model predicted that maximum TN-removal efficiencies of 90.2% would be achieved at a C/N ratio of 6, a temperature of 10.2°C, pH 7.2, and a shaking speed of 156 rpm. Under the optimal conditions, a 3-day cultivation in the verification experiment yielded a TN-removal efficiency of 89.8 ± 1.8%%, which closely approached the theoretically predicted maximum RE for TN. This confirmed the capability of the model to accurately optimize cultivation conditions. Nitrogen metabolic pathways in NDC-6 Nitrogen balance analysis The single functional strains, heterotrophic nitrifying consortium, aerobic denitrifying consortium, and NDC-6 were inoculated into optimized CBM (containing 52.5 ± 0.2 mg/L NH 4 + -N and 52.6 ± 0.2 mg/L NO 3 − -N) at optimal inoculation ratios. Following 3-day incubation at 10.2°C with shaking at 156 rpm, different forms of nitrogen were determined to establish nitrogen mass balance (Table 3 ). Notably, a distinct accumulation of NO 2 − -N (> 5 mg/L) was consistently observed during NR by P. veronii HN1, P. poae HN2, P. peli HN3, and the nitrifying consortium NC1, suggestive of the absence of significant denitrification capability of these strains. Furthermore, only the individual denitrifier, denitrifying consortium, and NDC-6 were capable of converting TN into gaseous-N. Of the initial TN content, 40.2 ± 1.6% was converted to gaseous-N by NDC-6, which was significantly higher than that in the case of Aeromonas sp. AD1 (25.5 ± 1.4%), P. extremaustralis AD2 (28.2 ± 1.8%), S. liquefaciens AD3 (24.9 ± 2.1%), and the denitrifying consortium DC1 (28.4 ± 1.2%). Gas chromatography revealed N 2 O concentrations of 0.26 ± 0.02 mg/L for Aeromonas sp. AD1, 0.33 ± 0.02 mg/L for P. extremaustralis AD2, 0.29 ± 0.02 mg/L for S. liquefaciens AD3, 0.15 ± 0.01 mg/L for DC1, and 0.07 ± 0.01 mg/L for NDC-6, indicating that the gaseous-N produced during denitrification by these strains was predominantly N 2 instead of N 2 O. These observations indicate enhanced completeness of denitrification and metabolic efficiency by NDC-6, wherein a higher proportion of NO 3 − -N or NO 2 − -N was fully reduced to the terminal product N 2 rather than accumulating as intermediate gases (e.g., N 2 O) or residual ions. Similar results were reported by Fang et al. [ 58 ], who found that coculturing the yeast Kazachstania exigua T14-1 with the bacterium Methylobacterium sp. T5-6 substantially improved the nitrogen metabolism capacity compared with monocultures of either strain. Furthermore, following 3 days of cultivation at 10.2°C, > 30 mg/L of intracell-N was detected in all the tested microbial combinations, indicating that > 30% of the initial TN content was assimilated into cellular nitrogen via biosynthetic assimilation. Among these, NDC-6 exhibited the highest intracell-N conversion rate at 49.5 ± 1.4%. In contrast to the reported microbial NR pathways primarily relying on assimilation [ 59 ], NDC-6 achieved NR under low-temperature aerobic conditions primarily via dual pathways of bacterial assimilation and dissimilation converting inorganic N (mainly NH 4 + -N and NO 3 − -N) into intracell-N and N 2 . These findings indicated that the composite consortium NDC-6 exhibited exceptional NR performance under low-temperature conditions, attributable to synergistic interactions among constituent strains that significantly enhance nitrogen metabolism efficiency, highlighting its greater potential for application in wastewater treatment. Table 3 Nitrogen balance analysis during nitrogen removal by different strain combinations Strain combinations Nitrogen concentration (mg/L) RE TN (%) NH 4 + -N NO 3 ― -N NO 2 ― -N Organic-N Intracell-N Gaseous-N TN Pseudomonas veronii HN1 7.21 ± 0.34 50.67 ± 1.65 7.64 ± 0.52 1.53 ± 0.31 37.79 ± 1.05 – 67.29 ± 1.53 36.19 ± 1.28 Pseudomonas poae HN2 7.67 ± 0.42 51.12 ± 1.78 5.85 ± 1.03 1.78 ± 0.13 38.66 ± 0.92 – 66.42 ± 1.24 37.01 ± 1.16 Pseudomonas peli HN3 7.13 ± 0.38 50.08 ± 2.14 7.35 ± 1.05 1.94 ± 0.21 38.58 ± 1.07 – 66.50 ± 1.56 36.94 ± 1.03 Aeromonas sp. AD1 31.5 ± 1.03 7.51 ± 0.36 2.24 ± 0.87 2.51 ± 0.22 34.43 ± 1.21 26.89 ± 1.59 43.76 ± 1.25 58.52 ± 2.05 Pseudomonas extremaustralis AD2 29.81 ± 1.22 7.26 ± 0.13 2.06 ± 0.73 2.27 ± 0.46 33.91 ± 1.06 29.77 ± 2.31 41.40 ± 1.37 60.73 ± 2.14 Serratia liquefaciens AD3 31.64 ± 1.74 7.68 ± 0.29 2.78 ± 0.46 2.64 ± 0.38 34.07 ± 1.23 26.27 ± 1.69 44.74 ± 1.59 57.57 ± 1.98 Nitrifying consortium NC1 6.77 ± 0.09 50.48 ± 1.71 5.99 ± 1.22 2.08 ± 0.18 40.31 ± 1.02 – 65.32 ± 2.11 38.06 ± 1.56 Denitrifying consortium DC1 30.22 ± 1.39 7.12 ± 0.48 1.21 ± 0.05 2.08 ± 0.24 34.47 ± 1.25 29.98 ± 1.47 40.63 ± 1.72 61.47 ± 1.78 NDC-6 4.61 ± 0.55 4.13 ± 0.38 0.48 ± 0.17 1.64 ± 0.38 52.15 ± 1.69 42.37 ± 2.04 10.88 ± 0.57 89.68 ± 1.35 Functional genes related to NR The genes hao , nap A, ni rS, ni rK, cnor B, and nosZ are generally considered as the functional genes encoding key enzymes involved in microbial nitrogen metabolism. In this study, PCR amplification of these six functional genes was conducted across all bacterial strains to further validate their nitrification and denitrification capabilities, thereby elucidating the potential metabolic pathways of NR under low-temperature conditions. The amplification results are presented in Fig. 5 and summarized in Table S5. The hao gene, encoding hydroxylamine oxidase (HAO), is a critical biomarker for nitrification. HAO catalyzes the oxidation of hydroxylamine (NH 2 OH), a key intermediate in nitrification, to NO 2 − -N [ 60 ]. In this study, the successful amplification and expression of the hao gene in P. veronii HN1 (282 bp), P. poae HN2 (265 bp), and P. peli HN3 (1752 bp) confirmed the involvement of hao in nitrification in these three strains. The hao gene was likewise amplified in other nitrification-capable Pseudomonas strains, such as P. mendocina SCZ-2 [ 61 ] and P. citronellolis YN-21 [ 27 ]. Furthermore, PCR amplification revealed the absence of functional genes encoding nitrite reductases (NIR) in these three strains. As NIR catalyzes the reduction of NO 2 − -N to gaseous NO or N 2 O [ 62 ], this genetic characteristic explained distinct accumulation of NO 2 − -N observed during the NR process. Notably, the nos Z gene, encoding nitrous oxide reductase (NOS), was successfully amplified from strains HN1 (396 bp), HN2 (352 bp), and HN3 (298 bp). Given the well-established catalytic role of NOS in reducing N 2 O to N 2 , as demonstrated by Guo et al. [ 63 ], this finding signified that these three strains can convert N 2 O generated from ancillary denitrification processes into N 2 , thereby mitigating the emission of greenhouse gases. As illustrated in Fig. 5 and Table S5, typical denitrification genes ( nap A, nir S, nir K, cnor B, and nos Z) were PCR-amplified from Aeromonas sp. AD1, P. extremaustralis AD2, and S. liquefaciens AD3. The nap A gene, a biomarker involved in aerobic denitrification, encodes the periplasmic nitrate reductase (NAP), which catalyzes the reduction of NO 3 − -N to NO 2 − -N as demonstrated by Zheng et al. [ 64 ]. The nap A gene has also been amplified from other psychrotolerant denitrifiers, such as Priestia aryabhattai KX-3 [ 65 ] and Psychrobacter cryohalolentis strain F5-6 [ 66 ]. Concurrently, nir S and nir K were identified as functional genes encoding NIR. These two types of functional genes in this study could simultaneously exist in the same strain and promoted the removal of NO 2 − -N. This genetic configuration correlated with the observed low NO 2 − -N accumulation during the removal of NO 3 − -N by strains AD1, AD2, and AD3, and their composite consortium, indicating synergistic functionality of multiple NIR isoforms in nitrite metabolism. The cnorB gene encoding nitric oxide reductase (NOR) for the conversion of NO to N 2 O and the nos Z gene responsible for the reduction of N 2 O to N 2 were detected in Aeromonas sp. AD1, P. extremaustralis AD2, and S. liquefaciens AD3, respectively, indicating that these three strains prevented NO and N 2 O emission by directly reducing them to N 2 during denitrification. These findings aligned with the previously observed low concentrations of N 2 O produced by these three strains during denitrification. In summary, successful amplification of nap A, nir S, nir K, cnor B, and nos Z in strains AD1, AD2, and AD3 indicated that NAP, NIR, NOR, and NOS are involved in different steps of the dissimilatory nitrate reduction pathway. This genetic evidence confirms their capability to achieve complete denitrification without intermediate accumulation. This denitrification pathway was consistent with the nitrogen-removal mechanism in P. aeruginosa WS-03 reported by Wei et al. [ 28 ]. Pathways for nitrogen metabolism Based on the results of nitrogen balance analysis and PCR amplification of functional genes related to NR, the proposed pathways for nitrogen metabolism in NDC-6 are depicted in Fig. 6 . The composite consortium NDC-6, constructed from heterotrophic nitrifiers and aerobic denitrifiers, was speculated to possess a complete pathway for nitrogen metabolism and to achieve synchronous and efficient removal of NH 4 + -N and NO 3 − -N through dual metabolic pathways: assimilatory conversion into intracell-N and dissimilatory transformation (heterotrophic nitrification and aerobic denitrification) to N 2 . The initial step of heterotrophic nitrification in the composite consortium NDC-6 involved the oxidation of NH 4 + -N to NH 2 OH using sodium succinate as the carbon source and electron donor, mediated by constituent strains P. veronii HN1, P. poae HN2, and P. peli HN3. Subsequently, HAO from these nitrifying strains catalyzed further oxidation of NH 2 OH into NO 2 − -N. Concurrently, during aerobic denitrification, NAP expressed by Aeromonas sp. AD1, P. extremaustralis AD2, and S. liquefaciens AD3 reduced NO 3 − -N to NO 2 − -N. In the following steps, the NO 2 − -N generated through nitrification and initial denitrification steps underwent sequential enzymatic conversions: NIR catalyzed the reduction of NO 2 − -N to NO, followed by NOR-mediated transformation of NO to N 2 O, and final reduction to N 2 via NOS. The functional enzymes involved in these steps were collectively expressed by Aeromonas sp. AD1, P. extremaustralis AD2, and S. liquefaciens AD3. Overall, the composite consortium NDC-6 achieved efficient removal of NH 4 + -N, NO 3 − -N, and NO 2 − -N through synergistic metabolism and complementary functions among its constituent strains, which facilitated a broad ecological niche for this CMA and enhanced its environmental adaptability, resulting in superior metabolic capabilities for diverse nitrogen substrates in wastewater treatment. Conclusion Three heterotrophic nitrifying bacteria and three aerobic denitrifying bacteria were isolated from activated sludge, river sediment, and frozen soil samples in winter. The composite consortium NDC-6, constructed by coculturing these six strains, exhibited exceptional biomass proliferation capacity and efficient NR performance at 10°C. The optimal conditions for TN removal were determined to be sodium succinate as carbon source, C/N ratio of 6, temperature of 10.2°C, pH of 7.2, and shaking speed of 156 rpm. Nitrogen balance analysis revealed that NDC-6 achieved the highest gaseous-N conversion rate (42.4%) and intracell-N assimilation rate (49.5%) among all the tested groups. Under low-temperature aerobic conditions, NDC-6 primarily facilitated NR via dual pathways of dissimilation and assimilation. Functional gene amplification confirmed that the complete NR mechanism in NDC-6 relied on synergistic interactions and metabolic complementarity between the hao gene in the nitrifying consortium and denitrification genes ( nap A, nir S, nir K, cnor B, and nos Z) in the denitrifying consortium. These findings provide a theoretical foundation for applying psychrotolerant CMA in the treatment of nitrogen-containing wastewater under low-temperature conditions. Declarations Supplementary Information The online version contains supplementary material available at Acknowledgements This research was financially supported by the Basic Research Project of Educational Department of Liaoning Province (No. LJ212411035027) and the National Natural Science Foundation of China (No. 52300023). Authors contribution Yihua Dong: Writing – original, Review & editing, Conceptualization, Funding acquisition. Jing Xu: Methodology, Investigation, Visualization. Feng Chen: Methodology, Investigation, Validation, Data curation. Liang Li: Conceptualization, Formal analysis, Project administration. Guangsheng Qian: Supervision, Conceptualization, Funding acquisition. Peng Zhang: Methodology, Investigation, Validation. Data availability The data used in this study are available on request. Conflict of interest The authors declare that there are no competing interests. Ethics approval and consent to participate Not applicable. 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Process Biochem 130:674–684. https:// doi. org/ 10. 1016/ j. procbio. 2023. 05. 030 Hou Y, Zhang DY, Cao HR, Zhang YL, Zhao DD, Zeng WM, Lei H, Bai Y (2022) Identification of aerobic-denitrifying Psychrobacter cryohalolentis strain F5-6 and its nitrate removal at low temperature. Int Biodeter Biodegr 172:105426. https:// doi. org/ 10. 1016/ j. ibiod. 2022. 105426 Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 24 Oct, 2025 Reviews received at journal 24 Oct, 2025 Reviewers agreed at journal 04 Oct, 2025 Reviews received at journal 14 Sep, 2025 Reviewers agreed at journal 03 Sep, 2025 Reviewers agreed at journal 01 Sep, 2025 Reviewers agreed at journal 01 Sep, 2025 Reviewers invited by journal 15 Aug, 2025 Editor assigned by journal 15 Aug, 2025 Submission checks completed at journal 15 Aug, 2025 First submitted to journal 14 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Carbon source: sodium citrate (SC), sodium acetate (SA), glucose (Glu), sucrose (Suc), and sodium succinate (SS).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7370617/v1/2b834dfa8cd20541751ba3dc.png"},{"id":89669524,"identity":"3526c1ba-29dd-4d7d-8dfd-373d630a60d5","added_by":"auto","created_at":"2025-08-22 12:39:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3074417,"visible":true,"origin":"","legend":"\u003cp\u003eThree-dimensional (3D) response surface plots of the interaction of C/N, temperature, pH and shaking speed for TN removal efficiency by NDC-1. (\u003cstrong\u003eA\u003c/strong\u003e) C/N and temperature; (\u003cstrong\u003eB\u003c/strong\u003e) C/N and pH; (\u003cstrong\u003eC\u003c/strong\u003e) C/N and shaking speed; (\u003cstrong\u003eD\u003c/strong\u003e) temperature and pH; (\u003cstrong\u003eE\u003c/strong\u003e) temperature and shaking speed; (\u003cstrong\u003eF\u003c/strong\u003e) pH and shaking speed\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7370617/v1/e3f41b0d87968348c7e97c2b.png"},{"id":89669509,"identity":"1edf9a19-57bf-410e-86ec-0da51b6ef9b9","added_by":"auto","created_at":"2025-08-22 12:39:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1048223,"visible":true,"origin":"","legend":"\u003cp\u003ePCR amplification offunctional genes for key enzyme of isolated strains\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7370617/v1/fbc87811fc7dd59b65391f02.png"},{"id":89670073,"identity":"f556f6c0-c77c-48f3-b9e3-7b622d3f276f","added_by":"auto","created_at":"2025-08-22 12:47:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1549440,"visible":true,"origin":"","legend":"\u003cp\u003eNitrogen removal pathways of composite consortia NDC-1\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7370617/v1/1d998b03d1768c666c46ee93.png"},{"id":89672691,"identity":"e3a6f482-08aa-43fd-b87b-2c398aa72b91","added_by":"auto","created_at":"2025-08-22 13:11:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15707120,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7370617/v1/d7a13645-8d23-4e00-a1ba-d296cf698d50.pdf"},{"id":89670072,"identity":"2fd3a9fa-6bca-48f2-9bc6-9bd7c269449f","added_by":"auto","created_at":"2025-08-22 12:47:06","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1013595,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7370617/v1/ff30567cd7ed30a117474991.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of a psychrotolerant composite microbial agent for nitrogen removal and its nitrogen metabolism pathways","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSubstantial amounts of nitrogen-containing pollutants have been persistently entering the aquatic systems over the recent decades, owing to extensive application of fertilizers in farmlands, direct discharge of untreated domestic sewage, and inadequate treatment of industrial effluents from sectors, such as chemical manufacturing, pharmaceuticals, and food processing [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Excessive nitrogen causes eutrophication of natural water bodies and seriously threatens ecosystem functioning and human health [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Therefore, efficient treatment of nitrogen-contaminated wastewater is currently a critical environmental issue that needs to be solved urgently. Biological nitrogen removal (BNR) has emerged as a prominent research focus worldwide in the field of wastewater treatment in view of the powerful functions of microbial metabolism and transformation, including ammoniation, nitrification, and denitrification. Compared with traditional physical or chemical methods, BNR exhibits remarkable advantages, such as higher efficiency, lower energy consumption, and superior ecological benefits [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, environmental temperature is a key factor that limits the actual application of BNR in wastewater purification [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Currently, most known functional microorganisms carry out nitrogen removal (NR) under mesophilic conditions, and the optimal temperature for microbial growth and physiological metabolism is 25\u0026ndash;37\u0026deg;C, with the best metabolic activity occurring at 20\u0026ndash;25\u0026deg;C [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Low-temperature environment (\u0026lt;\u0026thinsp;15\u0026deg;C) can damage the cell membrane of microorganisms, seriously inhibit the metabolic activity of enzymes and the protein synthesis rate, and slow down the growth rate of functional microbes, ultimately resulting in unsatisfactory NR performance [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. For example, reducing the temperature from 35 to 10\u0026deg;C drastically decreased the aerobic denitrification rate of ammonia-oxidizing archaea from 20.64 to 7.75 mg N/(L\u0026middot;h), with a concomitant decrease in total nitrogen (TN) removal efficiency from 98.5\u0026ndash;60.9% [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Similar effects have been reported for \u003cem\u003eGlutamicibacter arilaitensis\u003c/em\u003e EM-H8 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and \u003cem\u003eRaoultella ornithinolytica strain\u003c/em\u003e YX-4 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Compared with mesophilic nitrogen-removing bacteria, psychrotolerant bacteria can proliferate extensively over broad low-temperature ranges while maintaining high NR activity. Therefore, developing psychrotolerant bacteria with enhanced NR capabilities could be a potential strategy for improving wastewater purification efficiency under low-temperature conditions.\u003c/p\u003e\u003cp\u003eIn recent years, several researchers have isolated psychrotolerant functional microorganisms from low-temperature environment and used them for treating nitrogen-containing wastewater, achieving certain removal effects. Yang et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] obtained \u003cem\u003eBacillus simplex\u003c/em\u003e H-b, a psychrotrophic heterotrophic nitrification and aerobic denitrification (HNAD) bacterium, from the frozen soil in Northeast China. \u003cem\u003eB. simplex\u003c/em\u003e H-b exhibited excellent NR capabilities, with 82.2% ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, 60 mg/L), 67.3% nitrate nitrogen (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, 63 mg/L), and 78.7% nitrite nitrogen (NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, 10 mg/L) removal efficiencies at 10\u0026deg;C. \u003cem\u003ePseudomonas fragi\u003c/em\u003e EH-H1 exhibited high-efficiency HNAD performance at 15\u0026deg;C with NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, and TN removal efficiencies of 100%, 99.1%, and 97.7%, respectively [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Ma et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] found that psychrotolerant bacteria employed diverse strategies, including maintenance of membrane fluidity, expression of cold shock proteins, structural regulation of enzymes, and accumulation of compatible solutes, to counteract cold-induced stress. Despite promising application prospects in wastewater purification, single microbial species poses inherent limitations in degrading complex pollutants because of metabolic constraints and stringent cultivation requirements. Consequently, single microbial strains achieve much lower NR efficiency when treating actual wastewater with complex and unstable composition [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Fluctuating wastewater treatment efficiency and substandard effluent quality constrains practical implementation of psychrotolerant functional bacteria in full-scale wastewater treatment plants [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. To improve the treatment efficacy of nitrogen-containing wastewater, some researchers have focused on developing composite microbial agent (CMA) by coculturing two or more functionally distinct but compatible (i.e., non-antagonistic) microorganisms [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. CMA has been shown to exhibit excellent synergistic effects in wastewater treatment, which can improve the pollutant-removal efficiencies and broaden metabolic pathways by leveraging the metabolic complementarity and interspecies cooperation among different strains [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Compared with single strains, CMA exhibits superior operational stability and ecological adaptability, especially for actual wastewater treatment. The composite consortium B-Cl, comprising \u003cem\u003eB. marisflavi\u003c/em\u003e B1 and \u003cem\u003eB. marisflavi\u003c/em\u003e B5 in a volume ratio of 3:2, achieved a removal efficiency of 86.9% for 2,4-dichlorophenol, higher than that of B1 (69.9%) and B5 (75.5%) alone [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Shi et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] used acclimated CMA, consisting of indigenous sediment microorganisms, functional bacteria (\u003cem\u003eB. natto\u003c/em\u003e ADT), and supplemental bacteria (\u003cem\u003eLactobacillus bulgaricus\u003c/em\u003e, \u003cem\u003eEnterococcus\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e, etc.) in a volume ratio of 10:1:1, for \u003cem\u003ein-situ\u003c/em\u003e remediation of polluted black-odorous streams. After treatment, the concentrations of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N decreased by 74.0% and 76.3%, underscoring the effectiveness of the CMA. While CMA has demonstrated broad applicability in bioremediation of polluted environment, including petroleum hydrocarbon degradation in oil-contaminated soil [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], organic matter decomposition of kitchen waste [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and immobilization of heavy metal [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], its potential for treating nitrogen-contaminated wastewater under low-temperature conditions remains underexplored. Thus, it is imperative to screen psychrotolerant NR-functional bacteria, construct high-efficiency microbial consortia with optimized functions, and further explore the synergistic metabolic mechanisms among strains to enhance bioremediation performance in cold climates.\u003c/p\u003e\u003cp\u003eIn this study, a psychrotolerant CMA with efficient NR performance was developed to improve the NR efficacy at low-temperature conditions. The specific objectives of this study were to (i) isolate and screen a series of psychrotolerant bacteria with NR capacities from river sediment, frozen soil, and activated sludge in cold regions during winter; (ii) construct a psychrotolerant CMA for efficient removal of nitrogen based on principles of ecological niche separation and synergistic metabolisms; (iii) optimize the key factors affecting NR by CMA using response surface methodology (RSM) based on the Box\u0026ndash;Behnken design; (iv) elucidate the NR pathways employed by CMA via polymerase chain reaction (PCR) amplification of key genes encoding nitrifying and denitrifying enzymes, together with nitrogen mass balance analysis. The development and characterization of psychrotolerant CMA provides mechanistic insights into the treatment of nitrogen-contaminated wastewater in regions with cold climate and offers novel solutions for it.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMaterials\u003c/h2\u003e\u003cp\u003eSamples for isolating and screening functional strains with NR capability were obtained from river sediment, frozen soil, and activated sludge from a sewage treatment plant in Shenyang, Liaoning Province, China during winters with temperatures below 5\u0026deg;C. The collected samples were stored in a refrigerator at 4\u0026deg;C until use.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCulture medium\u003c/h3\u003e\n\u003cp\u003eA mineral salt medium (MSM) containing 0.2 g/L MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 0.01 g/L CaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, 0.001 g/L FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, and 2 mL/L of trace element solution, pH 7\u0026ndash;7.2, was used. The trace element solution was as described by Yang et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAn enrichment medium (EM) was used to enrich psychrotolerant nitrifying and denitrifying bacteria from samples under low-temperature conditions. The EM consisted of an MSM base supplemented with 2.5 g/L C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eNa\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, 0.32 g/L NaNO\u003csub\u003e3\u003c/sub\u003e, 0.25 g/L (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, and 0.044 g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eThe Luria broth (LB) medium, used for isolating and purifying specific functional strains, contained 10 g/L peptone, 5 g/L yeast extract, and 10 g/L NaCl and had a pH of 7\u0026ndash;7.2.\u003c/p\u003e\u003cp\u003eThe psychrotolerant nitrifying bacteria were screened using a nitrification medium (NM), which consisted of an MSM base supplemented with 2.5 g/L C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eNa\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, 0.5 g/L (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;SO\u003csub\u003e4\u003c/sub\u003e (equivalent to 0.105 g/L NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N), and 0.044 g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eThe psychrotolerant denitrifying bacteria were screened using a denitrification medium (DM), which consisted of an MSM base supplemented with 2.5 g/L C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eNa\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, 0.64 g/L NaNO\u003csub\u003e3\u003c/sub\u003e (equivalent to 0.105 g/L NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N), 0.044 g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, and 1 mL/L bromothymol blue (BTB, 1% w/v).\u003c/p\u003e\u003cp\u003eA composite bacterial medium (CBM), serving as the basal medium for cultivating the CMA and optimizing key influencing factors, was prepared by supplementing MSM with the following components: 2.5 g/L C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eNa\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, 0.25 g/L (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;SO\u003csub\u003e4\u003c/sub\u003e (equivalent to 0.0525 g/L NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N), 0.32 g/L NaNO\u003csub\u003e3\u003c/sub\u003e (equivalent to 0.0525 g/L NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N), and 0.044 g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eAll the above-mentioned chemical reagents were purchased from the Sinopharm Chemical Reagent Co., Ltd. (China). Unless otherwise specified, these chemicals were of analytical or higher purity grade. The agar plate medium was prepared by adding 20 g agar per liter of liquid medium. All culture media used in this study were sterilized at 121\u0026deg;C for 30 min before use.\u003c/p\u003e\n\u003ch3\u003eIsolation and screening of psychrotolerant functional bacteria\u003c/h3\u003e\n\u003cp\u003eEnrichment and isolation of psychrotolerant bacteria\u003c/p\u003e\u003cp\u003eFifty milliliters of activated sludge or 5 g of river sediment/frozen soil samples was added to 100 mL EM in 250 mL sterilized conical flasks. Several sterilized glass beads were put in the flasks, followed by incubation at 10\u0026deg;C with shaking at 150 rpm for 3 h. After complete suspension and dispersion of the sludge/sediment, 10 mL aliquots were transferred to 100 mL fresh EM and cultivated at 10\u0026deg;C for 5 days with shaking at 150 rpm to facilitate bacterial proliferation and enrichment. The same process was repeated several times until significant NR effects were detected in the EM. Subsequently, the enriched cultures were serially diluted with sterile deionized water in concentration gradients of 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, and 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e. Thereafter, 1 mL of diluted bacterial suspensions were coated onto the LB agar plates and incubated in an inverted position in a constant-temperature incubator at 10\u0026deg;C for 7 days. Colony morphology and bacterial growth were regularly monitored. Distinct individual colonies were selected for streak purification on fresh LB agar plates. The purification process was repeated through multiple streaking cycles until pure cultures were confirmed under an optical microscope (Leica DMi1, GER).\u003c/p\u003e\u003cp\u003eScreening of psychrotolerant nitrification bacteria\u003c/p\u003e\u003cp\u003ePurified isolates were prepared as cell suspensions in sterile deionized water (optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;1.0). A 10% (v/v) inoculum of cell suspensions was added to 100 mL NM and cultured at 10\u0026deg;C with shaking at 150 rpm for 3 days. The NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N-removal performance of the isolates was evaluated by calculating the removal efficiency. The strains with superior NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N-removal efficiency (\u0026gt;\u0026thinsp;85% at 10\u0026deg;C)) were screened as candidates for subsequent investigation.\u003c/p\u003e\u003cp\u003eScreening of psychrotolerant aerobic denitrification bacteria\u003c/p\u003e\u003cp\u003eA 100 \u0026micro;L cell suspension (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.0) of purified isolates was coated onto the DM agar plates and incubated in an inverted position in a constant-temperature incubator at 10\u0026deg;C for 7 days. Distinct individual colonies exhibiting intrinsic blue coloration or blue halo were selectively transferred to 100 mL liquid DM. The cultures were incubated at 10\u0026deg;C for 3 days with shaking at 150 rpm. The strains demonstrating exceptional NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N removal capabilities (\u0026gt;\u0026thinsp;85% efficiency at 10\u0026deg;C) were prioritized for subsequent investigation.\u003c/p\u003e\n\u003ch3\u003eIdentification of psychrotolerant functional bacteria\u003c/h3\u003e\n\u003cp\u003eColony characteristics of the isolated strains were observed after 4 days of cultivation on respective agar plates. Gram staining for all pure screened strains was performed using the Gram Strain Kit (Bestbio, China), and the cellular morphology was analyzed via scanning electron microscopy (SEM, Zeiss Geminis, GER). Genomic DNA was extracted from each strain using an Ezup Column Bacterial Genomic DNA Extraction Kit (SK8255, Sangon Biotech, Shanghai, China), followed by PCR amplification of the 16S rRNA gene with the universal primers 27F and 1492R (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The amplification product was sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). The isolated strains were taxonomically identified by comparing the 16S rRNA gene sequence homology with other registered strains in the GenBank of the National Centre for Biotechnology Information using the BLAST algorithm. The neighbor-joining algorithm (1000 replicates) was employed to construct a phylogenetic tree of the isolates with the MEGA 11.0 software.\u003c/p\u003e\n\u003ch3\u003eConstruction of psychrotolerant CMA\u003c/h3\u003e\n\u003cp\u003eThe Oxford cup agar diffusion assay was performed to investigate the antagonistic interactions among the isolated bacterial strains on LB agar plates [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Non-antagonistic functional strains were cultivated at 10\u0026deg;C for 2 days with shaking at 150 rpm. Following centrifugation at 8000 rpm for 10 min, the bacterial cells were washed with sterile deionized water and resuspended to obtain bacterial suspensions (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.0) of each functional strain. The suspensions of strains sharing an identical metabolic function (nitrification or denitrification) were combined in equal volume ratios to form the nitrifying consortium NC1 and denitrifying consortium DC1. Subsequently, these functional consortia were pooled in equal volumes to form the CMA. The cell suspensions of each individual strain, single-functional consortium, and the CMA were inoculated at an inoculum volume of 10% (v/v) into 100 mL CBM and cultivated at 10\u0026deg;C for 3 days with shaking at 150 rpm. By comparing the biomass growth (OD\u003csub\u003e600\u003c/sub\u003e) and the NR performance of different inoculated strain combinations, the optimal culture strategy was determined. The nitrifying (NC1) and denitrifying (DC1) consortiums were inoculated at varying ratios (4:1, 3:1, 2:1, 1:1, 1:2, 1:3, and 1:4) with a total inoculum volume of 10% (v/v) into 100 mL CBM. Based on the comparison of the biomass growth and NR performance of CMA after 3-day cultivation, the optimal inoculation ratio was determined to construct the CMA. The CMA under the optimal inoculation ratio was named NDC-6.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eInfluence of carbon source\u003c/h2\u003e\u003cp\u003eSodium citrate (SC; 2.5 g/L), sodium acetate (SA; 2.09 g/L), sucrose (Suc; 1.45 g/L), glucose (Glu; 1.53 g/L), and sodium succinate (SS; 2.07 g/L) were individually supplemented into CBM as sole carbon sources. Each carbon source was adjusted to provide an equivalent carbon content of 612 mg/L in the CBM to evaluate its effect on the NR performance of NDC-6 at low temperature. Cell suspensions (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.0) of the NC1 and DC1 were inoculated at optimal ratios with a total inoculum volume of 10% (v/v) into the 100 mL CBM containing different carbon sources, followed by aerobic cultivation at 10\u0026deg;C for 3 days with shaking at 150 rpm. Comparative analyses of biomass growth and NR efficiencies of NDC-6 were used to determine the optimal carbon source.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eOptimization of culture condition for NR by NDC-6\u003c/h3\u003e\n\u003cp\u003eThe key cultivation factors influencing NR by NDC-6 were optimized using RSM based on a Box\u0026ndash;Behnken design. The C/N ratio (A), temperature (B), pH value (C), and shaking speed (D) were selected as independent variables and were coded low (\u0026minus;\u0026thinsp;1) or high (+\u0026thinsp;1) by keeping 0 as the mid-point. The coded and actual levels of these process variables are represented in Table S2. The initial TN concentration in the CBM medium was maintained at 0.105 g/L, with C/N ratios adjusted by varying the concentration of the optimal carbon source. The shaking speed was modulated to regulate the dissolved oxygen (DO) content in the culture medium. The TN removal efficiency was considered a response variable. A total of 29 experimental runs with a four-factor, three-level Box\u0026ndash;Behnken design were conducted in 250 mL conical flasks containing 100 mL CBM and 10 mL mixing inoculum of consortia NC1 and DC1 to optimize variable ranges and levels. After 3 days of cultivation, TN- removal efficiency in each experimental run was calculated. A quadratic polynomial regression model was implemented to characterize the experimental response data, facilitating identification of optimal conditions and assessment of the significance of variable interactions.\u003c/p\u003e\n\u003ch3\u003eNitrogen metabolism genes\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was exacted from the isolated strains using a DNA extraction kit (B518255, Sangon Biotech, Shanghai, China). PCR amplification was performed targeting functional genes, namely \u003cem\u003ehao\u003c/em\u003e, \u003cem\u003enap\u003c/em\u003eA, \u003cem\u003enir\u003c/em\u003eS, \u003cem\u003enir\u003c/em\u003eK, \u003cem\u003ecnor\u003c/em\u003eB, and \u003cem\u003enosZ\u003c/em\u003e, which encoded key enzymes involved in nitrogen metabolism. The specific target primers used in PCR are described in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and PCR reaction system and process are detailed in Table S3.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAnalytical methods\u003c/h2\u003e\u003cp\u003eBiomass growth was characterized by measuring the OD\u003csub\u003e600\u003c/sub\u003e of culture broth using a spectrophotometer (Agilent UV-Vis, USA) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. After centrifuging the culture broth at 8000 rpm for 10 min, the concentrations of COD, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, and TN in the supernatant were quantified using standard spectrophotometric methods [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Gas chromatography (Agilent 8850, USA) was used to detect the concentration of N\u003csub\u003e2\u003c/sub\u003eO in gas samples. The organic nitrogen (organic-N) in the culture broth primarily comprised extracellular proteins synthesized during bacterial growth and intracellular proteins released upon cell lysis. The concentration of organic-N was calculated by subtracting the concentrations of inorganic nitrogen (including NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N) from the TN concentration in the supernatant after centrifugation. The concentration of intracellular nitrogen (intracell-N) was calculated by subtracting the TN concentration in the centrifuged supernatant from that in the culture broth [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Gaseous nitrogen (gaseous-N) loss was determined by subtracting the TN concentration in the culture broth after 3-day incubation from the initial TN concentration.\u003c/p\u003e\u003cp\u003eThe pollutant removal efficiency (\u003cem\u003eRE\u003c/em\u003e) was determined as Eq. (1):\u003c/p\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u0026nbsp;(1)\u003c/h2\u003e\n \u003cp\u003eThe pollutant removal rate (\u003cem\u003eRR\u003c/em\u003e) was calculated as Eq.\u0026nbsp;(2):\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e(2)\u003c/h2\u003e\n \u003cp\u003ewhere, \u003cem\u003ec\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e and \u003cem\u003ec\u003c/em\u003e\u003csub\u003et\u003c/sub\u003e (mg/L) represent the pollutant (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e−\u003c/sup\u003e-N, TN, and COD) concentrations in the initial culture medium and centrifugal culture broth after \u003cem\u003et\u003c/em\u003e hours of cultivation, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eTriplicate measurements were performed for all experiments, and results are presented as mean value ± standard deviation (SD). Experimental data were plotted using the Origin software (Pro 2021, Origin Lab Corporation, USA). The Design-Expert software (v 13.1.0, Stat-Ease Inc., USA) was employed to implement the Box–Behnken response surface experimental design, analysis of the acquired regression and graphical presentation of experimental data, and to generate three-dimensional (3D) response surface plots. Statistical analysis was performed using one-way analysis of variance (ANOVA) in IBM SPSS Statistics (v. 27.0, Chicago, USA) to evaluate the interaction between response surface variables and the response value and to determine the intergroup difference (significance threshold: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eIsolation, screening, and identification of functional bacteria\u003c/h2\u003e\u003cp\u003eEighteen psychrotolerant nitrogen-removing bacterial strains were isolated and preliminarily screened from activated sludge, river sediments, and frozen soil in northern China during winter when temperatures were consistently below 5\u0026deg;C. These strains exhibited capabilities for removing NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, and TN. After re-screening, three strains each of nitrifying and aerobic denitrifying bacteria were obtained. The colony morphology and cellular characteristics of these six strains are detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eColonial and cellular morphological characteristics of six isolated psychrotolerant bacteria\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFunction\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eStrain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eColony morphology\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGram staining\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCell morphology\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGenBank Accession No.\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eNitrification\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHN1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLight yellow and transparent, circular morphology with irregular edges, smooth and moist surface with a center protrusion, and 2\u0026ndash;3 mm in diameter.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eG\u0026ndash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eStraight long rod, (1.5\u0026ndash;3.0) \u0026micro;m \u0026times; (0.6\u0026ndash;0.8) \u0026micro;m\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePP913934\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHN2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSlightly yellow and transparent, circular morphology with regular edges, smooth and moist surface with a center protrusion, and 3\u0026ndash;4 mm in diameter.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eG\u0026ndash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eStraight rod, (1.0\u0026ndash;2.5) \u0026micro;m \u0026times; (0.6\u0026ndash;0.8) \u0026micro;m\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePP911455\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHN3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLight yellow and semi-transparent, irregular in morphology, smooth, moist and glossy surface with a center protrusion, and 1\u0026ndash;2 mm in diameter.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eG\u0026ndash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCurved rod, (1.0\u0026ndash;2.0) \u0026micro;m \u0026times; (0.8\u0026ndash;1.0) \u0026micro;m\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePP911456\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eDenitrification\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAD1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWhite and transparent, circular morphology with regular edges, smooth and moist surface with a center high protrusion, and 2\u0026ndash;4 mm in diameter.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eG\u0026ndash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eShort rod, (0.6\u0026ndash;0.8) \u0026micro;m \u0026times; (0.6\u0026ndash;0.8) \u0026micro;m\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePP913935\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAD2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLight yellow and transparent, irregular in morphology, smooth and moist surface with a center protrusion, and 2\u0026ndash;3 mm in diameter.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eG\u0026ndash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eStraight long rod, (2.0\u0026ndash;3.0) \u0026micro;m \u0026times; (0.6\u0026ndash;0.8) \u0026micro;m\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePP911450\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAD3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWhite and opaque, circular morphology with regular edges, smooth and moist surface with a center high protrusion, and 2\u0026ndash;3 mm in diameter.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eG\u0026ndash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCurved rod, (1.5\u0026ndash;2.5) \u0026micro;m \u0026times; (0.8\u0026ndash;1.0) \u0026micro;m\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePP913933\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThree screened nitrifying strains were taxonomically identified and designated as \u003cem\u003ePseudomonas veronii\u003c/em\u003e HN1, \u003cem\u003eP. poae\u003c/em\u003e HN2, and \u003cem\u003eP. peli\u003c/em\u003e HN3 based on the results of 16S rRNA gene sequence alignment against the GenBank database and phylogenetic tree analysis (Fig. S2A-C). After culture in NM at 10\u0026deg;C for 3 days with shaking at 150 rpm, these isolates exhibited significant removal of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, achieving REs of 86.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7%, 85.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8%, and 86.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1%, with average removal rate of 1.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15, 1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13, and 1.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 mg/L/h, respectively, higher than that of \u003cem\u003eB. simplex\u003c/em\u003e H-b (0.74 mg/L/h) at 10\u0026deg;C [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The \u003cem\u003ePseudomonas\u003c/em\u003e species have been extensively documented in previous studies for their significant nitrification capabilities under diverse environmental settings. Yang et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] isolated a novel acid-resistant bacterium, \u003cem\u003eP. citronellolis\u003c/em\u003e YN-21, which exhibited exceptional heterotrophic nitrification ability under acidic conditions with NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N RR of 7.84 mg/L/h at pH 5.0. \u003cem\u003eP. aeruginosa\u003c/em\u003e WS-03 could effectively remove NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N at 30\u0026deg;C with an RR of 8.96 mg/L/h [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. According to several reports, \u003cem\u003ePseudomonas\u003c/em\u003e species, characterized by exceptional growth rates, remarkable environmental adaptability, and superior NR efficiency, exhibit significant competitive advantages over conventional nitrifying bacteria [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, we selected these three isolated \u003cem\u003ePseudomonas\u003c/em\u003e species as candidates for CMA construction.\u003c/p\u003e\u003cp\u003eIsolated aerobic denitrifying strains exhibited the highest sequence identity (\u0026ge;\u0026thinsp;99%) with \u003cem\u003eAeromonas\u003c/em\u003e sp. strain J223, \u003cem\u003eP. extremaustralis\u003c/em\u003e 14\u0026thinsp;\u0026minus;\u0026thinsp;3, and \u003cem\u003eSerratia liquefaciens\u003c/em\u003e strain ATCC 27592 in the GenBank database (Fig. S2D-F). Thus, the three strains were identified and designated as \u003cem\u003eAeromonas\u003c/em\u003e sp. AD1, \u003cem\u003eP. extremaustralis\u003c/em\u003e AD2, and \u003cem\u003eSerratia liquefaciens\u003c/em\u003e AD3, which exhibited remarkable NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N removal performance during 3-day cultivation in DM at 10\u0026deg;C and shaking at 150 rpm, with corresponding REs of 85.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6%, 86.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8%, and 85.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8%. Furthermore, the average RR of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N by these three strains were 1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21, 1.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17, and 1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 mg/L/h, respectively, surpassing that of \u003cem\u003eAcinetobacter tandoii\u003c/em\u003e MZ-5 (1.04 mg/L/h at 25\u0026deg;C) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and \u003cem\u003eArthrobacter arilaitensis\u003c/em\u003e Y-10 (0.35 mg/L/h at 15\u0026deg;C) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. During cultivation, all three bacterial strains accumulated detectable levels of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, with concentrations of 2.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 mg/L (\u003cem\u003eAeromonas\u003c/em\u003e sp. AD1), 2.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 mg/L (\u003cem\u003eP\u003c/em\u003e. \u003cem\u003eextremaustralis\u003c/em\u003e AD2), and 2.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 mg/L (\u003cem\u003eS\u003c/em\u003e. \u003cem\u003eliquefaciens\u003c/em\u003e AD3). Chen et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] demonstrated that \u003cem\u003eAeromonas\u003c/em\u003e sp. HN-02, an HNAD bacterium, could maintain activity at 5\u0026deg;C, with RRs of 0.9 and 22.3 mg/L/h for ammonia and COD, respectively. \u003cem\u003eP. extremaustralis\u003c/em\u003e, a psychrophilic bacterium native to the extreme environments in Antarctica, has been reported as an antibacterial agent [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and a phenacetin-degrading strain [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]; however, its potential application in NR, especially under low-temperature conditions, has received limited attention. In addition, this study potentially represents the first documented evidence of the denitrification capability of \u003cem\u003eS. liquefaciens\u003c/em\u003e, revealing a previously uncharacterized metabolic trait of this species.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eConstruction of psychrotolerant CMA\u003c/h2\u003e\u003cp\u003ePairwise antagonism assays conducted on the six screened bacterial isolates did not reveal any detectable antagonistic interactions, which indicated that these strains did not inhibit the growth of each other and can be cocultured for the preparation of CMA. Therefore, the CMA developed in this study comprised a psychrotolerant nitrifying consortium NC1 (including \u003cem\u003eP. veronii\u003c/em\u003e HN1, \u003cem\u003eP. poae\u003c/em\u003e HN2, and \u003cem\u003eP. peli\u003c/em\u003e HN3) and an aerobic denitrifying consortium DC1 (including \u003cem\u003eAeromonas\u003c/em\u003e sp. AD1, \u003cem\u003eP. extremaustralis\u003c/em\u003e AD2, and \u003cem\u003eS. liquefaciens\u003c/em\u003e AD3). The results of comparative experiment illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e indicate that the CMA achieved REs of 89.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8%, 88.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2%, 85.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3%, and 95.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7% for NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, TN, and COD, respectively, in CBM, which were obviously improved compared with those of the individual strains and single-functional consortia cultivated under identical conditions. Furthermore, the CMA exhibited the highest OD\u003csub\u003e600\u003c/sub\u003e of 1.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 among different strain combinations. These results indicate that when phylogenetically distinct microorganisms coexist in the same environment but occupy different ecological niches, they can establish mutualistic, symbiotic relationships and leverage the synergistic metabolic interactions to improve microbial proliferation and contaminant removal efficacy [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Jing et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] reported similar findings, demonstrating that the composite microbial consortium ECT, formed by combining three screened petroleum-degrading strains, \u003cem\u003eRalstonia\u003c/em\u003e sp. CP, \u003cem\u003eRhizobium\u003c/em\u003e sp. BX, and \u003cem\u003eAcinetobacter\u003c/em\u003e sp. FL, achieved superior petroleum-degrading efficiency compared with individual strains. During CMA-mediated NR, the lowest concentration of accumulated NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N was 0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 mg/L, indicating complete denitrification. Overall, the CMA exhibited excellent NR performance under low-temperature conditions, which indicated that the two functional bacterial consortia could be cocultured for synergistic elimination of nitrogen. Consequently, the cocultivation-derived CMA, integrating both nitrifying and denitrifying consortia, was selected for subsequent studies.\u003c/p\u003e\u003cp\u003eThe biomass growth of CMA and its NR performance at varying inoculation ratios of nitrifying and denitrifying consortia after 3 days of cultivation at 10\u0026deg;C with shaking at 150 rpm are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. At 1:1 inoculation ratio of NC1 and DC1 consortia, the CMA achieved the highest REs of 89.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5% for NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, 88.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8% for NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, 85.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1% for TN, and 95.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3% for COD. Concurrently, it exhibited maximal biomass growth (OD\u003csub\u003e600\u003c/sub\u003e, 1.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03) and minimal NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N accumulation (0.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 mg/L). The inoculation ratio significantly affected the growth of CMA and its NR efficiency. As reported by Guo et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], the inoculation ratio determined the initial relative abundance of functionally different microorganisms, thereby influencing synergistic metabolic interactions within the cocultured microorganisms. Ebadi et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] proposed that the decontamination performance of composite bacterial consortium is regulated by a dynamic equilibrium involving competitive relationships for nutrients (e.g., carbon, nitrogen, and phosphorus) and synergistic metabolic interactions among functionally distinct microorganisms. Notably, interspecies synergistic metabolism played a decisive role in the overall decontamination capacity of the system. Interspecies synergistic metabolism can be fully exploited only when functionally diverse bacterial strains are combined at their optimal inoculation ratios, thereby maximizing the RE of pollutants [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Therefore, the CMA cultured under the optimal inoculation ratio was designated NDC-6.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eEffects of carbon sources on NR\u003c/h2\u003e\u003cp\u003eThe biomass growth and NR performance of NDC-6 using different carbon sources were evaluated, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. When sodium succinate was used as the sole carbon source, with incubation at 10\u0026deg;C for 3 days and shaking at 150 rpm, NDC-6 achieved peak values for both biomass growth (OD\u003csub\u003e600\u003c/sub\u003e, 1.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01) and TN-removal efficiency (87.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1%). When cultured with sodium acetate or sodium citrate as sole carbon sources, NDC-6 maintained secondary biomass amount and NR efficacy relative to that achieved with the optimal carbon source, exhibiting OD\u003csub\u003e600\u003c/sub\u003e values of 1.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 and 1.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01, and TN-removal efficiencies of 79.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6% and 83.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4%, respectively. Relative to other carbon sources, NDC-6 exhibited the poorest growth (OD\u003csub\u003e600\u003c/sub\u003e: sucrose, 0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01; glucose, 0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01) and TN removal (sucrose 37.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3%; glucose 52.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1%) when cultured with sucrose or glucose as a carbon source. Numerous studies have indicated that sodium succinate and sodium citrate\u0026mdash;intermediate metabolites in the tricarboxylic acid cycle during bacterial respiration\u0026mdash;were more readily utilized by nitrifying or denitrifying bacteria and enhanced the activity of nitrate reductase [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Moreover, Wei et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] demonstrated that sodium acetate, a small molecule organic compound with a simple metabolic pathway, induced higher efficiency of electron generation, transfer, and competition, thereby promoting rapid bacterial proliferation and complete denitrification. Collectively, sodium succinate, sodium citrate, and sodium acetate have been identified as optimal carbon sources for various nitrifying and denitrifying bacteria. For instance, the use of sodium succinate as the carbon source yielded the highest NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N-removal efficiency in HNAD mixed bacteria HY-1 [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Sodium citrate served as an optimal carbon source for \u003cem\u003eAcinetobacter calcoaceticus\u003c/em\u003e TY1 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Wang et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] reported significant enhancement of the denitrification efficiency of \u003cem\u003eParacoccus versutus\u003c/em\u003e JUST-3 by sodium acetate. In contrast, when macromolecular organic compounds, such as glucose or sucrose, were used as sole carbon source, they underwent hydrolysis into small organic acids prior to microbial utilization. This process reduced the carbon assimilation efficiency, inhibited bacterial growth and proliferation, and consequently compromised the NR performance. Similar findings were reported in studies on \u003cem\u003eRhizobium\u003c/em\u003e sp. WS7 [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and \u003cem\u003eThauera linaloolentis\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] wherein sucrose was more unfavorable for denitrification. Therefore, based on its maximal promotion of NDC-6 growth and NR synthesis, sodium succinate was identified as the preferred carbon source and employed in subsequent experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eOptimization of culture conditions for NR by NDC-6\u003c/h2\u003e\u003cp\u003eTwenty-nine experimental Box\u0026ndash;Behnken design matrices for independent variables (C/N ratio, temperature, pH, and shaking speed) and corresponding response variable (\u003cem\u003eY\u003c/em\u003e) for TN-removal efficiency are presented in Table S4. Following second-order polynomial regression fitting, the quadratic polynomial regression equation in terms of coded independent variables was obtained (Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e3\u003c/span\u003e)).\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{Y}\\text{}\\text{=}\\text{}\\text{84.4}\\text{-1.59}\\text{A}\\text{+1.31}\\text{B}\\text{-5.05}\\text{C}\\text{-1.64}\\text{D}\\text{+0.72}\\text{AB}\\text{-0.32}\\text{AC}\\text{-0.18}\\text{AD}\\text{+0.18}\\text{BC}\\text{+0.83}\\text{BD}$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\text{-1.01CD-2.21}{\\text{A}}^{\\text{2}}\\text{-7.28}{\\text{B}}^{\\text{2}}\\text{-8.26}{\\text{C}}^{\\text{2}}\\text{-3.65}{\\text{D}}^{\\text{2}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAs presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the significance test value (\u003cem\u003eF-\u003c/em\u003evalue, 208.26) and low probability value (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) for the established quadratic regression model in ANOVA indicated that the model could effectively describe the impact of various factors on the TN-removal efficiency. The determination coefficient \u003cem\u003eR\u003c/em\u003e\u0026sup2; and adjusted \u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e were 0.987 and 0.983, respectively, which were close to the predicted \u003cem\u003eR\u003c/em\u003e\u0026sup2; of 0.983. The model exhibited nonsignificant lack of fit (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating negligible pure error and good agreement between the model-predicted and experimental values [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Collectively, these findings indicated that the fitted quadratic polynomial model for TN removal by NDC-6 had high accuracy and reliability in predicting the optimal values for the independent variables and analyzing the interactive effects of these variables on the TN-removal efficiency.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eANOVA of the fitted quadratic polynomial model for TN removal by NDC-6\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSource\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSum of squares\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMean square\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eF\u003c/em\u003e value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value (prob\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eF\u003c/em\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSignificance\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eModel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e995.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e71.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e73.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA-C/N\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eB-Temperature\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e26.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e23.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC-pH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e258\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e258\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e267\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eD-Shaking speed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e23.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e23.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.166\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.528\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0723\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.727\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.622\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e33.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e33.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e35.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eB\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e376\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e376\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e390\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e386.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e386\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eD\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e73.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e73.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e75.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eResidual\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLack of Fit\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.358\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003enot significant\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePure Error\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCor Total\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1008.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003eNote: ** very significant, \u003cem\u003eP\u003c/em\u003e value\u0026lt;0.01; * significant, \u003cem\u003eP\u003c/em\u003e value\u0026lt;0.05\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe ANOVA results revealed that all linear terms (A, B, C, and D) in the established model exhibited \u003cem\u003ep\u003c/em\u003e-values less than 0.01, signifying that each of the four factors significantly affected the TN removal by NDC-6. This result was consistent with previous reports that C/N ratio, temperature, pH, and DO are critical environmental factors for BNR [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Using the \u003cem\u003eF\u003c/em\u003e-value analysis, the factors influencing TN removal were ranked according to their influence degree as follows: pH\u0026thinsp;\u0026gt;\u0026thinsp;shaking speed\u0026thinsp;\u0026gt;\u0026thinsp;C/N ratio\u0026thinsp;\u0026gt;\u0026thinsp;temperature. pH had the most significant impact on TN removal. Similar findings were reported for the optimization of conditions for coculture of \u003cem\u003eB. cereus\u003c/em\u003e G2 and \u003cem\u003eB. pumilus\u003c/em\u003e G5 by Peng et al. [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], who demonstrated that excessively acidic or alkaline environments remarkably compromised the NR performance of CMA. Notably, temperature had a relatively weaker influence on TN removal compared with the other three factors. The reason might be that NDC-6 was composed of multiple psychrotolerant strains, which had a certain tolerance to low-temperature environments and a broad temperature-adaptation range. In addition, temperature and shaking speed significant influenced TN removal (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), signifying that the NR performance of NDC-6 was susceptible to the interactions of these two environmental parameters.\u003c/p\u003e\u003cp\u003eThe 3D response surface plots illustrating the interactive effects of the four independent variables (C/N ratio, temperature, pH, and shaking speed) on the removal of TN by NDC-6 are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. With increasing C/N ratio and temperature, the TN-removal efficiency by NDC-6 initially increased and subsequently decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The highest TN-removal efficiencies were achieved at a C/N ratio of approximately 5\u0026ndash;6 and a temperature around 10\u0026deg;C. Carbon sources serve as electron donors essential for denitrification [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Carbon deficiency triggers competitive inhibition between heterotrophic nitrifying bacteria and aerobic denitrifying bacteria for limited carbon resources, causing concurrent decline in NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N- and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N-removal efficiencies [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In contrast, excessive carbon availability stimulates the overproliferation of heterotrophic bacteria, which consumes substantial amounts of DO, resulting in the deterioration of water quality and carbon wastage. Therefore, effective enhancement of NR can only be achieved when the C/N ratio is maintained within an optimal range. Temperature stress induces microbial oxidative stress and cellular damage, thereby impairing the nutrient-removal capacity [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. At lower temperatures, enzymatic activity is inhibited, reducing metabolic rates and substantially diminishing the NR performance [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. On the contrary, excessively high temperatures induce enzyme denaturation [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Optimal metabolic rates can only be maintained within a defined temperature range to enhance NR.\u003c/p\u003e\u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF with the increase in initial pH of CBM and shaking speed of the oscillating incubator, the efficiency of TN removal by NDC-6 exhibited parabolic variation trends, characterized by an initial increase followed by a decrease. The highest efficiency of TN removal was achieved at initial pH in the range of 7.0\u0026ndash;7.5 and a shaking speed of 150\u0026ndash;160 rpm. Similar to other NR-functional strains, such as \u003cem\u003eParacoccus versutus\u003c/em\u003e JUST-3 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] and \u003cem\u003eGlutamicibacter halophytocola\u003c/em\u003e MD1 [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], NDC-6 preferred a neutral or weakly alkaline environment (pH 6\u0026ndash;9). Exposure to strongly acidic or alkaline conditions induces aggregation of surface charge on bacterial cells, disrupting the integrity of cell structure and inhibiting microbial proliferation capacity and enzymatic activity, thereby impairing nitrogen transformation [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Consequently, precise pH regulation is a critical determinant in optimizing microbial NR.\u003c/p\u003e\u003cp\u003eIn this study, DO level in the culture medium was adjusted by regulating the shaking speed of the oscillating incubator. Jin et al. [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] found that the DO content increased linearly with shaking speed within a certain range. Preliminary experiments in this study indicated that the DO content in the culture medium in 250 mL conical flask was approximately 3.3 mg/L at a shaking speed of 100 rpm. Each 10 rpm increment in shaking speed increased the DO content by approximately 0.4 mg/L. The highest TN-removal efficiency was observed for the shaking speed of 150\u0026ndash;160 rpm, corresponding to a DO content of 5.3\u0026ndash;5.7 mg/L. Li et al. [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] reported that excessively low DO content inhibited the activity of ammonia monooxygenase in nitrifying bacteria, reducing the nitrification performance. Moreover, low DO concentrations suppress the catalytic efficiency of denitrifying enzymes, thereby slowing down mass transfer and substrate utilization [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Conversely, an excessive high DO content not only inhibits the activity of nitrite reductase in the denitrification process but also induces competition for electrons between O\u003csub\u003e2\u003c/sub\u003e and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, leading to incomplete nitrate reduction [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. This highlights the importance of optimizing the shaking speed to ensure efficient removal of nitrogen by NDC-6.\u003c/p\u003e\u003cp\u003eThe cultivation conditions optimized for maximizing TN removal were as follows: A\u0026thinsp;=\u0026thinsp;0, B\u0026thinsp;=\u0026thinsp;0.075, C\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.3, and D\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.2. Accordingly, the model predicted that maximum TN-removal efficiencies of 90.2% would be achieved at a C/N ratio of 6, a temperature of 10.2\u0026deg;C, pH 7.2, and a shaking speed of 156 rpm. Under the optimal conditions, a 3-day cultivation in the verification experiment yielded a TN-removal efficiency of 89.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8%%, which closely approached the theoretically predicted maximum RE for TN. This confirmed the capability of the model to accurately optimize cultivation conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eNitrogen metabolic pathways in NDC-6\u003c/h2\u003e\u003cp\u003eNitrogen balance analysis\u003c/p\u003e\u003cp\u003eThe single functional strains, heterotrophic nitrifying consortium, aerobic denitrifying consortium, and NDC-6 were inoculated into optimized CBM (containing 52.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 mg/L NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and 52.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 mg/L NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N) at optimal inoculation ratios. Following 3-day incubation at 10.2\u0026deg;C with shaking at 156 rpm, different forms of nitrogen were determined to establish nitrogen mass balance (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Notably, a distinct accumulation of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N (\u0026gt;\u0026thinsp;5 mg/L) was consistently observed during NR by \u003cem\u003eP. veronii\u003c/em\u003e HN1, \u003cem\u003eP. poae\u003c/em\u003e HN2, \u003cem\u003eP. peli\u003c/em\u003e HN3, and the nitrifying consortium NC1, suggestive of the absence of significant denitrification capability of these strains. Furthermore, only the individual denitrifier, denitrifying consortium, and NDC-6 were capable of converting TN into gaseous-N. Of the initial TN content, 40.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6% was converted to gaseous-N by NDC-6, which was significantly higher than that in the case of \u003cem\u003eAeromonas\u003c/em\u003e sp. AD1 (25.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4%), \u003cem\u003eP. extremaustralis\u003c/em\u003e AD2 (28.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8%), \u003cem\u003eS. liquefaciens\u003c/em\u003e AD3 (24.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1%), and the denitrifying consortium DC1 (28.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2%). Gas chromatography revealed N\u003csub\u003e2\u003c/sub\u003eO concentrations of 0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 mg/L for \u003cem\u003eAeromonas\u003c/em\u003e sp. AD1, 0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 mg/L for \u003cem\u003eP. extremaustralis\u003c/em\u003e AD2, 0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 mg/L for \u003cem\u003eS. liquefaciens\u003c/em\u003e AD3, 0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mg/L for DC1, and 0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mg/L for NDC-6, indicating that the gaseous-N produced during denitrification by these strains was predominantly N\u003csub\u003e2\u003c/sub\u003e instead of N\u003csub\u003e2\u003c/sub\u003eO. These observations indicate enhanced completeness of denitrification and metabolic efficiency by NDC-6, wherein a higher proportion of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N or NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N was fully reduced to the terminal product N\u003csub\u003e2\u003c/sub\u003e rather than accumulating as intermediate gases (e.g., N\u003csub\u003e2\u003c/sub\u003eO) or residual ions. Similar results were reported by Fang et al. [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], who found that coculturing the yeast \u003cem\u003eKazachstania exigua\u003c/em\u003e T14-1 with the bacterium \u003cem\u003eMethylobacterium\u003c/em\u003e sp. T5-6 substantially improved the nitrogen metabolism capacity compared with monocultures of either strain. Furthermore, following 3 days of cultivation at 10.2\u0026deg;C, \u0026gt;\u0026thinsp;30 mg/L of intracell-N was detected in all the tested microbial combinations, indicating that \u0026gt;\u0026thinsp;30% of the initial TN content was assimilated into cellular nitrogen via biosynthetic assimilation. Among these, NDC-6 exhibited the highest intracell-N conversion rate at 49.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4%. In contrast to the reported microbial NR pathways primarily relying on assimilation [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], NDC-6 achieved NR under low-temperature aerobic conditions primarily via dual pathways of bacterial assimilation and dissimilation converting inorganic N (mainly NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N) into intracell-N and N\u003csub\u003e2\u003c/sub\u003e. These findings indicated that the composite consortium NDC-6 exhibited exceptional NR performance under low-temperature conditions, attributable to synergistic interactions among constituent strains that significantly enhance nitrogen metabolism efficiency, highlighting its greater potential for application in wastewater treatment.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eNitrogen balance analysis during nitrogen removal by different strain combinations\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eStrain combinations\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"7\" nameend=\"c8\" namest=\"c2\"\u003e\u003cp\u003eNitrogen concentration (mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eRE\u003c/em\u003e\u003csub\u003eTN\u003c/sub\u003e (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e―\u003c/sup\u003e-N\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e―\u003c/sup\u003e-N\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOrganic-N\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eIntracell-N\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eGaseous-N\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eTN\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas veronii\u003c/em\u003e HN1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e7.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e50.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e7.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e37.79\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026ndash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e67.29\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e\u003cp\u003e36.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas poae\u003c/em\u003e HN2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e7.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e51.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e5.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e38.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026ndash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e66.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e\u003cp\u003e37.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas peli\u003c/em\u003e HN3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e7.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e50.08\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e7.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e38.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026ndash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e66.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e\u003cp\u003e36.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAeromonas\u003c/em\u003e sp. AD1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e31.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e7.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e34.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e26.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e43.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e\u003cp\u003e58.52\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas extremaustralis\u003c/em\u003e AD2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e29.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e7.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e33.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e29.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e41.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e\u003cp\u003e60.73\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eSerratia liquefaciens\u003c/em\u003e AD3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e31.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e7.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e34.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e26.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e44.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e\u003cp\u003e57.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNitrifying consortium NC1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e6.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e50.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e5.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e40.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026ndash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e65.32\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e\u003cp\u003e38.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDenitrifying consortium DC1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e30.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e7.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e1.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e34.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e29.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e40.63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e\u003cp\u003e61.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNDC-6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e4.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e4.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e1.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e52.15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e42.37\u0026thinsp;\u0026plusmn;\u0026thinsp;2.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e\u003cp\u003e10.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e\u003cp\u003e89.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eFunctional genes related to NR\u003c/h2\u003e\u003cp\u003eThe genes \u003cem\u003ehao\u003c/em\u003e, \u003cem\u003enap\u003c/em\u003eA, \u003cem\u003eni\u003c/em\u003erS, \u003cem\u003eni\u003c/em\u003erK, \u003cem\u003ecnor\u003c/em\u003eB, and \u003cem\u003enosZ\u003c/em\u003e are generally considered as the functional genes encoding key enzymes involved in microbial nitrogen metabolism. In this study, PCR amplification of these six functional genes was conducted across all bacterial strains to further validate their nitrification and denitrification capabilities, thereby elucidating the potential metabolic pathways of NR under low-temperature conditions. The amplification results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and summarized in Table S5.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ehao\u003c/em\u003e gene, encoding hydroxylamine oxidase (HAO), is a critical biomarker for nitrification. HAO catalyzes the oxidation of hydroxylamine (NH\u003csub\u003e2\u003c/sub\u003eOH), a key intermediate in nitrification, to NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. In this study, the successful amplification and expression of the \u003cem\u003ehao\u003c/em\u003e gene in \u003cem\u003eP. veronii\u003c/em\u003e HN1 (282 bp), \u003cem\u003eP. poae\u003c/em\u003e HN2 (265 bp), and \u003cem\u003eP. peli\u003c/em\u003e HN3 (1752 bp) confirmed the involvement of \u003cem\u003ehao\u003c/em\u003e in nitrification in these three strains. The \u003cem\u003ehao\u003c/em\u003e gene was likewise amplified in other nitrification-capable \u003cem\u003ePseudomonas\u003c/em\u003e strains, such as \u003cem\u003eP. mendocina\u003c/em\u003e SCZ-2 [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] and \u003cem\u003eP. citronellolis\u003c/em\u003e YN-21 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Furthermore, PCR amplification revealed the absence of functional genes encoding nitrite reductases (NIR) in these three strains. As NIR catalyzes the reduction of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N to gaseous NO or N\u003csub\u003e2\u003c/sub\u003eO [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], this genetic characteristic explained distinct accumulation of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N observed during the NR process. Notably, the \u003cem\u003enos\u003c/em\u003eZ gene, encoding nitrous oxide reductase (NOS), was successfully amplified from strains HN1 (396 bp), HN2 (352 bp), and HN3 (298 bp). Given the well-established catalytic role of NOS in reducing N\u003csub\u003e2\u003c/sub\u003eO to N\u003csub\u003e2\u003c/sub\u003e, as demonstrated by Guo et al. [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], this finding signified that these three strains can convert N\u003csub\u003e2\u003c/sub\u003eO generated from ancillary denitrification processes into N\u003csub\u003e2\u003c/sub\u003e, thereby mitigating the emission of greenhouse gases.\u003c/p\u003e\u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Table S5, typical denitrification genes (\u003cem\u003enap\u003c/em\u003eA, \u003cem\u003enir\u003c/em\u003eS, \u003cem\u003enir\u003c/em\u003eK, \u003cem\u003ecnor\u003c/em\u003eB, and \u003cem\u003enos\u003c/em\u003eZ) were PCR-amplified from \u003cem\u003eAeromonas\u003c/em\u003e sp. AD1, \u003cem\u003eP. extremaustralis\u003c/em\u003e AD2, and \u003cem\u003eS. liquefaciens\u003c/em\u003e AD3. The \u003cem\u003enap\u003c/em\u003eA gene, a biomarker involved in aerobic denitrification, encodes the periplasmic nitrate reductase (NAP), which catalyzes the reduction of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N to NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N as demonstrated by Zheng et al. [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. The \u003cem\u003enap\u003c/em\u003eA gene has also been amplified from other psychrotolerant denitrifiers, such as \u003cem\u003ePriestia aryabhattai\u003c/em\u003e KX-3 [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e] and \u003cem\u003ePsychrobacter cryohalolentis\u003c/em\u003e strain F5-6 [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Concurrently, \u003cem\u003enir\u003c/em\u003eS and \u003cem\u003enir\u003c/em\u003eK were identified as functional genes encoding NIR. These two types of functional genes in this study could simultaneously exist in the same strain and promoted the removal of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N. This genetic configuration correlated with the observed low NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N accumulation during the removal of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N by strains AD1, AD2, and AD3, and their composite consortium, indicating synergistic functionality of multiple NIR isoforms in nitrite metabolism. The \u003cem\u003ecnorB\u003c/em\u003e gene encoding nitric oxide reductase (NOR) for the conversion of NO to N\u003csub\u003e2\u003c/sub\u003eO and the \u003cem\u003enos\u003c/em\u003eZ gene responsible for the reduction of N\u003csub\u003e2\u003c/sub\u003eO to N\u003csub\u003e2\u003c/sub\u003e were detected in \u003cem\u003eAeromonas\u003c/em\u003e sp. AD1, \u003cem\u003eP. extremaustralis\u003c/em\u003e AD2, and \u003cem\u003eS. liquefaciens\u003c/em\u003e AD3, respectively, indicating that these three strains prevented NO and N\u003csub\u003e2\u003c/sub\u003eO emission by directly reducing them to N\u003csub\u003e2\u003c/sub\u003e during denitrification. These findings aligned with the previously observed low concentrations of N\u003csub\u003e2\u003c/sub\u003eO produced by these three strains during denitrification. In summary, successful amplification of \u003cem\u003enap\u003c/em\u003eA, \u003cem\u003enir\u003c/em\u003eS, \u003cem\u003enir\u003c/em\u003eK, \u003cem\u003ecnor\u003c/em\u003eB, and \u003cem\u003enos\u003c/em\u003eZ in strains AD1, AD2, and AD3 indicated that NAP, NIR, NOR, and NOS are involved in different steps of the dissimilatory nitrate reduction pathway. This genetic evidence confirms their capability to achieve complete denitrification without intermediate accumulation. This denitrification pathway was consistent with the nitrogen-removal mechanism in \u003cem\u003eP. aeruginosa\u003c/em\u003e WS-03 reported by Wei et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003ePathways for nitrogen metabolism\u003c/h2\u003e\u003cp\u003eBased on the results of nitrogen balance analysis and PCR amplification of functional genes related to NR, the proposed pathways for nitrogen metabolism in NDC-6 are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The composite consortium NDC-6, constructed from heterotrophic nitrifiers and aerobic denitrifiers, was speculated to possess a complete pathway for nitrogen metabolism and to achieve synchronous and efficient removal of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N through dual metabolic pathways: assimilatory conversion into intracell-N and dissimilatory transformation (heterotrophic nitrification and aerobic denitrification) to N\u003csub\u003e2\u003c/sub\u003e. The initial step of heterotrophic nitrification in the composite consortium NDC-6 involved the oxidation of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N to NH\u003csub\u003e2\u003c/sub\u003eOH using sodium succinate as the carbon source and electron donor, mediated by constituent strains \u003cem\u003eP. veronii\u003c/em\u003e HN1, \u003cem\u003eP. poae\u003c/em\u003e HN2, and \u003cem\u003eP. peli\u003c/em\u003e HN3. Subsequently, HAO from these nitrifying strains catalyzed further oxidation of NH\u003csub\u003e2\u003c/sub\u003eOH into NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N. Concurrently, during aerobic denitrification, NAP expressed by \u003cem\u003eAeromonas\u003c/em\u003e sp. AD1, \u003cem\u003eP. extremaustralis\u003c/em\u003e AD2, and \u003cem\u003eS. liquefaciens\u003c/em\u003e AD3 reduced NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N to NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N. In the following steps, the NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N generated through nitrification and initial denitrification steps underwent sequential enzymatic conversions: NIR catalyzed the reduction of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N to NO, followed by NOR-mediated transformation of NO to N\u003csub\u003e2\u003c/sub\u003eO, and final reduction to N\u003csub\u003e2\u003c/sub\u003e via NOS. The functional enzymes involved in these steps were collectively expressed by \u003cem\u003eAeromonas\u003c/em\u003e sp. AD1, \u003cem\u003eP. extremaustralis\u003c/em\u003e AD2, and \u003cem\u003eS. liquefaciens\u003c/em\u003e AD3.\u003c/p\u003e\u003cp\u003eOverall, the composite consortium NDC-6 achieved efficient removal of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N through synergistic metabolism and complementary functions among its constituent strains, which facilitated a broad ecological niche for this CMA and enhanced its environmental adaptability, resulting in superior metabolic capabilities for diverse nitrogen substrates in wastewater treatment.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThree heterotrophic nitrifying bacteria and three aerobic denitrifying bacteria were isolated from activated sludge, river sediment, and frozen soil samples in winter. The composite consortium NDC-6, constructed by coculturing these six strains, exhibited exceptional biomass proliferation capacity and efficient NR performance at 10\u0026deg;C. The optimal conditions for TN removal were determined to be sodium succinate as carbon source, C/N ratio of 6, temperature of 10.2\u0026deg;C, pH of 7.2, and shaking speed of 156 rpm. Nitrogen balance analysis revealed that NDC-6 achieved the highest gaseous-N conversion rate (42.4%) and intracell-N assimilation rate (49.5%) among all the tested groups. Under low-temperature aerobic conditions, NDC-6 primarily facilitated NR via dual pathways of dissimilation and assimilation. Functional gene amplification confirmed that the complete NR mechanism in NDC-6 relied on synergistic interactions and metabolic complementarity between the \u003cem\u003ehao\u003c/em\u003e gene in the nitrifying consortium and denitrification genes (\u003cem\u003enap\u003c/em\u003eA, \u003cem\u003enir\u003c/em\u003eS, \u003cem\u003enir\u003c/em\u003eK, \u003cem\u003ecnor\u003c/em\u003eB, and \u003cem\u003enos\u003c/em\u003eZ) in the denitrifying consortium. These findings provide a theoretical foundation for applying psychrotolerant CMA in the treatment of nitrogen-containing wastewater under low-temperature conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e The online version contains supplementary material available at\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThis research was financially supported by the\u0026nbsp;Basic Research Project of Educational Department of Liaoning Province (No. LJ212411035027) and the National Natural Science Foundation of China (No. 52300023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eYihua Dong: Writing \u0026ndash; original, Review \u0026amp; editing, Conceptualization, Funding acquisition. Jing Xu:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMethodology, Investigation, Visualization. Feng Chen: Methodology, Investigation, Validation, Data curation. Liang Li: Conceptualization, Formal analysis, Project administration. Guangsheng Qian: Supervision, Conceptualization, Funding acquisition. Peng Zhang: Methodology, Investigation, Validation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e The data used in this study are available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe authors declare that there are no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAll the authors consent to publish this research paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHao Z, Shi YY, Zhan XY, Yu BW, Fan Q, Zhu J, Liu LH, Zhang QW, Zhao GX (2024) Quantifying and assessing nitrogen sources and transport in a megacity water supply watershed: insights for effective non-point source pollution management with mixSIAR and SWAT models. 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J Environ Manage 388:125972. https:// doi. org/ 10. 1016/ j. jenvman. 2025. 125972\u003c/li\u003e\n\u003cli\u003eKang X, Zhao XX, Song XS, Wang DH, Shi GT, Duan XF, Chen XH, Shen GX (2023) Nitrogen removal by a novel strain \u003cem\u003ePriestia aryabhattai\u003c/em\u003e KX-3 from East Antarctica under alkaline pH and low-temperature conditions. Process Biochem 130:674\u0026ndash;684. https:// doi. org/ 10. 1016/ j. procbio. 2023. 05. 030\u003c/li\u003e\n\u003cli\u003eHou Y, Zhang DY, Cao HR, Zhang YL, Zhao DD, Zeng WM, Lei H, Bai Y (2022) Identification of aerobic-denitrifying \u003cem\u003ePsychrobacter cryohalolentis\u003c/em\u003e strain F5-6 and its nitrate removal at low temperature. Int Biodeter Biodegr 172:105426. https:// doi. org/ 10. 1016/ j. ibiod. 2022. 105426\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bioprocess-and-biosystems-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bioprocess and Biosystems Engineering](https://www.springer.com/journal/449)","snPcode":"449","submissionUrl":"https://submission.nature.com/new-submission/449/3","title":"Bioprocess and Biosystems Engineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Psychrotolerant bacteria, Composite microbial agent, Biological nitrogen removal, Nitrification, Denitrification, Metabolic pathway","lastPublishedDoi":"10.21203/rs.3.rs-7370617/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7370617/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLow temperature can suppress biological nitrogen removal (NR) efficiency. Although NR characteristics of single psychrotolerant bacteria have been extensively studied, synergistic interactions between functionally distinct psychrotolerant nitrogen-removing consortia remain unexplored. In this study, a composite microbial agent, designated NDC-6, was generated by coculturing a psychrotolerant nitrifying consortium NC1 (\u003cem\u003ePseudomonas veronii\u003c/em\u003e HN1, \u003cem\u003eP. poae\u003c/em\u003e HN2, and \u003cem\u003eP. peli\u003c/em\u003e HN3) and an aerobic denitrifying consortium DC1 (\u003cem\u003eAeromonas\u003c/em\u003e sp. AD1, \u003cem\u003eP. extremaustralis\u003c/em\u003e AD2, and \u003cem\u003eSerratia liquefaciens\u003c/em\u003e AD3) at a 1:1 inoculation ratio, and its NR performance was systematically evaluated. After 3 days of incubation at 10\u0026deg;C, NDC-6 achieved removal efficiencies of 89.3%, 88.1%, 85.5%, and 95.3% for NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, total nitrogen (TN), and chemical oxygen demand (COD), which were significantly higher than those of individual strains or single-function consortia. Sodium succinate was identified as the optimal carbon source, which simultaneously improved biomass growth and NR efficacy of NDC-6. Optimal culture conditions determined using response surface methodology were as follows: C/N ratio, 6; temperature, 10.2\u0026deg;C; pH, 7.2; and shaking speed, 156 rpm. Under these conditions, the maximum TN removal efficiency reached 89.8%. Nitrogen balance and functional gene expression (\u003cem\u003ehao\u003c/em\u003e, \u003cem\u003enap\u003c/em\u003eA, \u003cem\u003enir\u003c/em\u003eS, \u003cem\u003enir\u003c/em\u003eK, \u003cem\u003ecnor\u003c/em\u003eB, and \u003cem\u003enos\u003c/em\u003eZ) analyses revealed that NDC-6 achieved complete NR through both assimilatory and dissimilatory pathways. The dissimilatory mechanism relied on synergistic metabolism and functional complementation between NC1 and DC1, mediated by their respective functional genes. This study provides mechanistic insights into the biological treatment of nitrogen-containing wastewater, particularly under low-temperature conditions, and offers a novel strategy for such treatment.\u003c/p\u003e","manuscriptTitle":"Development of a psychrotolerant composite microbial agent for nitrogen removal and its nitrogen metabolism pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 12:38:54","doi":"10.21203/rs.3.rs-7370617/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-24T13:34:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-24T13:23:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"18517127691014392563773306832303678508","date":"2025-10-04T12:43:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-14T11:49:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"174205172161006627491359518881239989742","date":"2025-09-03T15:43:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"269267779069887439800228027104167827162","date":"2025-09-02T02:15:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"67535874432288096084336669665265012919","date":"2025-09-01T15:46:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-15T08:56:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-15T07:01:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-15T06:08:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bioprocess and Biosystems Engineering","date":"2025-08-14T06:46:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bioprocess-and-biosystems-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bioprocess and Biosystems Engineering](https://www.springer.com/journal/449)","snPcode":"449","submissionUrl":"https://submission.nature.com/new-submission/449/3","title":"Bioprocess and Biosystems Engineering","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0b733ce4-2199-46c5-a88c-91898ec7cd7e","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-16T10:23:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-22 12:38:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7370617","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7370617","identity":"rs-7370617","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}