A low-temperature aerobic denitrifying Pseudomonas sp. H2-1 and its application

Abstract

The application relates to a low-temperature aerobic denitrifying Pseudomonas sp. H2-1 and application thereof, and belongs to the technical field of microorganisms. Pseudomonas The strain expands the resource library of aerobic denitrifying bacteria in a polar low-temperature environment, and provides theoretical support for revealing the aerobic denitrifying mechanism under cold conditions and optimizing the sewage treatment process in cold regions.

Description

Technical Field

[0001] This application belongs to the field of microbial technology, specifically relating to a strain of low-temperature aerobic denitrifying Pseudomonas. Pseudomonas sp. H2-1 and its applications. Background Technology

[0002] Nitrogen is an essential nutrient element in water bodies, but with the intensification of human activities, large amounts of nitrogen-containing wastewater are continuously discharged into the environment, leading to increasingly prominent ecological and environmental problems such as eutrophication, frequent red tides, and hypoxia. Therefore, the efficient removal of nitrogen pollutants from emissions is of great significance for maintaining the stability of aquatic ecosystems and ensuring water environmental safety. Compared with physical and chemical methods, biological denitrification is considered the main approach to wastewater denitrification due to its advantages such as high efficiency, economy, and environmental friendliness. Traditional biological denitrification processes usually rely on sequential nitrification and denitrification processes, but due to the differences in oxygen and carbon source requirements between the two, they often need to be operated in stages in practical applications, thus increasing process complexity and operating costs.

[0003] Since the 1980s, a method capable of simultaneously performing nitrification and denitrification under aerobic conditions has been first isolated from activated sludge. Thiosphaera pantotropha (Now named) Paracoccus denitrificans Since the advent of [unclear text - likely a typo, should be removed], aerobic denitrification (HN-AD) has gradually attracted widespread attention. However, existing research largely focuses on mesophilic conditions, and the isolated strains are mainly derived from activated sludge, soil, and freshwater environments. Under low-temperature conditions, the efficiency of conventional nitrification-denitrification processes decreases significantly, severely limiting the nitrogen removal efficiency of wastewater treatment plants in cold regions. Although a few low-temperature-tolerant aerobic denitrifying bacteria (ADB) have been reported, most reports focus on the removal efficiency of individual strains under specific conditions, while in-depth mechanistic studies surrounding their molecular mechanisms, enzyme activity regulation, and cold adaptation mechanisms of gene expression remain lacking. Therefore, screening strains capable of efficient nitrogen removal under low-temperature conditions is of great significance for overcoming the bottleneck in wastewater treatment in cold regions.

[0004] The polar environment, subjected to prolonged periods of low temperatures and multiple environmental stresses, harbors a rich diversity of microorganisms with unique metabolic characteristics. These microorganisms may not only exhibit excellent nitrogen removal capabilities but also carry unique low-temperature adaptation genes. Therefore, screening for refractory aerobic denitrifying bacteria under the perpetually low temperatures of the polar regions presents a significant opportunity. Summary of the Invention

[0005] To address the technical challenges of decreased nitrogen removal efficiency and nitrite accumulation in existing aerobic denitrifying bacteria under low-temperature conditions, this application provides a low-temperature, high-efficiency aerobic denitrifying strain derived from typical Antarctic environmental samples and its applications. A total of 449 strains with aerobic denitrification capabilities were isolated using a modified denitrification medium. Seven representative strains exhibiting the highest denitrification efficiency at 15℃ were screened through a 48-hour nitrogen removal performance test. These seven strains all achieved nitrate removal rates exceeding 99.8% and nitrite accumulation below 1.5 μmol / L within 48 hours, demonstrating excellent nitrogen removal capabilities. Further whole-genome sequencing and functional annotation of these seven strains revealed that four strains—D2-1a, W2-12b, H2-1, and ND2-8—possess complete denitrification pathways, and all strains carry multiple low-temperature adaptation-related genes, providing a molecular basis for maintaining efficient metabolism in low-temperature environments. Comparative genomic analysis showed that strain H2-1 has an ANI < 95% and DDH < 70% compared to its most similar type strain, indicating potential for novel species characteristics. Nitrogen balance analysis showed that the denitrification process of this strain was mainly completed through a combination of assimilation and aerobic denitrification. Based on genome annotation, enzyme activity assays, and nitrogen balance analysis, the metabolic pathway for nitrate nitrogen in this strain is: NO3- - → NO2 - → NO → N2O → N2. The nitrate reductase and nitrite reductase maintained high activity at 15℃, indicating a stable low-temperature enzymatic reaction capability. This strain not only expands the resource pool of aerobic denitrifying bacteria in polar low-temperature environments but also provides theoretical support for revealing the aerobic denitrification mechanism under cold conditions and optimizing wastewater treatment processes in cold regions.

[0006] In view of this, this application provides, in its first aspect, a strain of Pseudomonas H2-1, which is classified and named as follows: Pseudomonas sp., with accession number CGMCC NO.37029, was deposited at the China General Microbiological Culture Collection Center on December 11, 2025.

[0007] In a second aspect, this application provides a microbial preparation comprising the aforementioned Pseudomonas H2-1, or its spore suspension, its fermentation broth, or its lyophilized powder.

[0008] In a third aspect, this application provides a wastewater treatment agent comprising the aforementioned Pseudomonas H2-1, or its spore suspension, its fermentation broth, or its lyophilized powder.

[0009] This application provides, in its fourth aspect, the application of the aforementioned Pseudomonas H2-1 in aerobic denitrification.

[0010] This application provides, in its fifth aspect, the application of the aforementioned Pseudomonas H2-1 in nitrate nitrogen removal.

[0011] This application provides, in its sixth aspect, the application of the aforementioned Pseudomonas H2-1 in wastewater treatment.

[0012] This application provides, in its seventh aspect, the application of the aforementioned Pseudomonas H2-1 in wastewater treatment in cold regions.

[0013] Compared with the prior art, the beneficial effects that at least one technical solution adopted in the embodiments of this specification can achieve include at least: (1) Strain H2-1 has good resistance to low temperature and high efficiency in denitrification. It can still maintain high efficiency in denitrification at 15℃. The nitrate removal rate within 20h is more than 89.57%. The average nitrate removal rate in this process is 2.28 mg / L / h, and the maximum removal rate is 4.78 mg / L / h. (2) Nitrite only reached its peak value of 0.39 mg / L at 8 h, and the reaction process was stable; (3) The denitrification process of this strain is mainly completed through assimilation and aerobic denitrification. The denitrification pathway is complete, achieving the complete conversion of nitrate nitrogen to nitrogen gas. Based on genome annotation, enzyme activity assays, and nitrogen balance analysis, the denitrification metabolic pathway of this strain for nitrate nitrogen is: NO3... - → NO2 - → NO → N2O → N2, the assimilation pathway is: NO3 → N2O → N2. - →NO2 - → NH4 + At 16 hours, the dissimilatory efficiency reached 23.63%, the assimilation efficiency 65.41%, and the total nitrogen removal rate reached 89.04%. (4) It carries a variety of low-temperature adaptation-related genes, including cold shock protein genes, fatty acid desaturase genes and antioxidant stress-related genes, which provide a molecular basis for maintaining efficient metabolism in low-temperature environments; (5) Strain H2-1 has an ANI of <95% and DDH of <70% with the most similar type strain, and has the characteristics of a potential new species, which expands the resource pool of polar low-temperature aerobic denitrifying bacteria; (6) Nitrate reductase and nitrite reductase maintained high activity at 15℃, at 0.03 and 0.21 U / mg prot, respectively, indicating that they have stable low-temperature enzymatic reaction capabilities.

[0014] (7) It provides new functional bacterial strains for optimizing wastewater treatment processes under low temperature conditions. Attached Figure Description

[0015] Figure 1Electrophoresis images of the 16S rRNA gene amplification products of the seven Pseudomonas strains involved in this application.

[0016] Figure 2 The denitrification effect of the seven Pseudomonas strains involved in this application within 48 h is shown.

[0017] Figure 3 These are colony morphology photographs of the seven Pseudomonas strains involved in this application, where ag in the figures represent colony morphology photographs of Pseudomonas strains D2-1a, W2-12b, H2-1C, H2-1, D3-6a, D3B-25, and ND2-8, respectively.

[0018] Figure 4 The phylogenetic tree is constructed based on the whole genome sequences of the 7 isolates involved in this application and the 21 most closely related type strains screened in NCBI. The scale bar represents the average number of base substitutions at each site, reflecting the evolutionary distance between different strains. The isolates in this application are marked in bold.

[0019] Figure 5 This study describes the nitrate reduction pathway and related genes of the pseudomonad strains inferred from the KEGG annotations in this application.

[0020] Figure 6 The following is a circular map of the genomes of four Pseudomonas strains D2-1a, W2-12b, H2-1 and ND2-8 with complete denitrification metabolic pathways involved in this application. It shows the complete denitrification pathway based on KEGG annotation. The map is presented from the outside to the inside as follows: the outermost and fourth rings show the distribution of protein-coding genes annotated by Prokka, the second ring shows the distribution of GC content in the whole genome, the third ring shows the GC skewness characteristics, and the innermost ring is the genome size marker scale.

[0021] Figure 7 This paper reflects the growth curves and denitrification properties of the seven strains involved in this application, where (a)-(d) show the OD values ​​of the seven strains during growth. 600 The data shows the changes in nitrate nitrogen, nitrite nitrogen, and ammonia nitrogen, which intuitively reflects the transformation process of inorganic nitrogen during the growth of these seven bacteria. Detailed Implementation

[0022] The following embodiments are intended to enable those skilled in the art to more fully understand the technical solutions and implementation effects of this application, but the scope of protection of this application is not limited thereto. This section provides a more detailed description of this application in conjunction with specific implementation examples, and its technical features and advantages will be clearly demonstrated in the description. It should be noted that the embodiments described are merely illustrative examples and are not intended to limit the scope of the claims of this application. Any detailed adjustments, equivalent substitutions, or adaptive improvements based on the core principles of this application fall within the substantive protection scope of this application.

[0023] Example 1: Sample source and culture medium: The samples used in this application were obtained from dung samples from Penguin Island in Antarctica and soil samples from Yellow River Station in the Arctic. Specific station information is shown in Table 1.

[0024] Table 1 Information on samples from the North and South Poles Note: Since the four soil samples collected from the Arctic Yellow River Station have similar latitude and longitude, they were combined for processing.

[0025] The enrichment culture of the strains in this application used three different types of culture media: ① aerobic denitrification liquid medium (DM) + 10% (v / v) landfill leachate (source: Suzhou Qizishan landfill, main water quality indicators are COD: 801.5 mg / L, TN: 1780.4 mg / L, NH4+). + 56.1 mg / L, NO3 - ① 7.5 mg / L); and ② simultaneous nitrification-denitrification liquid medium (HNDM) + 10% (v / v) landfill leachate (source: Suzhou Qizishan Landfill, main water quality indicators are COD: 801.5 mg / L, TN: 1780.4 mg / L, NH4+: ...). + 56.1 mg / L, NO3 - : 7.5 mg / L); ③ Landfill leachate.

[0026] The strains were isolated using BTB agar plates, prepared by adding 0.1% (v / v) bromothymol blue solution (1% dissolved in ethanol) and 2% (w / v) agar powder to DM medium. Strawberry purification was performed using nutrient agar (NA) plates. NA plates were prepared by adding 2% (w / v) agar powder to liquid medium (Nutrient Broth, NB; Difco™, Becton Dickinson, USA). All media were initially pHed to 7.0–7.5 before use and autoclaved at 121 °C for 30 min before use.

[0027] Example 2: Isolation and Preservation of Denitrifying Bacteria The isolation of the denitrifying bacteria involved in this application was carried out according to the following steps: 10 g of sample was transferred to an Erlenmeyer flask containing 100 mL of enrichment medium and cultured at 15 °C and 150 rpm for 21 days. During the enrichment period, 10 mL of bacterial suspension was transferred to 100 mL of fresh enrichment medium every 7 days. After enrichment, 100 μL of culture medium was taken for 10... -1 -10 -7 Serial dilutions were performed, and bacterial solutions of different dilution gradients were spread onto BTB agar plates and incubated at 15 °C. Colonies with different morphological characteristics and color development (turning blue) were selected and purified by repeated streak plating on NA agar plates to obtain single strains. Through Examples 1 and 2, a total of 449 culturable aerobic denitrifying bacteria were obtained in this application.

[0028] The 449 bacterial strains were inoculated onto the surface of NA slant agar and streaked. After the bacterial growth was good, an appropriate amount of sterile 20% (v / v) glycerol solution was added, and the mixture was thoroughly mixed. 1.5 mL of the bacterial suspension was then transferred to sterile cryovials, and three replicates were prepared for each strain. The samples were pre-frozen overnight at -20°C and then transferred to a -80°C freezer for long-term storage.

[0029] Example 3: Sequencing analysis of the 16S rRNA gene of the strain After obtaining 449 culturable aerobic denitrifying bacteria, this application further screened representative strains with efficient nitrogen removal capabilities by comparing and eliminating duplicates of the 16S rRNA gene sequence. Specifically, DNA was extracted from single colonies using a rapid boiling method. Colonies were picked from the plate and mixed into 1.5 mL EP tubes containing 20 μL of sterilized ultrapure water. The tubes were boiled for 2-3 min and centrifuged at 12000 rpm for 3 min. The supernatant was retained as a DNA template. The full-length 16S rRNA gene was amplified using the universal bacterial forward primer sequence 27F, i.e., 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No. 1), and the universal bacterial reverse primer sequence 1492R, i.e., 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID No. 2). The blank control group used sterilized ultrapure water instead of DNA. Amplification program: pre-denaturation (95℃, 5 min), denaturation (94℃, 45 s), annealing (55℃, 45 s), extension (72℃, 2 min), 25 cycles, final extension (72℃, 10 min), stored at 10℃ after the program. The obtained PCR products were examined by agarose gel electrophoresis to confirm whether they were the target fragments. The agarose gel concentration was 1%, and the electrophoresis conditions were 120V for 20 min. Electrophoresis observation and photography were performed using a gel imaging system. Figure 1The image shown below is an electrophoresis photograph of the 16S rRNA gene amplification products of seven representative Pseudomonas strains. The amplification products were bidirectionally sequenced by Shanghai Sangon Biotech Co., Ltd. The obtained sequences were preliminarily classified and identified using the NCBI-BLAST tool (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The identification results showed that 119 pure culture strains were identified as different species based on the 16S rRNA gene alignment. The 16S rRNA sequence of strain H2-1 in this application is specifically (SEQ ID No. 3): Example 4: Secondary screening of denitrifying bacteria The 119 pure culture strains identified as different species by 16S rRNA gene alignment were used to determine their nitrate removal capacity under aerobic conditions (15°C, 48 h). Each of the 119 strains was inoculated into 100 mL of DM medium at an inoculum size of 10% (v / v). Therefore, this application selected conditions of 15 °C and 150 rpm for shaking culture of each strain for 48 h, and culture medium samples were collected before inoculation (0 h) and after culture (48 h) to determine the nitrate (NO3) content. - ) and nitrite (NO2) - The concentration changes of nitrate nitrogen in the water samples were analyzed to assess the denitrification capacity of the strain under low-temperature conditions. Nitrate nitrogen in the water samples was determined by ultraviolet spectrophotometry, referring to the standard "Determination of Nitrate in Water - Ultraviolet Spectrophotometry" (HJ / T 346-2007). The detection range of this method is 0.08–4.00 mg N / L. Nitrite nitrogen in the water samples was determined by the N-(1-naphthyl)-ethylenediamine spectrophotometric method, referring to the standard "Determination of Nitrite Nitrogen in Water - N-(1-naphthyl)-ethylenediamine spectrophotometric method" (GB 7493-87 or HJ 535-2009). The detection range of this method is 0.003–0.20 mg N / L.

[0030] The results showed that 48 strains exhibited significant growth activity during cultivation, with 23 strains achieving a nitrate removal rate exceeding 99.8% within 48 hours, demonstrating excellent aerobic denitrification performance. After intraspecific weight removal treatment, seven representative strains with the highest nitrate removal efficiency were finally obtained (Table 2), namely D2-1a, W2-12b, H2-1C, and H2-1 (Antarctic penguin island dung soil samples); D3-6a and D3B-25 (Antarctic Zhongshan Station soil samples); and ND2-8 (Arctic Yellow River Station soil samples). Figure 2 As shown, these seven strains all exhibited nitrate removal rates exceeding 99.8% and nitrite accumulation levels below 1.5 μmol / L within 48 hours, indicating their highly efficient and stable denitrification capacity. Figure 3 In the image, ag shows Pseudomonas colony plate photographs of D2-1a, D3-6a, D3B-25, H2-1, H2-1C, W2-12b, and ND2-8, respectively. For strain H2-1 involved in this application, its colonies are round and opaque, smaller than other bacteria, with a diameter of 1~1.5mm, smooth surface, flat edges, and a beige color.

[0031] In this rescreening process, more than half of the strains belonged to the genus *Pseudomonas* (…). Pseudomonas ). PseudomonasThis genus is known for its extensive carbon source utilization capabilities, flexible electron transport pathways, and multi-level stress response mechanisms. Existing research indicates that some... Pseudomonas The strains carry complete denitrification pathway genes, enabling them to maintain good nitrogen removal activity under low-temperature conditions. The seven highly efficient strains screened in this application still exhibited significant nitrogen removal capacity under low-temperature conditions, indicating that they possess a unique low-temperature adaptation and highly efficient nitrogen metabolism mechanism. Experimental results showed that all seven strains exhibited extremely low nitrite accumulation during nitrate removal, indicating that their denitrification pathway is complete and electron transfer is highly efficient. This characteristic is particularly important in wastewater treatment applications in cold regions, because nitrite accumulation not only increases effluent toxicity but may also inhibit other functional microorganisms.

[0032] Table 2. Basic information and nitrate removal rate of 7 Pseudomonas strains Example 5: DNA Extraction, Genome Sequencing and Assembly Seven representative isolates with high denitrification efficiency were selected, and genomic DNA was extracted using the FastPure Bacteria DNAIsolation MiniKit-Box 2 (Vazyme, China). After constructing PE150 libraries, single-strain genome draft sequencing was performed on an Illumina high-throughput sequencing platform (Majorbio, China). Raw data underwent quality checks and splicing using fastQC (v0.12.1) and trimmomatic (v0.39), followed by assembly of the spliced ​​data using SPAdes (v4.1.0) to obtain contig and scaffold sequence files. To assess the genome assembly quality of the seven Pseudomonas isolates, assembly statistical analysis was performed using QUAST (v5.3.0), and CheckM (v1.2.3) was used to evaluate genome integrity and contamination levels.

[0033] The high-quality genome drafts of the seven potential aerobic denitrifying bacteria obtained in this application are shown in Table 3. QUAST analysis results show that the genome sizes of the strains are relatively consistent, ranging from 5.94 to 7.00 Mb, and the number of coding genes is also similar, ranging from 5342 to 6403. The GC content of each genome ranges from 58.55% to 59.85%. The N50 values ​​range from 194,358 to 815,300 bp, and the L50 values ​​range from 3 to 10, indicating high assembly quality. CheckM assessment results show that the genome integrity of each strain ranges from 98.56% to 100%, and the contamination level ranges from 0.11% to 1.27%, indicating reliable genome assembly quality suitable for subsequent comparative genome and functional annotation analysis.

[0034] Table 3. Basic genomic information of 7 Pseudomonas strains Example 6: Phylogenetic Analysis After performing draft genome sequencing on the seven isolated strains with high denitrification efficiency, the genomes were assembled using high-throughput sequencing data. Subsequently, GTDB-Tk (v 2.4.1) (Genome Taxonomy Database Toolkit) was used to perform more accurate species classification annotation on the assembled genomes. To further elucidate the phylogenetic relationships of these strains, the complete genomes of all publicly available type strains of the same genus were downloaded from the NCBI database as reference data. The genomes of the above strains were compared and analyzed with the reference genomes downloaded from NCBI, and maximum likelihood phylogenetic analysis was performed using IQ-Tree (v2.3.6). The average nucleotide homology ANI-blast (ANIb) value and DNA-DNA hybridization (dDDH) value between the genomes of the strain and its closely related type strains were calculated using the online server JSpeciesWS (http: / / jspecies.ribohost.com / jspeciesws) and the online inter-genome distance calculator version 3.0 (https: / / ggdc.dsmz.de / ggdc.php), respectively, focusing on whether the similarity was below the thresholds of 95-96% and 70%.

[0035] The results showed that, according to annotations in the GTDB-Tk database, the seven strains with high denitrification efficiency screened all belonged to [the relevant group / organism / etc.]. Pseudomonas Genus. To further clarify its phylogenetic position, 544 type specimens were downloaded from the NCBI database. Pseudomonas The complete genome sequence of the strain was obtained, and a phylogenetic tree was constructed using the strains mentioned above. Based on the phylogenetic clustering results, 21 type strains most closely related to these 7 strains were selected for further comparative analysis. The results are as follows: Figure 4 As shown, strains D3-6a, W2-12b, and D3B-25 are directly clustered on the same phylogenetic branch as their corresponding reference strains. Their DNA-DNA hybridization (dDDH) values ​​are all higher than 70%, and their average nucleotide similarity (ANI) values ​​are also higher than the species classification threshold of 95–96%, indicating that they should be classified as existing named species. In contrast, the remaining four strains, D2-1a, H2-1C, H2-1, and ND2-8, do not form close clusters with known strains on the phylogenetic tree, and their dDDH and ANI values ​​with their closest reference strains are all lower than the species classification threshold, suggesting that they may be potential new species.

[0036] Example 7: Genome Annotation To investigate the denitrification mechanisms of seven potential aerobic denitrifying bacteria, this application used genome functional annotation results and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway reconstruction analysis. Open Reading Frames (ORFs) prediction was performed using Prodigal (v2.6.3). The obtained protein-coding sequences were functionally annotated using eggNOG-mapper (v2.1.12). The annotation database used was eggNOG (v5.0.2), which integrates multiple functional classification systems, including: Clusters of Orthologous Groups (COG), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Carbohydrate-Active Enzymes (CAZy), Biochemical, Genetic and Genomic Reaction (BiGG), and Protein Families (PFAMs). Nitrogen metabolism pathways were reconstructed using the KEGG Mapper-Reconstruct Pathway tool (https: / / www.genome.jp / kegg / mapper / reconstruct.html) from the KEGG database. Basic genome information was annotated using prokka. A genome loop map was generated using CGView v2.

[0037] The results show that... Figure 5 As shown, D2-1a, W2-12b, H2-1, and ND2-8 possess a complete denitrification metabolic pathway (NO3). - → NO2 - → NO → N2O → N2). and Figure 6 The genome maps of these four bacterial strains are shown. Typical key denitrification functional genes were detected in the genomes of all four strains, including... nar GHI, nap AB nir S, nor BC and nos Z indicates that it has the potential to complete the entire denitrification process at the genomic level.

[0038] It is noteworthy that these four strains exhibit differences in the nitrate reduction phase. For example, W2-12b lacks the pericellular nitrate reductase NapAB but retains the membrane-bound nitrate reductase NarGHI, which differs from the traditional aerobic denitrifying enzyme theory. This suggests that different strains may differ in their oxygen concentration gradients or electron acceptor utilization mechanisms. This difference in enzyme structure may reflect their adaptation mechanisms to fluctuations in oxygen availability under low-temperature environments. Previous studies have indicated that many organisms typically possess both Nar and Nap systems: the former plays a major role under hypoxic or high-nitrate conditions, while the latter is more advantageous in aerobic or low-nitrate environments. Therefore, W2-12b may favor denitrification in hypoxic environments, while other strains can flexibly switch metabolic systems according to environmental changes. This difference in the Nar / Nap system may reflect the energy optimization strategies of bacteria under different redox gradients.

[0039] Furthermore, genome annotation results also show that D2-1a, W2-12b, H2-1, and ND2-8, in addition to possessing complete denitrification pathways, also include assimilation of nitrate reduction and dissimilation of nitrate reduction to ammonia. For example, nas AB-coded nitrate reductase and nir The BD-encoded nitrite reductases were all detected in the genome, indicating that these strains possess strong metabolic flexibility under different nitrogen source conditions. This multi-pathway coexistence characteristic... Pseudomonas The genera already have genomic annotations and some physiological studies to support their existence. For example... P.putida Y 9. Simultaneously possesses three routes; Arctic Pseudomonas Three pathway genes were also annotated in strains PMCC200344 / 200367. Therefore, this can be considered a potential strategy for maintaining nitrogen cycling in this genus under variable environments. In these strains, the denitrification pathway is mainly accomplished through a typical enzyme system: nitrate (NO3) - Nitrate is converted to nitrite (NO2) by nitrate reductase (NarGHJI or NapAB). - Nitrite (NO) is then converted to nitric oxide (NO) by nitrite reductase (NirS), which in turn converts it to nitrous oxide (N₂O) by nitric oxide reductase (NorBC), and finally reduced to nitrogen gas (N₂) by nitrous oxide reductase (NosZ). Simultaneously, in the DNRA pathway, nitrite reductase NirBD converts nitrite (NO₂) into nitrogen gas. - ) is reduced to ammonium ions (NH4) + In the nitrate assimilation pathway, nitrite is catalyzed by the assimilatory nitrite reductase NirA to generate NH4. + The generated NH4 +Subsequently, it can be converted to L-glutamine by glutamine synthase (GlnA), and then to L-glutamate by the glutamate synthase complex (GltBD), ultimately entering the glutamate metabolic pathway to provide nitrogen for bacterial growth and metabolism. Furthermore, genes related to the ammonia oxidation pathway were detected in the genomes of all four bacterial strains. nxr (B) indicates that these bacteria have the ability to oxidize nitrite to nitrate.

[0040] Example 8: Nitrogen Balance of Strains To elucidate the fate of nitrate nitrogen in four strains with intact nitrogen metabolism pathways under low-temperature conditions, this application systematically analyzed the nitrogen balance of D2-1a, W2-12b, H2-1, and ND2-8 after incubation at 15 °C for 16 h. These strains are currently deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession numbers CGMCC No. 37028, CGMCC No. 37026, CGMCC No. 37029, and CGMCC No. 37027. This application used nitrate nitrogen as the sole nitrogen source and substrate for batch experiments. The strains were inoculated into DM medium and incubated at 15 °C and 150 r / min for 16 h. Bacterial samples were collected, and OD values ​​were measured. 600 Ammonia nitrogen (NH4) + -N), nitrate nitrogen (NO3) - -N), nitrite nitrogen (NO2) - The concentrations of nitrogen (N), dissolved total nitrogen (DTN), and total nitrogen (TN) in the system are measured. Based on these measured concentration parameters, the intracellular nitrogen content, gaseous nitrogen content, denitrification efficiency of the strain for nitrogen transformation, assimilation efficiency, and total nitrogen removal efficiency can be calculated.

[0041] Intracellular nitrogen content was calculated by subtracting 0-hour cellular nitrogen from 16-hour cellular nitrogen content. The gaseous nitrogen content (nitrogen loss) is calculated by the difference between total nitrogen (TN) at 0 h and 16 h: The formulas for calculating the denitrification efficiency and assimilation efficiency of the strain for nitrogen transformation are as follows: Total nitrogen removal efficiency is the sum of assimilation efficiency and denitrification efficiency: The results are shown in Table 4. After 16 h of culture, approximately 65.41%–78.96% of the initial total nitrogen was assimilated into the cell biomass, while 9.30%–23.63% of the initial nitrogen was converted into gaseous nitrogen. The generation of gaseous nitrogen clearly indicates that these strains can utilize some nitrate nitrogen for denitrification under aerobic conditions at 15°C. Under these culture conditions, the total nitrogen (TN) removal rate of the four strains reached 85.02%–89.04%, with assimilation playing a dominant role. This result is consistent with previously reported... P. putida strain NP5 and P. plecoglossicida Aerobic denitrifying Pseudomonas such as Y-1 exhibit a consistent assimilation-based denitrification process. It should be noted that although nitrogen removal primarily relies on assimilation, the stable proportion of gaseous nitrogen production clearly demonstrates that the strain possesses a sustained aerobic denitrification flux under low-temperature conditions.

[0042] Table 4. Nitrogen balance analysis of the strains at 15℃ Example 9: Evaluation of the denitrification characteristics of the strain To verify the low-temperature denitrification performance of the strain, its aerobic denitrification ability at 15 °C was further verified using DM medium with nitrate nitrogen as the sole nitrogen source. Specifically, the strain was inoculated into DM medium and cultured at 15 °C and 150 r / min for 16 h. Then, it was inoculated into sterile DM medium at a 10% (volume ratio) inoculation rate and cultured in a shaker at 15 °C and 150 r / min for 28 h. Bacterial samples were collected periodically, and OD values ​​were measured. 600 Ammonia nitrogen (NH4) + -N), nitrate nitrogen (NO3) - -N), nitrite nitrogen (NO2) - -N) concentration.

[0043] Based on the concentration parameters measured above, the removal efficiency (RE) and removal rate (RR) of nitrogen compounds under different conditions can be calculated using the following formulas: Where RE and RR represent nitrogen removal efficiency and removal rate, C0 is the initial nitrogen concentration, and C t The value represents the nitrogen concentration at time t, where t represents the reaction time.

[0044] The results are as follows Figure 7As shown, (a)-(d) respectively show the OD values ​​of the seven strains involved in this application during their growth. 600 The changes in nitrate nitrogen, nitrite nitrogen, and ammonia nitrogen are shown, which visually demonstrates the inorganic nitrogen transformation process during the growth of these four bacterial strains. Among them, the four strains D2-1a, W2-12b, H2-1, and ND2-8, which possess complete denitrification metabolic pathways, all experienced a stasis period of approximately 8 hours in the early stages of culture, followed by the logarithmic growth phase, reaching maximum biomass at 20-24 hours, and then gradually entering a plateau phase. The nitrate nitrogen removal process showed a significant synchronicity with the increase in cell biomass. The main nitrate nitrogen removal phase occurred during the logarithmic growth phase of the strains (8–16 hours), with the maximum nitrate nitrogen removal rate reaching 3.67–5.37 mg / L·h during this phase. At 20 hours of culture, the average nitrate nitrogen removal rate was 1.82–2.28 mg / L·h, corresponding to a nitrate nitrogen removal rate of 87.52%–93.90%. The removal rates and efficiencies described above are superior to those of several previously reported strains of low-temperature aerobic denitrifying Pseudomonas (Table 5). For example, its average nitrate nitrogen removal rate is significantly higher than that of other strains. P. putida Y-9 P. putida Y-12 and P. taiwanensis J. Despite P. tolaasii The average nitrate removal rate of Y-11 at 15 °C was similar to that of the strain in this application, but its total nitrogen removal rate was only 41.9%, which was significantly lower than that of the strain in this application. Furthermore, Y-11 was only observed to have excellent nitrate removal efficiency after 4 days, which further highlights the advantage of the strain in this application in terms of nitrogen transformation integrity.

[0045] Only small amounts of nitrite nitrogen (0.19–0.65 mg / L) were detected during the culture process, and all of it was eventually completely removed, indicating that no significant accumulation of denitrification intermediates occurred. After the nitrite concentration reached its peak, a transient increase in ammonium nitrogen in the culture medium occurred, which may be related to the reduction of dissimilatory nitrate to ammonium (DNRA) or the reduction of assimilatory nitrate to ammonium (ANRA). Similar phenomena were also observed... Pseudomonas sp. JQ-H3 and Rhizobium This was observed in strains such as sp. WS7. The renewed increase in ammonium nitrogen concentration in the later stages of culture may be related to the release of intracellular nitrogen and organic nitrogen after partial cell death, a trend consistent with previous findings.

[0046] Table 5. Denitrification properties of *Pseudomonas* strains described in this application and other previously reported *Pseudomonas* strains at 15 °C. Example 10: Enzyme Activity Assay Previous studies have shown that the presence of nitrogen metabolism functional genes alone is insufficient to fully demonstrate the actual denitrification capacity of a strain. Therefore, the determination of the activities of key denitrifying enzymes can serve as important supplementary physiological evidence. This application further determined the activities of nitrate reductase (NR) and nitrite reductase (NIR) in four Pseudomonas strains. The activities of nitrate reductase (NR) and nitrite reductase (NIR) were determined using a commercial enzyme activity assay kit (Sangon Biotech, Shanghai, China) combined with spectrophotometry. Specific operating procedures were performed according to the kit's instructions. NR activity was determined based on the change in absorbance of NADH at 340 nm, a method that uses NADH as an electron donor to catalyze the reduction of nitrate to nitrite. NIR activity was characterized by monitoring the decrease in nitrite concentration at 540 nm. After culturing the strains in DM medium at 15 °C for 16 h, the bacterial cells were collected, centrifuged (12,000 rpm, 3 min, 4 °C) to obtain the bacterial pellet, and resuspended using NR / NIR extraction buffer. Subsequently, the enzyme was sonicated under ice bath conditions (300 W, 3 s sonication, 7 s interval, total duration 3 min), and centrifuged again (12,000 rpm, 3 min, 4 ℃). The supernatant was collected as crude enzyme solution for enzyme activity assay. Protein content was quantitatively determined using a Bradford protein assay kit (Beyotime, Shanghai, China). Enzyme activity units were defined as follows: NR activity was defined as 1 μmol NADH consumed per milligram of protein per hour (U / mg), and NIR activity was defined as 1 μmol NO2 reduced per milligram of protein per hour. - -N represents 1 active unit (U / mg).

[0047] The results showed that the activities of NR and NIR were 0.02-0.05 U / mg and 0.17-0.25 U / mg, respectively, indicating that these strains could still express and maintain the activity of key denitrifying enzymes under aerobic and low-temperature conditions. Therefore, the enzyme activity detection results of NR and NIR provide strong support for the aforementioned nitrogen balance characteristics and nitrate removal performance.

[0048] in conclusion: This application isolated 449 potential aerobic denitrifying bacteria from typical environmental samples from the Arctic and Antarctic. Seven representative strains obtained after secondary screening were all... PseudomonasFour strains exhibited complete denitrification pathways: D2-1a, W2-12b, H2-1, and ND2-8. These strains are currently deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession numbers CGMCC No. 37028, CGMCC No. 37026, CGMCC No. 37029, and CGMCC No. 37027. Genomic analysis revealed that these strains generally carry multiple low-temperature adaptation-related genes, such as cold shock proteins, RNA helicases, and membrane lipid-modifying enzymes, providing the molecular basis for maintaining metabolic activity in cold environments. These strains all demonstrated excellent denitrification performance, achieving maximum nitrate removal rates of 3.67–5.37 mg / L·h at 15°C with nitrate nitrogen as the sole nitrogen source. After 20 h of culture, the average nitrate removal rate was 1.82–2.28 mg / L·h, corresponding to nitrate removal rates of 87.52%–93.90%. Nitrogen balance analysis results indicate that these strains primarily denitrify through assimilation and aerobic denitrification. Based on genome annotation, enzyme activity assays, and nitrogen balance analysis, the metabolic pathway for nitrate nitrogen in these four strains is presumed to be: NO3- - → NO2 - → NO → N2O → N2. These results provide theoretical support for the low-temperature adaptation and denitrification mechanism of polar aerobic denitrifying bacteria, and offer new insights for the development and application of microbial resources in wastewater treatment in cold regions.

[0049] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in this application, based on the technical solution and inventive concept of this application, should be included within the scope of protection of this application.

Claims

1. A strain of Pseudomonas H2-1, characterized in that, The pseudomonads H2-1 were classified and named Pseudomonas sp., with accession number CGMCC NO.37029, was deposited at the China General Microbiological Culture Collection Center on December 11, 2025.

2. A formulation, characterized in that, The formulation comprises Pseudomonas H2-1 as described in claim 1, or its spore suspension, its fermentation broth, or its lyophilized powder.

3. A wastewater treatment agent, characterized in that, The wastewater treatment agent comprises Pseudomonas H2-1 as described in claim 1, or its spore suspension, its fermentation broth, or its freeze-dried powder.

4. The application of the Pseudomonas H2-1 as described in claim 1 in aerobic denitrification.

5. The application of the Pseudomonas H2-1 as described in claim 1 in nitrate nitrogen removal.

6. The application of the Pseudomonas H2-1 as described in claim 1 in wastewater treatment.

7. The application of the Pseudomonas H2-1 of claim 1 in wastewater treatment in cold regions.

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