A heterotrophic nitrifying aerobic denitrifying Pseudomonas aeruginosa for Zn 2+ Wastewater denitrification methods

Abstract

A heterotrophic nitrifying aerobic denitrifying Pseudomonas aeruginosa for Zn 2+ Wastewater denitrification method, involving a Zn 2+ Wastewater denitrification method: This invention provides a strain resistant to heavy metal Zn. 2+ Heterotrophic nitrifying aerobic denitrifying Pseudomonas and its applications, this Pseudomonas is pseudomonas sp. SK-4, strain preservation number CCTCC NO: 2024163. This invention comprehensively analyzes the enrichment, isolation, screening, and identification of strain SK-4, the effects of different environmental factors on the strain's denitrification performance, the determination of the strain's denitrification performance, and Zn... 2+ The effect on the denitrification performance of the strain confirmed that this bacterium has excellent denitrification ability and is effective against Zn. 2+ It exhibits strong tolerance. This bacterium can thrive in high concentrations of heavy metal ions, such as Zn. 2+ Despite the influence of heavy metals, it can still maintain a high removal rate of ammonia nitrogen and nitrate nitrogen, providing a new option for biological denitrification of wastewater under heavy metal stress.

Description

Technical Field

[0001] This invention relates to a Zn 2+ Wastewater denitrification methods, particularly involving a heterotrophic nitrifying aerobic denitrifying Pseudomonas aeruginosa for Zn 2+ Wastewater denitrification methods. Background Technology

[0002] Biological denitrification is a key step in biological wastewater treatment. Controlling the entry of wastewater containing heavy metals into urban wastewater treatment processes is more complex, and the entry of large amounts of heavy metals into the biological denitrification system will greatly affect the denitrification effect.

[0003] Currently, many studies have focused on the negative impact of heavy metals on biological nitrogen removal. Heavy metals can alter the diversity and abundance of activated sludge microorganisms, thereby reducing the efficiency of biological systems in removing pollutants from water and affecting effluent quality.

[0004] The drawbacks of traditional biological denitrification under heavy metal stress are obvious, and this method is far from meeting the current treatment needs of industrial and domestic wastewater. Therefore, with the continuous in-depth research of scholars, novel biological denitrification has become the focus of many scholars' research due to its advantages such as being able to withstand extreme (heavy metal) environments and overcoming the defects of traditional biological denitrification. Summary of the Invention

[0005] The purpose of this invention is to provide a heterotrophic nitrifying aerobic denitrifying Pseudomonas aeruginosa for Zn 2+ A wastewater denitrification method that cultivates a strain tolerant to heavy metal Zn. 2+ This strain of Pseudomonas aeruginosa possesses heterotrophic nitrifying, aerobic, and denitrifying capabilities. pseudomonas sp. SK-4, in high concentrations of the heavy metal Zn 2+ Despite the impact of the weather, its denitrification capacity remains excellent, providing a new option for biological denitrification treatment of wastewater in harsh environments. It has broad application prospects, especially opening up new application directions for the treatment of high-concentration zinc-containing wastewater.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A heterotrophic nitrifying aerobic denitrifying Pseudomonas aeruginosa for Zn 2+ Wastewater denitrification method, the method comprising the following steps:

[0008] (1) Cultivation of heterotrophic nitrifying aerobic denitrifying Pseudomonas

[0009] S1. The mud-water mixture sample retrieved from the aerobic tank is stored at 2-5℃. 1-6 ml of the sample is transferred to an Erlenmeyer flask containing enrichment medium under a sterile operating table. After thorough mixing, it is incubated in a constant temperature shaker at 30-40℃ and 120-160 rpm / min for 1-3 days. After incubation, 1-6 ml of the bacterial culture is transferred to an Erlenmeyer flask containing enrichment medium, and the above operation is repeated three times.

[0010] S2. The seed solution obtained from the above enrichment was diluted using a gradient dilution method to obtain a 10-1 solution. -1 ~10 -9 Cell dilutions at varying concentrations (dilution steps: add 1 ml of seed culture to 9 ml of sterile water, and so on) were prepared. 0.1–0.3 ml of each concentration gradient of bacterial culture was evenly spread onto aerobic denitrification medium and incubated at 30–40°C for 2–3 days. After incubation, colonies near which the medium changed from yellow-green to blue were selected and inoculated onto heterotrophic nitrification medium for multiple three-part streak cultures to improve purity. Finally, the purified strain was inoculated onto heterotrophic nitrification medium and stored at 2–5°C.

[0011] S3. Inoculate 1-3 loops of the single colony obtained from the culture with an inoculation loop and culture in heterotrophic nitrification liquid medium for 12-20 h to obtain seed liquid. Transfer 1 ml of seed liquid into heterotrophic nitrification liquid medium and culture at 30-40℃ and 120-160 rpm / min for 12-20 h. Measure its ammonia nitrogen removal efficiency. Finally, screen strains with an ammonia nitrogen removal efficiency greater than 90% for further screening.

[0012] S4. Transfer the seed culture of the strain with a removal efficiency greater than 90% to a solution containing 50-80 mg / L Zn. 2+ In heterotrophic nitrification medium, the samples were cultured at 30–40℃ and 120–160 rpm / min for 12–20 h to screen for Zn. 2+ The most resistant Pseudomonas pseudomonas sp.SK-4, and then frozen for preservation.

[0013] (2) The Pseudomonas bacteria obtained from the above culture in the presence of Zn 2+ Application of denitrification in wastewater

[0014] This bacterium is present in Zn-containing environments. 2+ For nitrogen removal from wastewater, the above-mentioned heterotrophic nitrification liquid culture medium and aerobic denitrification liquid culture medium were respectively supplemented with Zn at concentrations of 0 mg / L, 5 mg / L, 10 mg / L, 30 mg / L, 50 mg / L, and 80 mg / L. 2+Seed culture of the strain at concentrations of 1%–3% was collected, and the concentration of ammonia nitrogen in the heterotrophic nitrification medium was measured at 12–16 h, while the concentration of nitrate nitrogen in the aerobic denitrification medium was measured at 18–20 h. This bacterium thrives in high concentrations of heavy metal ions (Zn). 2+ Despite the influence of heavy metal stress, it can still maintain a high removal rate of ammonia nitrogen and nitrate nitrogen, providing an option for biological denitrification of wastewater under heavy metal stress.

[0015] The aforementioned heterotrophic nitrifying aerobic denitrifying Pseudomonas aeruginosa for Zn 2+ Wastewater denitrification methods, this bacterium pseudomonas sp. SK-4 is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M2024163, and deposit date January 22, 2024. The morphological and physiological-biochemical characteristics of this heterotrophic nitrifying-aerobic denitrifying bacterium SK-4 include: SK-4 colonies are round, milky white overall, slightly raised in the center, and have a viscous surface; under a microscope, they appear as short rods; they stain red with Gram stain, indicating they are Gram-negative. After multiple centrifugations of the 12-hour culture seed culture, the sample was sent to a biotechnology company for gene sequencing. The sequencing results were compared for homology in the GenBank database, showing 99.93% similarity to known strains. A phylogenetic tree was constructed, revealing that this strain is a *Pseudomonas hunanense*, and it was named... pseudomonas sp. SK-4.

[0016] The aforementioned heterotrophic nitrifying aerobic denitrifying Pseudomonas aeruginosa for Zn 2+ In the wastewater denitrification method, the enrichment culture medium in step S1 comprises: ammonium chloride 0.2–0.6 g, sodium succinate 4–8 g, and Vickers salt solution 30–50 ml, diluted to 1–1.5 L. The Vickers salt solution consists of: dipotassium hydrogen phosphate 1.5–2 g, magnesium sulfate 0.2–0.5 g, sodium chloride 2–5 g, ferrous sulfate 0.05–0.1 g, and manganese sulfate 0.05–0.1 g, diluted to 1–2 L, with a pH of 7.0–8.0.

[0017] The aforementioned heterotrophic nitrifying aerobic denitrifying Pseudomonas aeruginosa for Zn 2+ In the wastewater denitrification method, in step S1:

[0018] 1) The aerobic denitrification medium consists of: NaNO3 0.5-2 g, sodium succinate 5-12 g, K2HPO4 0.5-1 g, KH2PO4 1-2 g, MgSO4·7H2O 0.1-0.2 g, FeSO4·7H2O 0.05-0.1 g, 1% bromothymol blue ethanol solution (BTB) 1-2 ml, pH 7.0-7.8, agar 20-50 g, trace element solution 1-2 ml, and distilled water 1-2 L;

[0019] 2) The heteroaerobic nitrification medium consists of: ammonium chloride 0.5-2 g, sodium succinate 5-12 g, K2HPO4 0.5-1 g, KH2PO4 1-2 g, MgSO4·7H2O 0.1-0.2 g, FeSO4·7H2O 0.05-0.1 g, pH 7.0-7.8, agar 20-50 g, and distilled water 1-2 L;

[0020] 3) Trace element solution: EDTA-2Na 40~70mg / L, ZnSO4·7H2O 2~6mg / L, CaCl2·2H2O 5~9mg / L, MnCl2·4H2O 6~8mg / L, FeSO4·7H2O 6~8mg / L, CuSO4·5H2O 1~5mg / L, COCl2 1~5mg / L.

[0021] The aforementioned heterotrophic nitrifying aerobic denitrifying Pseudomonas aeruginosa for Zn 2+ In the wastewater denitrification method, the heterotrophic nitrification liquid culture medium in S1 comprises: ammonium chloride 0.5–2 g, sodium succinate 5–12 g, K₂HPO₄ 0.5–1 g, KH₂PO₄ 1–2 g, MgSO₄·7H₂O 0.1–0.2 g, FeSO₄·7H₂O 0.05–0.1 g, pH 7.0–7.8, and distilled water 1–2 L.

[0022] The advantages and effects of this invention are:

[0023] 1. The strain of Pseudomonas of the present invention pseudomonas sp. SK-4 colonies are round, milky white overall, slightly raised in the center, and have a viscous surface; under a microscope, they appear as short rods; they stain red with Gram stain, indicating they are Gram-negative bacteria. (Strain) pseudomonas sp.SK-4 exhibits the best denitrification performance when sodium citrate is used as the carbon source, C / N=10, pH=7-8, temperature 30℃, and rotation speed 140 rpm / min, achieving a removal rate of over 99% for ammonia nitrogen within 24 hours.

[0024] 2. The strain of this invention pseudomonassp.SK-4 in Zn 0–100 mg / L 2+ Under stress, the removal efficiency of ammonia nitrogen and nitrate nitrogen in wastewater with total nitrogen = 100 mg / L did not fluctuate significantly, and the removal efficiency of ammonia nitrogen could reach more than 90%, and that of nitrate nitrogen could reach more than 70%.

[0025] 3. The strain of this invention pseudomonas sp.SK-4 has the ability to simultaneously perform heterotrophic nitrification and aerobic denitrification, and it has the characteristics of fast growth and reproduction rate and high nitrogen removal efficiency. Its logarithmic growth phase is 6-12 hours, and the ammonia nitrogen removal rate in wastewater is nearly 90% after 12 hours. It can be seen that this bacterium can be used immediately and efficiently to achieve biological nitrogen removal. Attached Figure Description

[0026] Figure 1 This is an phylogenetic tree diagram of strain SK-4;

[0027] Figure 2 The graph shows the ammonia nitrogen removal efficiency of strain SK-4 under different carbon source conditions.

[0028] Figure 3 The graph shows the ammonia nitrogen removal efficiency of strain SK-4 under different C / N conditions.

[0029] Figure 4 The graph shows the ammonia nitrogen removal efficiency of strain SK-4 under different pH conditions.

[0030] Figure 5 The graph shows the ammonia nitrogen removal efficiency of strain SK-4 under different rotation speeds.

[0031] Figure 6 The graph shows the ammonia nitrogen removal efficiency of strain SK-4 under different temperature conditions.

[0032] Figure 7 This is a graph showing the degradation capacity of strain SK-4 for ammonia nitrogen.

[0033] Figure 8 This is a graph showing the degradation capacity of strain SK-4 for nitrate nitrogen.

[0034] Figure 9 For strain SK-4 in Zn 2+ A graph showing the degradation capacity of ammonia nitrogen in wastewater. Detailed Implementation

[0035] The present invention will now be described in detail with reference to the embodiments shown in the accompanying drawings.

[0036] The strain SK-4 of this invention can be used in high concentrations of the heavy metal Zn 2+ It maintains excellent denitrification capacity under the impact of environmental factors, providing a new option for biological denitrification treatment of wastewater in harsh environments, and has broad application prospects.

[0037] The strain of Pseudomonas in this invention pseudomonas sp. SK-4, deposited on January 22, 2024, is deposited at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M2024163.

[0038] Example 1: Isolation, Screening and Purification of Strains

[0039] The culture medium formula is as follows:

[0040] 1) Enrichment medium: Ammonium chloride 0.2–0.6 g, sodium succinate 4–8 g, Vickers salt solution 30–50 ml, adjusted to 1–1.5 L. Vickers salt solution: dipotassium hydrogen phosphate 1.5–2 g, magnesium sulfate 0.2–0.5 g, sodium chloride 2–5 g, ferrous sulfate 0.05–0.1 g, manganese sulfate 0.05–0.1 g, adjusted to 1–2 L, pH 7.0–8.0;

[0041] 2) Heterotrophic nitrification solid medium: ammonium chloride 0.5-2 g, sodium succinate 5-12 g, K2HPO4 0.5-1 g, KH2PO4 1-2 g, MgSO4·7H2O 0.1-0.2 g, FeSO4·7H2O 0.05-0.1 g, pH 7.0-7.8, agar 20-50 g, trace element solution 1-2 ml, distilled water 1-2 L;

[0042] 3) Aerobic denitrification solid culture medium: NaNO3 0.5-2 g, sodium succinate 5-12 g, K2HPO4 0.5-1 g, KH2PO4 1-2 g, MgSO4·7H2O 0.1-0.2 g, FeSO4·7H2O 0.05-0.1 g, 1% bromothymol blue ethanol solution (BTB) 1-2 ml, pH 7.0-7.8, agar 20-50 g, trace element solution 1-2 ml, distilled water 1-2 L;

[0043] 4) Heterotrophic nitrification liquid culture medium: ammonium chloride 0.5-2 g, sodium succinate 5-12 g, K2HPO4 0.5-1 g, KH2PO4 1-2 g, MgSO4·7H2O 0.1-0.2 g, FeSO4·7H2O 0.05-0.1 g, pH 7.0-7.8, agar 20-50 g, distilled water 1-2 L;

[0044] 5) Aerobic denitrification liquid culture medium: 0.5-2 g, sodium succinate 5-12 g, K2HPO4 0.5-1 g, KH2PO4 1-2 g, MgSO4·7H2O 0.1-0.2 g, FeSO4·7H2O 0.05-0.1 g, pH 7.0-7.8, trace element solution 1-2 ml, distilled water 1-2 L;

[0045] 6) Trace element solution: EDTA-2Na 40-70 mg / L, ZnSO4·7H2O 2-6 mg / L, CaCl2·2H2O 5-9 mg / L, MnCl2·4H2O 6-8 mg / L, FeSO4·7H2O 6-8 mg / L, CuSO4·5H2O 1-5 mg / L, COCl2 1-5 mg / L;

[0046] Isolation and screening of strains:

[0047] S1. The mud-water mixture sample retrieved from the aerobic tank is stored at 2–5°C. Using a sterile operating table, 1–6 ml of the sample is transferred to an Erlenmeyer flask containing enrichment medium. After thorough mixing, it is incubated for 1–3 days in a constant-temperature shaker at 30–40°C and 120–160 rpm / min. After incubation, 1–6 ml of the bacterial culture is transferred to an Erlenmeyer flask containing enrichment medium, and the above operation is repeated three times.

[0048] S2. The seed solution obtained from the above enrichment was diluted using a gradient dilution method to obtain a 10-1 solution. -1 ~10 -9 Cell dilutions at varying concentrations (dilution steps: add 1 ml of seed culture to 9 ml of sterile water, and so on) were prepared. 0.1 ml of each concentration gradient was evenly spread onto aerobic denitrification medium and incubated at 30–40°C for 2–3 days. After incubation, colonies near which the medium had changed from yellow-green to blue were selected and inoculated onto heterotrophic nitrification medium for multiple three-part streak cultures to improve purity. Finally, the purified strain was inoculated onto heterotrophic nitrification medium and stored at 2–5°C.

[0049] S3. Inoculate 1-3 loops of the obtained single colonies with an inoculation loop and culture them in heterotrophic nitrification liquid medium for 12-20 h to obtain seed culture. Transfer 1 ml of seed culture to heterotrophic nitrification liquid medium and culture it at 30-40℃ and 120-160 rpm / min for 12-20 h. Measure its ammonia nitrogen removal efficiency. Finally, screen strains with an ammonia nitrogen removal efficiency greater than 90% for further screening.

[0050] S4. Transfer the seed culture of the strain with a removal efficiency greater than 90% to a solution containing 50 mg / L Zn. 2+In heterotrophic nitrification medium, the samples were cultured at 30–40℃ and 120–160 rpm / min for 12–20 h to screen for Zn. 2+ The most resistant Pseudomonas pseudomonas sp.SK-4, and then frozen for preservation.

[0051] Example 2: Identification and characteristics of fungal strains

[0052] This bacterium was deposited on January 22, 2024, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the accession number CCTCC NO: M2024163. The morphological and physiological-biochemical characteristics of this heterotrophic nitrifying-aerobic denitrifying bacterium SK-4 include: *Pseudomonas SK-4* colonies are round, milky white overall, slightly raised in the center, and have a viscous surface; under a microscope, they appear as short rods; they stain red with Gram stain, indicating they are Gram-negative. After multiple centrifugations of the 12-hour culture seed culture, gene sequencing was performed. The sequencing results were compared for homology in the GenBank database, showing 99.93% similarity to known strains. A phylogenetic tree was constructed (see attached). Figure 1 The results showed that the strain was a *Pseudomonas hunanense*, and it was named... pseudomonas sp.SK-4.

[0053] Example 3: Environmental factors affecting the heterotrophic nitrification capacity of Pseudomonas SK-4

[0054] Seed culture: Pick two loops of single colonies with an inoculation loop and place them in 100 ml of heterotrophic nitrification liquid medium. Incubate at 30–35 °C and 120–140 rpm / min for 12–14 h, and measure the OD of the bacterial culture. 600 If the value is greater than 1, the seed solution can be used normally.

[0055] (1) Effect of carbon source on heterotrophic nitrification of the strain: Sucrose, glucose, sodium succinate, sodium citrate, and sodium acetate were used as single carbon sources, with a uniform C / N ratio of 10. 1 ml of the cultured seed culture was pipetted into 100 ml of heterotrophic nitrification liquid culture medium with different carbon sources. The bottle mouths were sealed and the cultured in an incubator at 120 rpm / min and 30 ℃. Samples were taken every 4 h to measure the NH4 in the bacterial culture. + The concentration was determined, and the experiment was repeated three times. The results are as follows: Figure 2 As shown.

[0056] (2) Effect of C / N ratio on heterotrophic nitrification of the strain: The C / N ratio of the heterotrophic nitrification liquid medium was adjusted to 4, 6, 8, 10, 12 and 15 respectively. Sodium citrate was used as the single carbon source and the pH was adjusted to 7. 1 ml of the cultured seed solution was taken with a pipette and transferred to 100 ml of heterotrophic nitrification liquid medium with different C / N ratios. The bottle mouth was sealed and the culture was carried out in an incubator at 120 rpm / min and 30 ℃. Samples were taken every 4 h to measure the NH4 in the bacterial solution. + The concentration was determined, and the experiment was repeated three times. The results are as follows: Figure 3 As shown.

[0057] (3) Effect of pH on heterotrophic nitrification of the strain: The pH of the heterotrophic nitrification liquid medium was adjusted to 5, 6, 7, 8, and 9, respectively. Sodium citrate was used as the single carbon source, and the C / N ratio was uniformly 10. 1 ml of the cultured seed solution was pipetted into 100 ml of heterotrophic nitrification liquid medium with different C / N ratios. The bottle mouths were sealed, and the culture was carried out in an incubator at 120 rpm / min and 30℃. Samples were taken every 4 hours to measure the concentration of NH4+ in the bacterial solution. The experiment was repeated three times. The experimental results are shown below. Figure 4 As shown.

[0058] (4) Effect of rotation speed on heterotrophic nitrification of the strain: Sodium citrate was used as the single carbon source, with a uniform C / N ratio of 10 and pH adjusted to 7. 1 ml of the cultured seed culture was taken with a pipette and transferred to 100 ml of heterotrophic nitrification liquid culture medium with different C / N ratios. The bottle mouths were sealed, and the culture temperature was fixed at 30℃. The culture was carried out at 80 rpm / min, 100 rpm / min, 120 rpm / min, 140 rpm / min, and 160 rpm / min, respectively. Samples were taken every 4 hours to measure the NH4+ in the bacterial culture. + The concentration was determined, and the experiment was repeated three times. The results are as follows: Figure 5 As shown.

[0059] (5) Effect of temperature on heterotrophic nitrification of the strain: Sodium citrate was used as the single carbon source, with a uniform C / N ratio of 10 and pH adjusted to 7. 1 ml of the cultured seed culture was taken with a pipette and transferred to 100 ml of heterotrophic nitrification liquid culture medium with different C / N ratios. The bottle mouths were sealed, and the culture temperatures were set at 20℃, 25℃, 30℃, 35℃, and 40℃, respectively. The culture was carried out at 120 rpm / min, and samples were taken every 4 hours to measure the NH4+ in the bacterial culture. + The concentration was determined, and the experiment was repeated three times. The results are as follows: Figure 6 As shown.

[0060] Example 4: Pseudomonas pseudomonas Determination of nitrogen removal performance of sp.SK-4

[0061] Seed culture: Pick two loops of single colonies with an inoculation loop and place them in 100 ml of heterotrophic nitrification liquid medium. Incubate at 30–35 °C and 120–140 rpm / min for 12–14 h, and measure the OD of the bacterial culture. 600 If the value is greater than 1, the seed solution can be used normally.

[0062] Using sodium citrate as the sole carbon source, with a uniform C / N ratio of 10 and pH adjusted to 7, 1 ml of the cultured seed culture was pipetted into 100 ml of heterotrophic nitrification and aerobic denitrification liquid culture media with different C / N ratios. The bottle caps were sealed, and the cultures were incubated at 30°C and 120 rpm / min. Samples were taken every 3 hours to measure the NH4+ in the heterotrophic nitrification and aerobic denitrification bacterial cultures. + and NO3 - The concentration was determined, and the experiment was repeated three times. The results are as follows: Figure 7 As shown.

[0063] Example 5: Pseudomonas pseudomonas sp.SK-4 contains Zn 2+ Application in wastewater

[0064] Take 100 ml of heterotrophic nitrification liquid medium and add Zn at concentrations of 0 mg / L, 5 mg / L, 10 mg / L, 30 mg / L, 50 mg / L, and 80 mg / L, respectively. 2+ Collect 1 ml of seed culture and culture it at 30℃, 120 rpm / min, C / N=10, pH=7. Measure the ammonia nitrogen concentration in the heterotrophic nitrification medium from 0 to 24 h. This bacterium thrives in high concentrations of heavy metal ions (Zn). 2+ Despite the influence of the above, it can still maintain a high removal rate of ammonia nitrogen, and the test results are as follows: Figure 8 As shown.

[0065] This invention includes, but is not limited to, the above embodiments. Any technical substitutions made to some of the contents of the embodiments are still within the technical protection scope of this invention.

Claims

1. A heterotrophic nitrifying aerobic denitrifying Pseudomonas aeruginosa for Zn 2+ Wastewater denitrification method, characterized in that, The method includes the following steps: (1) Cultivation of heterotrophic nitrifying aerobic denitrifying Pseudomonas S1. The mud-water mixture sample retrieved from the aerobic tank is stored at 2-5℃. 1-6 ml of the sample is transferred to an Erlenmeyer flask containing enrichment medium under a sterile operating table. After thorough mixing, it is incubated in a constant temperature shaker at 30-40℃ and 120-160 rpm / min for 1-3 days. After incubation, 1-6 ml of the bacterial culture is transferred to an Erlenmeyer flask containing enrichment medium, and the above operation is repeated three times. S2. The seed solution obtained from the above enrichment was diluted using a gradient dilution method to obtain a 10-1 solution. -1 ~10 -9 Cell dilutions of varying concentrations were prepared by evenly spreading 0.1–0.3 ml of each concentration gradient onto aerobic denitrification medium and incubating at 30–40°C for 2–3 days. After incubation, colonies near which the medium changed from yellow-green to blue were selected and inoculated onto heterotrophic nitrification medium for multiple three-part streak cultures to improve purity. Finally, the purified strains were inoculated onto heterotrophic nitrification medium and stored at 2–5°C. S3. Inoculate 1-3 loops of the single colony obtained from the culture with an inoculation loop and culture in heterotrophic nitrification liquid medium for 12-20 h to obtain seed liquid. Transfer 1 ml of seed liquid into heterotrophic nitrification liquid medium and culture at 30-40℃ and 120-160 rpm / min for 12-20 h. Measure its ammonia nitrogen removal efficiency. Finally, screen strains with an ammonia nitrogen removal efficiency greater than 90% for further screening. S4. Transfer the seed culture of the strain with a removal efficiency greater than 90% to a solution containing 50-80 mg / L Zn. 2+ In heterotrophic nitrification medium, the samples were cultured at 30–40℃ and 120–160 rpm / min for 12–20 h to screen for Zn. 2+ The most resistant Pseudomonas pseudomonas sp. SK-4, and then frozen for preservation; the Pseudomonas pseudomonas sp.SK-4 is deposited at the China Center for Type Culture Collection (CCTCC), with the strain accession number CCTCC NO: M2024163; The above-mentioned Pseudomonas aeruginosa cultured pseudomonas sp.SK-4 contains Zn 2+ Application of denitrification in wastewater Pseudomonas pseudomonas sp.SK-4 contains Zn 2+ For denitrification of wastewater, the above-mentioned heterotrophic nitrification liquid culture medium was used to add Zn at concentrations of 0 mg / L, 5 mg / L, 10 mg / L, 30 mg / L, 50 mg / L, and 80 mg / L, respectively. 2+ Collect 1%–3% of the strain's seed culture and measure the ammonia nitrogen concentration in the heterotrophic nitrification medium over 0–24 hours. This bacterium thrives in high concentrations of heavy metal ions (Zn). 2+ Despite the influence of heavy metals, it can still maintain a high removal rate of ammonia nitrogen, providing an option for biological denitrification of wastewater under heavy metal stress.

2. The heterotrophic nitrifying aerobic denitrifying Pseudomonas aeruginosa according to claim 1 for Zn 2+ Wastewater denitrification method, characterized in that, The Pseudomonas pseudomonas sp. SK-4's morphological and physiological / biochemical characteristics include: Pseudomonas aeruginosa pseudomonas sp. SK-4 colonies are round, milky white overall, slightly raised in the center, and have a viscous surface; under a microscope, they appear as short rods; they stain red with Gram stain, indicating they are Gram-negative bacteria. After multiple centrifugations of the 12-hour culture seed culture, the bacteria were sent to a biotechnology company for gene sequencing. The sequencing results were compared for homology with known strains in the GenBank database, showing a 99.93% similarity. A phylogenetic tree was constructed, revealing that this strain is a *Pseudomonas hunanense*, and it was named *Pseudomonas hunanense*. pseudomonas sp.SK-4.

3. The heterotrophic nitrifying aerobic denitrifying Pseudomonas aeruginosa according to claim 1 for Zn 2+ Wastewater denitrification method, characterized in that, The enrichment culture medium consists of: 0.2–0.6 g ammonium chloride, 4–8 g sodium succinate, 30–50 ml Vickers salt solution, diluted to 1–1.5 L. The Vickers salt solution contains: 1.5–2 g dipotassium hydrogen phosphate, 0.2–0.5 g magnesium sulfate, 2–5 g sodium chloride, 0.05–0.1 g ferrous sulfate, 0.05–0.1 g manganese sulfate, diluted to 1–2 L, with a pH of 7.0–8.

0.

4. The heterotrophic nitrifying aerobic denitrifying Pseudomonas aeruginosa according to claim 1 for Zn 2+ Wastewater denitrification method, characterized in that, 1) The aerobic denitrification culture medium comprises: NaNO3 0.5-2 g, sodium succinate 5-12 g, K2HPO4 0.5-1 g, KH2PO4 1-2 g, MgSO4·7H2O 0.1-0.2 g, FeSO4·7H2O 0.05-0.1 g, 1% bromothymol blue ethanol solution (BTB) 1-2 ml, pH 7.0-7.8, agar 20-50 g, trace element solution 1-2 ml, and distilled water 1-2 L; 2) The heteroaerobic nitrification medium consists of: ammonium chloride 0.5–2 g, sodium succinate 5–12 g, K₂HPO₄ 0.5–1 g, KH₂PO₄ 1–2 g, MgSO₄·7H₂O 0.1–0.2 g, FeSO₄·7H₂O 0.05–0.1 g, pH 7.0–7.8, agar 20–50 g, and distilled water 1–2 L; 3) The trace element solution: EDTA-2Na 40-70 mg / L, ZnSO4·7H2O 2-6 mg / L, CaCl2·2H2O 5-9 mg / L, MnCl2·4H2O 6-8 mg / L, FeSO4·7H2O 6-8 mg / L, CuSO4·5H2O 1-5 mg / L, COCl2 1-5 mg / L.

5. The heterotrophic nitrifying aerobic denitrifying Pseudomonas aeruginosa according to claim 1 for Zn 2+ Wastewater denitrification method, characterized in that, The heterotrophic nitrification liquid culture medium consists of: 0.5–2 g ammonium chloride, 5–12 g sodium succinate, 0.5–1 g K₂HPO₄, 1–2 g KH₂PO₄, 0.1–0.2 g MgSO₄·7H₂O, 0.05–0.1 g FeSO₄·7H₂O, pH 7.0–7.8, and 1–2 L distilled water.

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