A method for treating high-salt and high-ammonia-nitrogen wastewater by using a heterotrophic nitrification and aerobic denitrification bacterium

Abstract

The application discloses a heterotrophic nitrification and aerobic denitrification bacteria for treating high-salt and high-ammonia-nitrogen wastewater, and relates to a high-salt and high-ammonia-nitrogen wastewater treatment method. Halomonas sp The bacteria strain is Halomonas sp. Halomonas , named MY-11, belongs to the genus Halomonas, is preserved in the China Center for Type Culture Collection, has a preservation number of CCTCC M 20242923 and a preservation date of December 27, 2024. The bacteria strain is a heterotrophic nitrification and aerobic denitrification bacteria, can efficiently remove inorganic nitrogen pollution in water bodies, has good denitrification performance under high-salt and high-ammonia-nitrogen stress, and has high tolerance to high-salt and high-ammonia-nitrogen. The bacteria strain can efficiently treat nitrogen-containing wastewater under the conditions of 2%-6% salinity and 100 mg / L-400 mg / L ammonia-nitrogen concentration, and the removal efficiency can be higher than 99%. The bacteria strain has great potential in the application of inorganic nitrogen pollution treatment of various water bodies such as breeding and chemical industry wastewater.

Description

Technical Field

[0001] This invention relates to a method for treating high-salt, high-ammonia-nitrogen wastewater, and more particularly to a method for treating high-salt, high-ammonia-nitrogen wastewater using heterotrophic nitrifying aerobic denitrifying bacteria. Background Technology

[0002] The HNAD strain can perform both heterotrophic nitrification and aerobic denitrification, allowing nitrification and denitrification to occur simultaneously. Furthermore, the strain exhibits advantages such as rapid growth rate and high nitrogen removal efficiency.

[0003] Currently, many researchers have conducted extensive studies on the nitrogen removal characteristics of HNAD strains, but their nitrogen metabolism pathways vary due to differences in strain species. Furthermore, the HNAD denitrifying strains isolated so far mainly originate from sludge or freshwater environments, making them difficult to effectively treat saline wastewater discharged from industries such as food processing, textile dyeing, pharmaceuticals, leather tanning, and oil refining. This is because high salt content in wastewater typically causes dehydration of microbial cells, disrupting the microbial treatment system. This study screens heterotrophic nitrifying aerobic denitrifying strains with high nitrogen removal efficiency in high-salt environments, providing a reference for the biological treatment of high-salt wastewater. Summary of the Invention

[0004] The purpose of this invention is to provide a method for treating high-salt, high-ammonia nitrogen wastewater using heterotrophic nitrifying-aerobic denitrifying bacteria. The salt-tolerant and high-ammonia nitrogen-tolerant heterotrophic nitrifying-aerobic denitrifying bacteria used in this method are *Haloxylon ammodendron*. Halomonas sp. MY11 exhibits excellent heterotrophic nitrification and aerobic denitrification capabilities under extreme conditions of high salinity and high ammonia nitrogen. It can utilize ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen as nitrogen sources, achieving efficient nitrogen removal regardless of whether a single or mixed nitrogen source is used. It also demonstrates strong tolerance to high salinity and high ammonia nitrogen levels. This strain can efficiently treat nitrogen-containing wastewater under conditions of 2%-6% salinity and ammonia nitrogen concentrations of 100 mg / L-400 mg / L, achieving a removal efficiency of over 99%. It shows great potential for application in the treatment of inorganic nitrogen pollution in various water bodies, including aquaculture and chemical wastewater, in my country.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A method for treating high-salt, high-ammonia nitrogen wastewater using heterotrophic nitrifying aerobic denitrifying bacteria, the method comprising the following steps:

[0007] (1) Enrichment, isolation and screening of heterotrophic nitrifying aerobic denitrifying halometa.

[0008] 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 solution is transferred to an Erlenmeyer flask containing enrichment medium, and the above operation is repeated three times.

[0009] 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 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 a heterotrophic nitrification medium and stored at 2–5°C.

[0010] S3. Inoculate 1-3 loops of the obtained single colonies with an inoculation loop and culture them in a heterotrophic nitrification liquid medium with a salinity of 4% for 2-20 h to obtain seed liquid. Transfer 1 mL of seed liquid into a heterotrophic nitrification liquid medium and culture it at 30-40℃ and 120-160 rpm / min for 12-48 h. Measure its ammonia nitrogen removal efficiency and finally screen strains with an ammonia nitrogen removal efficiency greater than 90% for further screening.

[0011] S4. Transfer the seed culture of the strains with a removal efficiency greater than 90% to an aerobic denitrification medium with a salinity of 4% and culture it at 30-40℃ and 120-160rpm / min for 12-48h. Measure its removal efficiency of nitrate nitrogen and finally screen out strains with a nitrate nitrogen removal efficiency greater than 90% for further screening.

[0012] S5. Based on the above analysis of the removal efficiency of ammonia nitrogen and nitrate nitrogen under high salinity conditions, the Halomonas bacteria with the strongest tolerance to high salinity conditions were identified. Halomonas sp MY-11, and freeze it;

[0013] (2) Denitrification by Halomonas in high-salinity wastewater

[0014] The denitrification of wastewater with a salinity of 4% by this bacterium was demonstrated by taking the above heterotrophic nitrification liquid culture medium and adding 0%, 2%, 4%, 6%, 8%, 10%, and 12% NaCl, KCl, Na2SO4, and K2SO4 respectively. 1% to 3% of the bacterial seed culture was then collected, and the concentrations of ammonia nitrogen and nitrate nitrogen in the heterotrophic nitrification medium and the aerobic denitrification medium were measured from 0 to 48 hours. The bacterium maintained the removal of ammonia nitrogen and nitrate nitrogen under the influence of high salinity.

[0015] The method for treating high-salt, high-ammonia nitrogen wastewater using heterotrophic nitrifying aerobic denitrifying bacteria comprises the following enrichment culture medium: 0.2-0.6g ammonium chloride, 4-8g sodium succinate, 30-50mL Vickers salt solution, 30-40g sodium chloride, diluted to 1-1.5L. The Vickers salt solution consists of: 1.5-2g dipotassium hydrogen phosphate, 0.2-0.5g magnesium sulfate, 2-5g sodium chloride, 0.05-0.1g ferrous sulfate, 0.05-0.1g manganese sulfate, diluted to 1-2L, with a pH of 7.0-8.0.

[0016] The method for treating high-salt, high-ammonia nitrogen wastewater using heterotrophic nitrifying aerobic denitrifying bacteria includes the following composition of the culture medium and trace elements:

[0017] 1) Composition of aerobic denitrification solid culture medium: NaNO3 0.5-3g, sodium succinate 5-40g, NaCl 30-40g, K2HPO4 0.5-1g, KH2PO4 1-2g, MgSO4·7H2O 0.1-0.2g, FeSO4·7H2O 0.05-0.1g, 1% bromothymol blue ethanol solution (BTB) 1-2mL, pH 7.0-7.8, agar 20-50g, trace element solution 1-2mL, distilled water 1-2L;

[0018] 2) Composition of aerobic denitrifying culture medium: NaNO3 0.5-3g, sodium succinate 5-40g, NaCl 30-40g, K2HPO4 0.5-1g, KH2PO4 1-2g, MgSO4·7H2O 0.1-0.2g, FeSO4·7H2O 0.05-0.1g, 1% bromothymol blue ethanol solution (BTB) 1-2mL, pH 7.0-7.8, trace element solution 1-2mL, distilled water 1-2L;

[0019] 3) Solid composition of heteroaerobic nitrification medium: ammonium chloride 0.5-2g, sodium succinate 5-40g, NaCl 30-40g, K2HPO 0.5-1g, KH2PO4 1-2g, MgSO·7H2O 0.1-0.2g, FeSO4·7H2O 0.05-0.1g, pH 7.0-7.8, agar 20-50g, distilled water 1-2L;

[0020] 4) Composition of heteroaerobic nitrification medium: ammonium chloride 0.5-2g, sodium succinate 5-40g, NaCl 30-40g, K2HPO 0.5-1g, KH2PO4 1-2g, MgSO4·7H2O 0.1-0.2g, FeSO4·7H2O 0.05-0.1g, pH 7.0-7.8, distilled water 1-2L;

[0021] 5) Trace element solution: EDTA-2Na 40~70mg / L, ZnSO·7H2O 2~6mg / L, CaCl2·2H2O 5~9mg / L, MnCl2·4H2O 6~8mg / L, FeSO·7H2O 6~8mg / L, CuSO·5H2O 1~5mg / L, COCl 1~5mg / L.

[0022] The method for treating high-salt, high-ammonia nitrogen wastewater using heterotrophic nitrifying aerobic denitrifying bacteria, wherein the heterotrophic nitrifying aerobic denitrifying bacteria are classified and named as Halomonas (Halomonas). Halomonas sp. The name is *Haloxylon ammodendron* MY11, belonging to the genus *Haloxylon ammodendron*. Halomonas (), deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, accession number CCTCC M 20242923, deposit date December 27, 2024.

[0023] The method for treating high-salt, high-ammonia nitrogen wastewater using heterotrophic nitrifying aerobic denitrifying bacteria involves the heterotrophic nitrifying aerobic denitrifying halomonas denitrifying bacteria removing nitrogen from ammonia nitrogen, nitrate nitrogen, nitrite nitrogen as the sole nitrogen source, and mixed nitrogen sources.

[0024] The method for treating high-salt, high-ammonia nitrogen wastewater using heterotrophic nitrifying aerobic denitrifying bacteria has the following optimal culture conditions: sodium succinate as the carbon source, C / N ratio of 10-14, temperature of 30℃-34℃, rotation speed of 160rpm / min-220rpm / min, salinity of 2%-6%, and pH of 8-10.

[0025] A method for treating high-salt, high-ammonia nitrogen wastewater using heterotrophic nitrifying aerobic denitrifying bacteria, wherein the heterotrophic nitrifying aerobic denitrifying halomonas bacteria denitrify in high-salt wastewater: take the above-mentioned heterotrophic nitrification liquid culture medium, and add 4%, 6%, 8%, 10%, and 12% of NaCl, KCl, Na2SO4, and K2SO4 respectively, and collect 1%-3% of the strain seed liquid. Measure the concentrations of ammonia nitrogen and nitrate nitrogen in the heterotrophic nitrification medium and the aerobic denitrification medium over 0-48 hours, and maintain the removal of ammonia nitrogen and nitrate nitrogen under the influence of high salt.

[0026] The advantages and effects of this invention are:

[0027] 1. The strain of this invention, *Haloxylon ammodendron* Halomonas sp MY-11 stains red with Gram, indicating it is a Gram-negative bacterium. (Strain) Halomonas sp The MY-11 strain exhibits the best denitrification performance when sodium citrate is used as the carbon source, C / N=14, pH=7-10, temperature 34℃, and rotation speed 220rpm / min, achieving a removal rate of over 99% for ammonia nitrogen within 30 hours.

[0028] 2. The strain of this invention Halomonas sp MY-11 showed good removal effects in wastewater of different salinity levels ranging from 4% to 12%, with a removal efficiency of over 99%.

[0029] 3. The strain of this invention Halomonas sp MY-11 possesses the ability to simultaneously heterotrophic nitrification and aerobic denitrification, and is characterized by rapid growth and reproduction rates and high nitrogen removal efficiency. Its logarithmic growth phase is 4-16 hours, and its ammonia nitrogen removal rate in wastewater reaches 100% within 28 hours. This demonstrates that the bacteria can be applied immediately and efficiently to achieve biological nitrogen removal. Attached Figure Description

[0030] Figure 1 Photograph of the culture medium for Halomonas MY11;

[0031] Figure 2 A microscopic photograph of Halomonas MY11 after Gram staining;

[0032] Figure 3 Phylogenetic tree of Halomonas MY11;

[0033] Figure 4 The nitrogen removal efficiency of Halomonas MY11 under different carbon sources (sucrose, glucose, sodium succinate, sodium citrate, sodium acetate) on different nitrogen sources (ammonia nitrogen, nitrate nitrogen);

[0034] Figure 5 The nitrogen removal efficiency of Halomonas MY11 for different nitrogen sources (ammonia nitrogen and nitrate nitrogen) under different C / N ratios (2, 6, 10, 14, 18, 22) is shown in the figure.

[0035] Figure 6 The nitrogen removal efficiency of Halomonas MY11 at different pH levels (5, 6, 7, 8, 9, 10) for different nitrogen sources (ammonia nitrogen and nitrate nitrogen) is shown in the graph.

[0036] Figure 7 The nitrogen removal effect of Halomonas MY11 on different nitrogen sources (ammonia nitrogen and nitrate nitrogen) at different inoculum amounts (1%, 2%, 3%, 4%, 5%) is shown in the figure.

[0037] Figure 8The nitrogen removal efficiency of Halomonas MY11 on different nitrogen sources (ammonia nitrogen and nitrate nitrogen) at different temperatures (24℃, 26℃, 28℃, 30℃, 32℃, 34℃, 36℃, 38℃, 40℃) is shown in the graph.

[0038] Figure 9 The nitrogen removal efficiency of Halomonas MY11 on different nitrogen sources (ammonia nitrogen and nitrate nitrogen) at different rotation speeds (100 rpm / min, 140 rpm / min, 180 rpm / min, 220 rpm / min, 260 rpm / min) is shown in the figure.

[0039] Figure 10 The nitrogen removal efficiency of Halomonas MY11 on different nitrogen sources (ammonia nitrogen and nitrate nitrogen) under different NaCl salinities (0%, 2%, 4%, 6%, 8%, 10%, 12%) is shown in the figure.

[0040] Figure 11 The graph shows the time-varying denitrification characteristics of Halomonas MY11 under different nitrogen source conditions (ammonia nitrogen, nitrate nitrogen, and mixed nitrogen source).

[0041] Figure 12 The nitrogen removal effect of Halomonas MY11 on different nitrogen sources (ammonia nitrogen and nitrate nitrogen) under different salinity KCl (0%, 2%, 4%, 6%, 8%, 10%, 12%) is shown in the figure.

[0042] Figure 13 The nitrogen removal efficiency of Halomonas MY11 on different nitrogen sources (ammonia nitrogen and nitrate nitrogen) under different salinities of Na2SO4 (0%, 2%, 4%, 6%, 8%, 10%, 12%) is shown in the figure.

[0043] Figure 14 The graph shows the nitrogen removal efficiency of Halomonas MY11 on different nitrogen sources (ammonia nitrogen and nitrate nitrogen) under different salinity K2SO4 conditions (0%, 2%, 4%, 6%, 8%, 10%, 12%). Detailed Implementation

[0044] The present invention will be further explained and illustrated below with reference to the embodiments. Example 1

[0045] The enrichment, isolation, screening, and purification of the strains were performed using the following culture medium formulation:

[0046] 1) The components of the aerobic denitrification solid culture medium include: NaNO3 0.5-3g, sodium succinate 5-40g, NaCl 30-40g, K2HPO4 0.5-1g, KH2PO4 1-2g, MgSO4·7H2O 0.1-0.2g, FeSO4·7H2O 0.05-0.1g, 1% bromothymol blue ethanol solution (BTB) 1-2mL, pH 7.0-7.8, agar 20-50g, trace element solution 1-2mL, and distilled water 1-2L;

[0047] 2) The aerobic denitrifying culture medium consists of: NaNO3 0.5-3g, sodium succinate 5-40g, NaCl 30-40g, K2HPO4 0.5-1g, KH2PO4 1-2g, MgSO4·7H2O 0.1-0.2g, FeSO4·7H2O 0.05-0.1g, 1% bromothymol blue ethanol solution (BTB) 1-2mL, pH 7.0-7.8, trace element solution 1-2mL, and distilled water 1-2L;

[0048] 3) The solid components of the heteroaerobic nitrification medium include: ammonium chloride 0.5-2g, sodium succinate 5-40g, NaCl 30-40g, K2HPO 0.5-1g, KH2PO4 1-2g, MgSO·7H2O 0.1-0.2g, FeSO4·7H2O 0.05-0.1g, pH 7.0-7.8, agar 20-50g, and distilled water 1-2L;

[0049] 4) The heteroaerobic nitrification medium consists of: ammonium chloride 0.5-2g, sodium succinate 5-40g, NaCl 30-40g, K2HPO 0.5-1g, KH2PO4 1-2g, MgSO·7H2O 0.1-0.2g, FeSO4·7H2O 0.05-0.1g, pH 7.0-7.8, and distilled water 1-2L;

[0050] 5) Trace element solution: EDTA-2Na 40~70mg / L, ZnSO·7H2O 2~6mg / L, CaCl2·2H2O 5~9mg / L, MnCl2·4H2O 6~8mg / L, FeSO·7H2O 6~8mg / L, CuSO·5H2O 1~5mg / L, COCl 1~5mg / L.

[0051] Enrichment, isolation and screening of strains

[0052] 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 solution is transferred to an Erlenmeyer flask containing enrichment medium, and the above operation is repeated three times.

[0053] 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.

[0054] S3. Inoculate 1-3 loops of the obtained single colonies with an inoculation loop and culture them in a heterotrophic nitrification liquid medium with a salinity of 4% for 2-20 h to obtain seed liquid. Transfer 1 mL of seed liquid into a heterotrophic nitrification liquid medium and culture it at 30-40℃ and 120-160 rpm / min for 12-48 h. Measure its ammonia nitrogen removal efficiency and finally screen strains with an ammonia nitrogen removal efficiency greater than 90% for further screening.

[0055] S4. Transfer the seed culture of the strains with a removal efficiency greater than 90% to an aerobic denitrification medium with a salinity of 4% and culture it at 30-40℃ and 120-160rpm / min for 12-48h. Measure its removal efficiency of nitrate nitrogen and finally screen out strains with a nitrate nitrogen removal efficiency greater than 90% for further screening.

[0056] S5. Based on the above analysis of the removal efficiency of ammonia nitrogen and nitrate nitrogen under high salinity conditions, the Halomonas bacteria with the strongest tolerance to high salinity conditions were identified. Halomonas sp MY-11, and freeze it; Example 2

[0057] Identification and characteristics of the strain: The morphological and physiological-biochemical characteristics of the heterotrophic nitrifying aerobic denitrifying bacterium MY-11 include: spherical colonies, milky white overall, with a viscous surface, such as... Figure 1 As shown; Gram staining is red, indicating Gram-negative bacteria, such as... Figure 2As shown. After multiple centrifugations of the bacterial seed culture cultured for 18 hours, gene sequencing was performed. The sequencing results were compared for homology with the GenBank database, showing a 99.98% similarity to known strains. A phylogenetic tree was then constructed, as shown. Figure 3 As shown, the results indicate that this strain is a Halomonas bacterium, and it is named... Halomonas sp MY-11.

[0058] Example 3:

[0059] Affecting Halomonas Halomonas sp Environmental factors affecting the heterotrophic nitrification capacity of MY-11

[0060] (1) Carbon source: Application of heterotrophic nitrifying aerobic denitrifying bacteria MY11 in the treatment of high-salt and high-ammonia nitrogen wastewater. The effects of different carbon sources (sucrose, glucose, sodium succinate, sodium citrate, sodium acetate) on the denitrification performance of this strain were investigated.

[0061] The heterotrophic nitrifying aerobic denitrifying bacteria obtained in Example 1 Halomonas sp MY-11 was activated and cultured in nitrifying medium at 32℃ and 160 rpm / min to obtain the bacterial OD. 600 The value was 1.0. The bacterial culture was inoculated at a rate of 1% (v / v) into different nitrogen source media (g / L) under the following conditions: C / N = 10, pH = 7, 32℃, 160 rpm / min, inoculum size: 1%, salinity (NaCl): 4%. After 60 h of culture, the concentrations of ammonia nitrogen and nitrate nitrogen were measured, and the results are as follows: Figure 4 As shown.

[0062] (2) C / N: Application of heterotrophic nitrifying aerobic denitrifying bacteria MY11 in the treatment of high-salt and high-ammonia nitrogen wastewater. The effects of different C / N ratios (2, 6, 10, 14, 18, 22) on the denitrification performance of this strain were investigated.

[0063] The heterotrophic nitrifying aerobic denitrifying bacteria obtained in Example 1 Halomonas sp MY-11 was activated and cultured in nitrifying medium at 32℃ and 160 rpm / min to obtain the bacterial OD. 600 The value was 1.0. The bacterial culture was inoculated at a rate of 1% (v / v) into different C / N media (g / L) under the following conditions: sodium succinate, pH=7, 32℃, 160 rpm / min, and salinity (NaCl): 4%. After 60 h of culture, the concentrations of ammonia nitrogen and nitrate nitrogen were measured, and the results are as follows: Figure 5 As shown.

[0064] (3) pH: Application of heterotrophic nitrifying aerobic denitrifying bacteria MY11 in the treatment of high-salt and high-ammonia nitrogen wastewater. The effect of different pH values ​​(5, 6, 7, 8, 9, 10) on the denitrification performance of this strain was investigated.

[0065] The heterotrophic nitrifying aerobic denitrifying bacteria obtained in Example 1 Halomonas sp MY-11 was activated and cultured in nitrifying medium at 32℃ and 160 rpm / min to obtain the bacterial OD. 600 The value was 1.0. The bacterial culture was inoculated at a 1% (v / v) inoculum into different pH culture media (g / L) under the following conditions: sodium succinate, C / N = 14, 32℃, 160 rpm / min, inoculum size: 1%, salinity (NaCl): 4%. After 60 h of culture, the concentrations of ammonia nitrogen and nitrate nitrogen were measured, and the results are as follows: Figure 6 As shown.

[0066] (4) Inoculum size: Application of heterotrophic nitrifying aerobic denitrifying bacteria MY11 in the treatment of high-salt and high-ammonia nitrogen wastewater. The effects of different inoculum sizes (1%, 2%, 3%, 4%, 5%) on the denitrification performance of this strain were investigated.

[0067] The heterotrophic nitrifying aerobic denitrifying bacteria obtained in Example 1 Halomonas sp MY-11 was activated and cultured in nitrifying medium at 32℃ and 160 rpm / min to obtain the bacterial OD. 600 The value was 1.0. The bacterial culture was inoculated at a rate of 1% (v / v) into culture media (g / L) at different temperatures under the following conditions: sodium succinate, C / N = 14, pH = 7, 160 rpm / min, and salinity (NaCl): 4%. After 60 h of incubation, the concentrations of ammonia nitrogen and nitrate nitrogen were measured, and the results are as follows: Figure 7 As shown.

[0068] (4) Temperature: Application of heterotrophic nitrifying aerobic denitrifying bacteria MY11 in the treatment of high-salt and high-ammonia nitrogen wastewater. The effects of different temperatures (24℃, 26℃, 28℃, 30℃, 32℃, 34℃, 36℃, 38℃, 40℃) on the denitrification performance of the strain were investigated.

[0069] The heterotrophic nitrifying aerobic denitrifying bacteria obtained in Example 1 Halomonas sp MY-11 was activated and cultured in nitrifying medium at 32℃ and 160 rpm / min to obtain the bacterial OD. 600The value was 1.0. The bacterial culture was inoculated at a 1% (v / v) inoculum into culture media (g / L) at different temperatures under the following conditions: sodium succinate, C / N = 14, pH = 7, 160 rpm / min, inoculum size: 1%, salinity (NaCl): 4%. After 60 h of culture, the concentrations of ammonia nitrogen and nitrate nitrogen were measured, and the results are as follows: Figure 8 As shown.

[0070] (5) Rotation speed: Application of heterotrophic nitrifying aerobic denitrifying bacteria MY11 in the treatment of high-salt and high-ammonia nitrogen wastewater. The effects of different rotation speeds (100 rpm / min, 140 rpm / min, 180 rpm / min, 220 rpm / min, 260 rpm / min) on the denitrification performance of the strain were investigated.

[0071] The heterotrophic nitrifying aerobic denitrifying bacteria obtained in Example 1 Halomonas sp MY-11 was activated and cultured in nitrifying medium at 34℃ and 160 rpm / min to obtain the bacterial OD. 600 The value was 1.0. The bacterial culture was inoculated at a rate of 1% (v / v) into culture media (g / L) at different rotation speeds under the following conditions: sodium succinate, C / N = 14, pH = 7, 34℃, inoculum size: 1%, salinity (NaCl): 4%. After 60 h of culture, the concentrations of ammonia nitrogen and nitrate nitrogen were measured, and the results are as follows: Figure 9 As shown.

[0072] (6) Salinity: Application of heterotrophic nitrifying aerobic denitrifying bacteria MY11 in the treatment of high-salt and high-ammonia nitrogen wastewater. The effects of different NaCl salinities (0%, 2%, 4%, 6%, 8%, 10%, 12%) on the denitrification performance of this strain were investigated.

[0073] The heterotrophic nitrifying aerobic denitrifying bacteria obtained in Example 1 Halomonas sp MY-11 was activated and cultured in nitrifying medium at 34℃ and 180 rpm / min to obtain the bacterial OD. 600 The inoculum was 1.0. The bacterial culture was inoculated at a rate of 1% (v / v) into culture media (g / L) at different rotation speeds under the following conditions: sodium succinate, C / N = 14, pH = 7, 34℃, 220 rpm / min, and inoculum size: 1%. After 60 h of culture, the concentrations of ammonia nitrogen and nitrate nitrogen were measured, and the results are as follows: Figure 10 As shown.

[0074] Example 4:

[0075] Halomonas Halomonas sp The denitrification characteristics of MY-11 under different nitrogen sources and its application in treating high-salt, high-ammonia nitrogen wastewater were investigated. The time-varying denitrification characteristics of this strain under different nitrogen source conditions were studied.

[0076] The Halomonas obtained in Example 1 Halomonas sp MY-11 was activated and cultured in nitrifying medium at 34℃ and 180 rpm / min for 60 h to obtain the bacterial OD. 600 The value was 1.0. Bacterial suspensions were inoculated at a 1% (v / v) inoculum into ammonia nitrogen, nitrate nitrogen, and mixed nitrogen source media, respectively. The initial ammonia nitrogen content in each single nitrogen source was 400 mg / L, and the nitrate nitrogen content was 300 mg / L. In the mixed nitrogen source media, the concentrations of ammonia nitrogen + nitrate nitrogen, ammonia nitrogen + nitrite nitrogen, and nitrate nitrogen + nitrite nitrogen were all 200 mg / L. In the ammonia nitrogen + nitrate nitrogen + nitrite nitrogen medium, the concentrations of ammonia nitrogen were 200 mg / L, nitrate nitrogen 100 mg / L, and nitrite nitrogen mg / L. Ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, and total nitrogen were measured every 6 hours. The results are as follows: Figure 11 As shown.

[0077] Example 5:

[0078] Halomonas Halomonas sp Nitrogen removal efficiency of MY-11 in different types and concentrations of salt

[0079] (1) KCl: The effects of different salinity KCl (0%, 2%, 4%, 6%, 8%, 10%, 12%) on the denitrification performance of the strain were investigated.

[0080] The heterotrophic nitrifying aerobic denitrifying bacteria obtained in Example 1 Halomonas sp MY-11 was activated and cultured in nitrifying medium at 34℃ and 180 rpm / min to obtain the bacterial OD. 600 The value was 1.0. The bacterial culture was inoculated at a rate of 1% (v / v) into culture media (g / L) at different rotation speeds under the following conditions: sodium succinate, C / N = 14, pH = 7, 34℃, and 220 rpm / min. After 60 h of incubation, the concentrations of ammonia nitrogen and nitrate nitrogen were measured, and the results are as follows: Figure 12 As shown.

[0081] (2) Na2SO4: The effects of different salinities of Na2SO4 (0%, 2%, 4%, 6%, 8%, 10%, 12%) on the denitrification performance of the strain were investigated.

[0082] The heterotrophic nitrifying aerobic denitrifying bacteria obtained in Example 1 Halomonas sp MY-11 was activated and cultured in nitrifying medium at 34℃ and 180 rpm / min to obtain the bacterial OD. 600The value was 1.0. The bacterial culture was inoculated at a rate of 1% (v / v) into culture media (g / L) at different rotation speeds under the following conditions: sodium succinate, C / N = 14, pH = 7, 34℃, and 220 rpm / min. After 60 h of incubation, the concentrations of ammonia nitrogen and nitrate nitrogen were measured, and the results are as follows: Figure 13 As shown.

[0083] (3) K2SO4: The effects of different salinities of K2SO4 (0%, 2%, 4%, 6%, 8%, 10%, 12%) on the denitrification performance of the strain were investigated.

[0084] The heterotrophic nitrifying aerobic denitrifying bacteria obtained in Example 1 Halomonas sp MY-11 was activated and cultured in nitrifying medium at 34℃ and 180 rpm / min to obtain the bacterial OD. 600 The value was 1.0. The bacterial culture was inoculated at a rate of 1% (v / v) into culture media (g / L) at different rotation speeds under the following conditions: sodium succinate, C / N = 14, pH = 7, 34℃, and 220 rpm / min. After 60 h of incubation, the concentrations of ammonia nitrogen and nitrate nitrogen were measured, and the results are as follows: Figure 14 As shown.

Claims

1. A method for treating high-salt, high-ammonia nitrogen wastewater using heterotrophic nitrifying aerobic denitrifying bacteria, characterized in that, The method includes the following steps: (1) Enrichment, isolation and screening of heterotrophic nitrifying aerobic denitrifying halometa. 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 for 1-3 days. After incubation, 1-6 mL of the bacterial solution 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 with concentration gradients: Dilution steps: Add 1 mL of seed culture to 9 mL of sterile water, and so on. Take 0.1–0.3 mL of each concentration gradient of bacterial culture and spread it evenly on aerobic denitrification solid medium. Incubate at 30–40°C for 2–3 days. After incubation, select colonies whose culture medium changes from yellow-green to blue and inoculate them onto heterotrophic nitrification solid medium for multiple three-zone streak cultures to improve their purity. Finally, inoculate the purified strains onto heterotrophic nitrification solid medium and store at 2–5°C. S3. Inoculate 1-3 loops of the obtained single colonies with an inoculation loop and culture them in a heterotrophic nitrification liquid medium with a salinity of 4% for 2-20 h to obtain seed liquid. Transfer 1 mL of seed liquid into a heterotrophic nitrification liquid medium and culture it at 30-40℃ and 120-160 rpm for 12-48 h. Measure its ammonia nitrogen removal efficiency and finally screen strains with an ammonia nitrogen removal efficiency greater than 90% for further screening. S4. Transfer the seed culture of the strains with ammonia nitrogen removal efficiency greater than 90% to aerobic denitrification liquid medium with a salinity of 4% and culture it at 30-40℃ and 120-160rpm for 12-48h. Measure its nitrate nitrogen removal efficiency and finally screen out strains with nitrate nitrogen removal efficiency greater than 90% for further screening. S5. Considering the combined removal efficiency of ammonia nitrogen and nitrate nitrogen under high salinity conditions, the Halomonas bacteria with the strongest tolerance to high salinity conditions were identified. Halomonas sp MY-11, and stored frozen; belongs to the genus *Halomonas* (…). Halomonas (), deposited at the China Center for Type Culture Collection, accession number CCTCC M 20242923; (2) Denitrification by Halomonas in high-salinity wastewater Take the above heterotrophic nitrification liquid culture medium and aerobic denitrification liquid culture medium respectively, and add 0%, 2%, 4%, 6%, 8%, 10%, and 12% of NaCl, KCl, Na2SO4, and K2SO4 respectively. Collect 1% to 3% of the bacterial seed liquid and measure the concentration of ammonia nitrogen and nitrate nitrogen in the heterotrophic nitrification liquid culture medium and aerobic denitrification liquid culture medium from 0 to 48 h. The bacteria maintain the removal of ammonia nitrogen and nitrate nitrogen under the influence of high salt.

2. The method for treating high-salt, high-ammonia nitrogen wastewater using heterotrophic nitrifying aerobic denitrifying bacteria according to claim 1, 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, 30–40 g sodium chloride, and a final volume of 1–1.5 L. The Vickers salt solution consists of: 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, and 0.05–0.1 g manganese sulfate, and a final volume of 1–2 L. The pH is 7.0–8.

0.

3. The method for treating high-salt, high-ammonia nitrogen wastewater using heterotrophic nitrifying aerobic denitrifying bacteria according to claim 1, characterized in that, The culture medium and trace elements consist of: 1) The aerobic denitrification solid culture medium consists of: NaNO3 0.5-3g, sodium succinate 5-40g, NaCl 30-40g, K2HPO4 0.5-1g, KH2PO4 1-2g, MgSO4·7H2O 0.1-0.2g, FeSO4·7H2O 0.05-0.1g, 1% bromothymol blue ethanol solution 1-2mL, pH 7.0-7.8, agar 20-50g, trace element solution 1-2mL, and distilled water 1-2L; 2) The aerobic denitrification liquid culture medium consists of: NaNO3 0.5-3g, sodium succinate 5-40g, NaCl 30-40g, K2HPO4 0.5-1g, KH2PO4 1-2g, MgSO4·7H2O 0.1-0.2g, FeSO4·7H2O 0.05-0.1g, 1% bromothymol blue ethanol solution 1-2mL, pH 7.0-7.8, trace element solution 1-2mL, and distilled water 1-2L; 3) The heterotrophic nitrification solid culture medium consists of: ammonium chloride 0.5-2g, sodium succinate 5-40g, NaCl 30-40g, K2HPO4 0.5-1g, KH2PO4 1-2g, MgSO4·7H2O 0.1-0.2g, FeSO4·7H2O 0.05-0.1g, pH 7.0-7.8, agar 20-50g, and distilled water 1-2L; 4) The heterotrophic nitrification liquid culture medium consists of: ammonium chloride 0.5-2g, sodium succinate 5-40g, NaCl 30-40g, K2HPO4 0.5-1g, KH2PO4 1-2g, MgSO4·7H2O 0.1-0.2g, FeSO4·7H2O 0.05-0.1g, pH 7.0-7.8, and distilled water 1-2L; 5) 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.

4. The method for treating high-salt, high-ammonia nitrogen wastewater using heterotrophic nitrifying aerobic denitrifying bacteria according to claim 1, characterized in that, The heterotrophic nitrifying aerobic denitrifying halomonas denitrifying bacteria denitrify in the presence of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen as the sole nitrogen source, and in the presence of mixed nitrogen sources.

5. The method for treating high-salt, high-ammonia nitrogen wastewater using heterotrophic nitrifying aerobic denitrifying bacteria according to claim 1, characterized in that, The optimal culture conditions are: sodium succinate as the carbon source, C / N ratio of 10-14, temperature of 30℃-34℃, rotation speed of 160rpm-220rpm, salinity of 2%-6%, and pH of 8-10.

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