Method for treating high-salt and high-ammonia-nitrogen sewage by heterotrophic nitrification and aerobic denitrification bacteria

Abstract

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

Description

Technical Field

[0001] The present invention relates to a method for treating high-salt and high-ammonia-nitrogen sewage, and particularly to a method for treating high-salt and high-ammonia-nitrogen sewage using heterotrophic nitrifying and aerobic denitrifying bacteria. Background Art

[0002] When the HNAD strain performs heterotrophic nitrification, it can also perform aerobic denitrification, enabling nitrification and denitrification to occur simultaneously. Moreover, the strain has advantages such as a fast growth rate and high denitrification efficiency.

[0003] Currently, many researchers have conducted extensive studies on the denitrification characteristics of the HNAD strain. However, due to differences in the strain types, their nitrogen metabolic pathways vary. And the currently isolated HNAD denitrifying strains mainly come from sludge or freshwater environments, making it difficult to effectively treat the saline wastewater discharged from industries such as food processing, printing and dyeing, medicine, leather making, and oil refining. Because when the salt content in the wastewater is too high, it usually causes dehydration of microbial cells and damages the microbial treatment system. Now, a heterotrophic nitrifying and aerobic denitrifying strain with high denitrification efficiency in a high-salt environment is screened to provide a reference for the biological treatment of high-salt wastewater. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for treating high-salt and high-ammonia-nitrogen sewage using heterotrophic nitrifying and aerobic denitrifying bacteria. The salt-tolerant and high-ammonia-nitrogen-tolerant heterotrophic nitrifying-aerobic denitrifying bacteria used in this method are Halomonas Halomonas sp. ) MY11, which has good heterotrophic nitrification and aerobic denitrification capabilities under the extreme conditions of high salt and high ammonia nitrogen. And it can utilize ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen as nitrogen sources, and can efficiently denitrify whether it is a single or mixed nitrogen source, with a strong tolerance to high salt and high ammonia nitrogen. This strain can efficiently treat nitrogen-containing wastewater under the conditions of a salinity of 2% - 6% and an ammonia nitrogen concentration of 100 mg / L - 400 mg / L, and the removal efficiency can reach more than 99%. It has great potential in the treatment of inorganic nitrogen pollution in various water bodies such as aquaculture and chemical industry sewage in China.

[0005] The purpose of the present invention is achieved through the following technical solutions: A method for treating high-salt and high-ammonia-nitrogen sewage using heterotrophic nitrifying and aerobic denitrifying bacteria, the method comprising the following steps: (1) Enrichment, isolation, and screening of heterotrophic nitrifying and aerobic denitrifying Halomonas S1. The mud - water mixture sample retrieved from the aerobic tank is stored at 2 - 5 °C. Under the aseptic operation table, 1 - 6 mL of the sample is taken and inoculated into a conical flask containing enrichment medium. After shaking well, it is cultured in a constant - temperature shaker at 30 - 40 °C and 120 - 160 rpm / min for 1 - 3 days. After the culture is completed, 1 - 6 mL of the bacterial liquid is taken and transferred into a conical flask containing enrichment medium, and the above operation is repeated three times. S2. The seed liquid obtained by the above enrichment is diluted by the gradient dilution method to obtain cell dilution liquids with concentration gradients of 10 -1 ~10 -9 . (Dilution steps: Take 1 mL of the seed liquid and add 9 mL of sterile water, and so on). Take 0.1 - 0.3 mL of the bacterial liquid of each concentration gradient and evenly spread it on the aerobic denitrification medium, and culture it in a constant - temperature incubator at 30 - 40 °C for 2 - 3 days. After the culture, select the colonies whose medium near the colonies changes from yellow - green to blue and inoculate them into the heterotrophic nitrification medium for multiple three - zone streak cultures to improve their purity. Finally, the purified strains are inoculated into the heterotrophic nitrification medium and stored at 2 - 5 °C. S3. Use an inoculation loop to pick 1 - 3 loops of the single colonies obtained by the culture into the heterotrophic nitrification liquid medium with a salinity of 4% and culture for 2 - 20 h to obtain the seed liquid. Take 1 mL of the seed liquid and culture it in the heterotrophic nitrification liquid medium at 30 - 40 °C and 120 - 160 rpm / min for 12 - 48 h, measure its ammonia - nitrogen removal efficiency, and finally screen out the strains with an ammonia - nitrogen removal efficiency greater than 90% for further screening. S4. Take the seed liquid of the strains with a removal efficiency greater than 90% and culture it in the aerobic denitrification medium with a salinity of 4% at 30 - 40 °C and 120 - 160 rpm / min for 12 - 48 h, measure its nitrate - nitrogen removal efficiency, and finally screen out the strains with a nitrate - nitrogen removal efficiency greater than 90% for further screening. S5. Based on the above ammonia - nitrogen and nitrate - nitrogen removal efficiencies under high - salt conditions, the halomonas with the strongest tolerance under high - salt conditions is obtained Halomonas sp . MY - 11, and it is cryopreserved. (2) Denitrification of halomonas in high - salt wastewater For the denitrification of this bacterium in the wastewater with a salinity of 4%, take the above heterotrophic nitrification liquid medium, and successively add 0%, 2%, 4%, 6%, 8%, 10%, 12% of NaCl, KCl, Na2SO4, K2SO4. Take 1% - 3% of the strain seed liquid, and measure the concentrations of ammonia - nitrogen and nitrate - nitrogen in the heterotrophic nitrification medium and the aerobic denitrification medium at 0 - 48 h. This bacterium maintains the removal of ammonia - nitrogen and nitrate - nitrogen under the influence of high salt.

[0006] The described method for treating high-salt and high-ammonia-nitrogen sewage using heterotrophic nitrifying and aerobic denitrifying bacteria. The composition of the enrichment medium is as follows: ammonium chloride 0.2 - 0.6 g, sodium succinate 4 - 8 g, vitamin solution 30 - 50 mL, sodium chloride 30 - 40 g, made up to 1 - 1.5 L. The vitamin 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, made up to 1 - 2 L, pH 7.0 - 8.0.

[0007] The described method for treating high-salt and high-ammonia-nitrogen sewage using heterotrophic nitrifying and aerobic denitrifying bacteria. The composition of the medium and trace elements is as follows: 1) Composition of the aerobic denitrifying solid medium: NaNO3 0.5 - 3 g, sodium succinate 5 - 40 g, NaCl 30 - 40 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; 2) Composition of the aerobic denitrifying liquid medium: NaNO3 0.5 - 3 g, sodium succinate 5 - 40 g, NaCl 30 - 40 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, trace element solution 1 - 2 mL, distilled water 1 - 2 L; 3) Composition of the heterotrophic nitrifying solid medium: ammonium chloride 0.5 - 2 g, sodium succinate 5 - 40 g, NaCl 30 - 40 g, K2HPO 0.5 - 1 g, KH2PO4 1 - 2 g, MgSO·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; 4) Composition of the heterotrophic nitrifying liquid medium: ammonium chloride 0.5 - 2 g, sodium succinate 5 - 40 g, NaCl 30 - 40 g, K2HPO 0.5 - 1 g, KH2PO4 1 - 2 g, MgSO·7H2O 0.1 - 0.2 g, FeSO4·7H2O 0.05 - 0.1 g, pH 7.0 - 7.8, distilled water 1 - 2 L; 5) Trace element solution: 40 - 70 mg / L of EDTA-2Na, 2 - 6 mg / L of ZnSO₄·7H₂O, 5 - 9 mg / L of CaCl₂·2H₂O, 6 - 8 mg / L of MnCl₂·4H₂O, 6 - 8 mg / L of FeSO₄·7H₂O, 1 - 5 mg / L of CuSO₄·5H₂O, 1 - 5 mg / L of CoCl₂.

[0008] The method for treating high-salt and high-ammonia-nitrogen sewage using the heterotrophic nitrifying and aerobic denitrifying bacteria described above, the heterotrophic nitrifying and aerobic denitrifying bacteria are classified and named as Halomonas ( Halomonas sp. ), named Halomonas MY11, belonging to the genus Halomonas ( Halomonas ), deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, deposit number CCTCC M 20242923, deposit date December 27, 2024.

[0009] The method for treating high-salt and high-ammonia-nitrogen sewage using the heterotrophic nitrifying and aerobic denitrifying bacteria described above, the heterotrophic nitrifying and aerobic denitrifying Halomonas bacteria carry out denitrification with ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen as the sole nitrogen source, as well as in a mixed nitrogen source.

[0010] The method for treating high-salt and high-ammonia-nitrogen sewage using the heterotrophic nitrifying and aerobic denitrifying bacteria described above, the optimal culture conditions are: the carbon source is sodium succinate, the C / N ratio is 10 - 14, the temperature is 30℃ - 34℃, the rotation speed is 160 rpm / min - 220 rpm / min, the salinity is 2% - 6%, and the pH is 8 - 10.

[0011] The method for treating high-salt and high-ammonia-nitrogen sewage using the heterotrophic nitrifying and aerobic denitrifying bacteria described above, the denitrification of the heterotrophic nitrifying and aerobic denitrifying Halomonas bacteria in high-salt wastewater: respectively take the above-mentioned heterotrophic nitrifying liquid culture medium, and sequentially add 4%, 6%, 8%, 10%, 12% of NaCl, KCl, Na₂SO₄, K₂SO₄, inoculate 1% - 3% of the strain seed liquid, and measure the concentrations of ammonia nitrogen and nitrate nitrogen in the heterotrophic nitrifying medium and aerobic denitrifying medium within 0 - 48 h, and maintain the removal of ammonia nitrogen and nitrate nitrogen under the influence of high salt.

[0012] The advantages and effects of the present invention are: 1. The strain Halomonas Halomonas sp . MY-11 of the present invention is Gram-stained red and is a Gram-negative bacterium. The strain Halomonas sp . MY-11 has the most excellent denitrification performance when sodium citrate is used as the carbon source, C / N = 14, PH = 7 - 10, temperature 34℃, and rotation speed 220 rpm / min, and can achieve an ammonia nitrogen removal rate of over 99% within 30 h.

[0013] 2. The strain of the present invention Halomonas sp . MY-11 has good removal effects in sewage with different salinity conditions of 4% - 12%, and the removal efficiency is above 99%.

[0014] 3. The strain of the present invention Halomonas sp . MY-11 has the ability of synchronous heterotrophic nitrification and aerobic denitrification, and is characterized by a fast growth and reproduction rate and a high denitrification efficiency. Its logarithmic growth phase is 4 - 16 h, and the ammonia nitrogen removal rate in the wastewater reaches 100% at 28 h. It can be seen that this bacterium can be put into use immediately and achieve efficient biological denitrification. Description of the Drawings

[0015] Figure 1 It is a photo of the medium of Halomonas MY11; Figure 2 It is a microscope photo of Halomonas MY11 after Gram staining; Figure 3 It is the phylogenetic tree of Halomonas MY11; Figure 4 It is the nitrogen removal effect diagram of Halomonas MY11 on different nitrogen sources (ammonia nitrogen, nitrate nitrogen) under different carbon sources (sucrose, glucose, sodium succinate, sodium citrate, sodium acetate); Figure 5 It is the nitrogen removal effect diagram of Halomonas MY11 on different nitrogen sources (ammonia nitrogen, nitrate nitrogen) under different C / N ratios (2, 6, 10, 14, 18, 22); Figure 6 It is the nitrogen removal effect diagram of Halomonas MY11 on different nitrogen sources (ammonia nitrogen, nitrate nitrogen) under different pH values (5, 6, 7, 8, 9, 10); Figure 7 It is the nitrogen removal effect diagram of Halomonas MY11 on different nitrogen sources (ammonia nitrogen, nitrate nitrogen) under different inoculation amounts (1%, 2%, 3%, 4%, 5%); Figure 8 It is the nitrogen removal effect diagram of Halomonas MY11 on different nitrogen sources (ammonia nitrogen, nitrate nitrogen) under different temperatures (24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C); Figure 9 It is the nitrogen removal effect diagram of Halomonas MY11 on different nitrogen sources (ammonia nitrogen, nitrate nitrogen) under different rotation speeds (100 rpm / min, 140 rpm / min, 180 rpm / min, 220 rpm / min, 260 rpm / min); Figure 10Removal efficiency diagrams of different nitrogen sources (ammonia nitrogen, nitrate nitrogen) by Halomonas MY11 at different NaCl salinities (0%, 2%, 4%, 6%, 8%, 10%, 12%); Figure 11 Time-varying diagrams of the denitrification characteristics of Halomonas MY11 under different nitrogen source conditions (ammonia nitrogen, nitrate nitrogen, mixed nitrogen source).

[0016] Figure 12 Removal efficiency diagrams of different nitrogen sources (ammonia nitrogen, nitrate nitrogen) by Halomonas MY11 at different KCl salinities (0%, 2%, 4%, 6%, 8%, 10%, 12%); Figure 13 Removal efficiency diagrams of different nitrogen sources (ammonia nitrogen, nitrate nitrogen) by Halomonas MY11 at different Na2SO4 salinities (0%, 2%, 4%, 6%, 8%, 10%, 12%); Figure 14 Removal efficiency diagrams of different nitrogen sources (ammonia nitrogen, nitrate nitrogen) by Halomonas MY11 at different K2SO4 salinities (0%, 2%, 4%, 6%, 8%, 10%, 12%). Detailed implementation mode

[0017] The present invention will be further explained below in conjunction with embodiments. Embodiment 1

[0018] Enrichment, isolation, screening and purification of strains. The culture medium formula is as follows: 1) The composition of the aerobic denitrification solid medium includes: 0.5 - 3 g of NaNO3, 5 - 40 g of succinate, 30 - 40 g of NaCl, 0.5 - 1 g of K2HPO4, 1 - 2 g of KH2PO4, 0.1 - 0.2 g of MgSO4·7H2O, 0.05 - 0.1 g of FeSO4·7H2O, 1 - 2 mL of 1% bromothymol blue ethanol solution (BTB), pH 7.0 - 7.8, 20 - 50 g of agar, 1 - 2 mL of trace element solution, 1 - 2 L of distilled water; 2) The composition of the aerobic denitrification medium includes: 0.5 - 3 g of NaNO3, 5 - 40 g of succinate, 30 - 40 g of NaCl, 0.5 - 1 g of K2HPO4, 1 - 2 g of KH2PO4, 0.1 - 0.2 g of MgSO4·7H2O, 0.05 - 0.1 g of FeSO4·7H2O, 1 - 2 mL of 1% bromothymol blue ethanol solution (BTB), pH 7.0 - 7.8, 1 - 2 mL of trace element solution, 1 - 2 L of distilled water; 3) The solid components of the heterotrophic nitrification medium include: 0.5 - 2 g of ammonium chloride, 5 - 40 g of sodium succinate, 30 - 40 g of NaCl, 0.5 - 1 g of K2HPO, 1 - 2 g of KH2PO4, 0.1 - 0.2 g of MgSO·7H2O, 0.05 - 0.1 g of FeSO4·7H2O, pH 7.0 - 7.8, 20 - 50 g of agar, and 1 - 2 L of distilled water; 4) The components of the heterotrophic nitrification medium include: 0.5 - 2 g of ammonium chloride, 5 - 40 g of sodium succinate, 30 - 40 g of NaCl, 0.5 - 1 g of K2HPO, 1 - 2 g of KH2PO4, 0.1 - 0.2 g of MgSO·7H2O, 0.05 - 0.1 g of FeSO4·7H2O, pH 7.0 - 7.8, and 1 - 2 L of distilled water; 5) Trace element solution: 40 - 70 mg / L of EDTA-2Na, 2 - 6 mg / L of ZnSO·7H2O, 5 - 9 mg / L of CaCl2·2H20, 6 - 8 mg / L of MnCl2·4H2O, 6 - 8 mg / L of FeSO·7H20, 1 - 5 mg / L of CuSO·5H2O, 1 - 5 mg / L of COCl.

[0019] Enrichment, isolation and screening of strains S1. The mud - water mixture sample taken from the aerobic tank is stored at 2 - 5°C. Transfer 1 - 6 mL of the sample to a conical flask containing the enrichment medium in a sterile operating table. After shaking well, culture it in a constant temperature shaker at 30 - 40°C and 120 - 160 rpm / min for 1 - 3 days; after the culture is completed, transfer 1 - 6 mL of the bacterial liquid to a conical flask containing the enrichment medium and repeat the above operation for three passages; S2. Dilute the seed liquid obtained by the above enrichment using the gradient dilution method to obtain cell dilution liquids with concentration gradients of 10 -1 ~10 -9 (Dilution steps: Take 1 mL of the seed liquid and add 9 mL of sterile water, and so on). Take 0.1 - 0.3 mL of the bacterial liquid of each concentration gradient and evenly coat it on the aerobic denitrification medium, and culture it in a constant temperature incubator at 30 - 40°C for 2 - 3 days; after the culture, select the colonies whose medium near the colonies changes from yellow - green to blue and inoculate them into the heterotrophic nitrification medium for multiple three - zone streak cultures to improve their purity; finally, inoculate the purified strains into the heterotrophic nitrification medium and store them at 2 - 5°C; S3. Use an inoculation loop to pick 1 - 3 loops of the single colonies obtained from the culture and inoculate them into a heterotrophic nitrification liquid medium with a salinity of 4%. After culturing for 2 - 20 h, a seed solution is obtained. Transfer 1 mL of the seed solution into the heterotrophic nitrification liquid medium and culture it at 30 - 40 °C and 120 - 160 rpm / min for 12 - 48 h. Measure its ammonia nitrogen removal efficiency, and finally screen out the strains with an ammonia nitrogen removal efficiency greater than 90% for further screening; S4. Transfer the seed solution of the strains with a removal efficiency greater than 90% into an aerobic denitrification medium with a salinity of 4%. Culture it at 30 - 40 °C and 120 - 160 rpm / min for 12 - 48 h. Measure its nitrate nitrogen removal efficiency, and finally screen out the strains with a nitrate nitrogen removal efficiency greater than 90% for further screening; S5. Based on the above ammonia nitrogen and nitrate nitrogen removal efficiencies under high - salt conditions, the halomonas with the strongest tolerance under high - salt conditions is obtained Halomonas sp . MY - 11, and it is cryopreserved; Example 2

[0020] Identification and characteristics of the strain. The morphological and physiological - biochemical characteristics of the heterotrophic nitrifying and aerobic denitrifying bacterium MY - 11 include: the colony is round, milky white as a whole, and the surface is viscous, as Figure 1 shown; the Gram - staining is red, and it is a Gram - negative bacterium, as Figure 2 shown. After centrifuging the seed solution of the strain cultured for 18 h multiple times, gene sequencing is carried out. The obtained sequencing results are compared for homology in the Genbank database, and the similarity with known strains is 99.98%. A phylogenetic tree is drawn, as Figure 3 shown. The results show that this strain is a halomonas, and it is named Halomonas sp . MY - 11.

[0021] Example 3: Environmental factors affecting the heterotrophic nitrification ability of halomonas Halomonas sp . MY - 11 (1) Carbon source: Application of a heterotrophic nitrifying and aerobic denitrifying bacterium MY11 in treating high - salt and high - ammonia - nitrogen wastewater. Investigate the effects of different carbon sources (sucrose, glucose, sodium succinate, sodium citrate, sodium acetate) on the denitrification performance of this strain.

[0022] Activate the heterotrophic nitrifying and aerobic denitrifying bacterium Halomonas sp . MY - 11 obtained in Example 1, and culture it in a nitrification medium at 32 °C and 160 rpm / min to obtain a bacterial liquid OD 600is 1.0. The bacterial solution was inoculated into different nitrogen source media (g / L) at an inoculation amount of 1% (v / v), with the conditions of C / N = 10, pH = 7, 32 °C, 160 rpm / min, inoculation amount: 1%, salinity (NaCl): 4%. After culturing for 60 h, the concentrations of ammonia nitrogen and nitrate nitrogen were measured respectively, and the results are as Figure 4 shown.

[0023] (2) C / N: Application of a heterotrophic nitrifying-aerobic denitrifying bacterium MY11 in treating 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.

[0024] The heterotrophic nitrifying-aerobic denitrifying bacterium obtained in Example 1 Halomonas sp . MY-11 was activated and cultured in a nitrification medium at 32 °C and 160 rpm / min to obtain a bacterial solution with an OD 600 of 1.0. The bacterial solution was inoculated into different C / N media (g / L) at an inoculation amount of 1% (v / v), with the conditions of succinate, pH = 7, 32 °C, 160 rpm / min, salinity (NaCl): 4%. After culturing for 60 h, the concentrations of ammonia nitrogen and nitrate nitrogen were measured respectively, and the results are as Figure 5 shown.

[0025] (3) pH: Application of a heterotrophic nitrifying-aerobic denitrifying bacterium MY11 in treating high-salt and high-ammonia-nitrogen wastewater. The effects of different pH values (5, 6, 7, 8, 9, 10) on the denitrification performance of this strain were investigated.

[0026] The heterotrophic nitrifying-aerobic denitrifying bacterium obtained in Example 1 Halomonas sp . MY-11 was activated and cultured in a nitrification medium at 32 °C and 160 rpm / min to obtain a bacterial solution with an OD 600 of 1.0. The bacterial solution was inoculated into different pH media (g / L) at an inoculation amount of 1% (v / v), with the conditions of succinate, C / N = 14, 32 °C, 160 rpm / min, inoculation amount: 1%, salinity (NaCl): 4%. After culturing for 60 h, the concentrations of ammonia nitrogen and nitrate nitrogen were measured respectively, and the results are as Figure 6 shown.

[0027] (4) Inoculation amount: Application of a heterotrophic nitrifying-aerobic denitrifying bacterium MY11 in treating high-salt and high-ammonia-nitrogen wastewater. The effects of different inoculation amounts (1%, 2%, 3%, 4%, 5%) on the denitrification performance of this strain were investigated.

[0028] The heterotrophic nitrifying-aerobic denitrifying bacterium obtained in Example 1 Halomonas sp. MY-11 was subjected to activation treatment and cultured in a nitrification medium at 32 °C and 160 rpm / min to obtain a bacterial liquid with an OD 600 of 1.0. The bacterial liquid was inoculated into different temperature media (g / L) at an inoculation amount of 1% (v / v). The conditions were sodium succinate, C / N = 14, pH = 7, 160 rpm / min, and salinity (NaCl): 4%. After culturing for 60 h, the concentrations of ammonia nitrogen and nitrate nitrogen were measured respectively, and the results are as Figure 7 shown.

[0029] (4) Temperature: Application of a heterotrophic nitrifying and aerobic denitrifying bacterium MY11 in treating high-salt and high-ammonia-nitrogen wastewater. The effects of different temperatures (24 °C, 26 °C, 28 °C, 30 °C, 32 °C, 34 °C, 36 °C, 38 °C, 40 °C) on the denitrification performance of this strain were investigated.

[0030] The heterotrophic nitrifying and aerobic denitrifying bacterium obtained in Example 1 Halomonas sp . MY-11 was subjected to activation treatment and cultured in a nitrification medium at 32 °C and 160 rpm / min to obtain a bacterial liquid with an OD 600 of 1.0. The bacterial liquid was inoculated into different temperature media (g / L) at an inoculation amount of 1% (v / v). The conditions were sodium succinate, C / N = 14, pH = 7, 160 rpm / min, inoculation amount: 1%, and salinity (NaCl): 4%. After culturing for 60 h, the concentrations of ammonia nitrogen and nitrate nitrogen were measured respectively, and the results are as Figure 8 shown.

[0031] (5) Rotation speed: Application of a heterotrophic nitrifying and aerobic denitrifying bacterium MY11 in treating 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 this strain were investigated.

[0032] The heterotrophic nitrifying and aerobic denitrifying bacterium obtained in Example 1 Halomonas sp . MY-11 was subjected to activation treatment and cultured in a nitrification medium at 34 °C and 160 rpm / min to obtain a bacterial liquid with an OD 600 of 1.0. The bacterial liquid was inoculated into different rotation speed media (g / L) at an inoculation amount of 1% (v / v). The conditions were sodium succinate, C / N = 14, pH = 7, 34 °C, inoculation amount: 1%, and salinity (NaCl): 4%. After culturing for 60 h, the concentrations of ammonia nitrogen and nitrate nitrogen were measured respectively, and the results are as Figure 9 shown.

[0033] (6)Salinity: Application of a heterotrophic nitrifying and aerobic denitrifying bacterium MY11 in treating high-salinity and high-ammonia-nitrogen wastewater. The effects of different salinities of NaCl (0%, 2%, 4%, 6%, 8%, 10%, 12%) on the denitrification performance of this strain were investigated.

[0034] The heterotrophic nitrifying and aerobic denitrifying bacterium obtained in Example 1 Halomonas sp . MY-11 was activated and cultured in a nitrification medium at 34 °C and 180 rpm / min to obtain a bacterial liquid OD 600 of 1.0. The bacterial liquid was inoculated into different rotational speed media (g / L) at an inoculation amount of 1% (v / v). The conditions were sodium succinate, C / N = 14, pH = 7, 34 °C, 220 rpm / min, and inoculation amount: 1%. After culturing for 60 h, the concentrations of ammonia nitrogen and nitrate nitrogen in them were measured respectively, and the results are as Figure 10 shown.

[0035] Example 4: Halomonas Halomonas sp . MY-11's denitrification characteristics in different nitrogen sources, application of the heterotrophic nitrifying and aerobic denitrifying bacterium MY11 in treating high-salinity and high-ammonia-nitrogen wastewater. The time variation of the denitrification characteristics of this strain under different nitrogen source conditions was studied.

[0036] The Halomonas obtained in Example 1 Halomonas sp . MY-11 was activated and cultured in a nitrification medium at 34 °C and 180 rpm / min for 60 h to obtain a bacterial liquid OD 600 of 1.0. The bacterial liquid was inoculated into ammonia nitrogen, nitrate nitrogen, and mixed nitrogen source media at an inoculation amount of 1% (v / v). The initial ammonia nitrogen content in the single nitrogen source was 400 mg / L, the nitrate nitrogen content in the single nitrogen source was 300 mg / L, and the concentrations of ammonia nitrogen + nitrate nitrogen, ammonia nitrogen + nitrite nitrogen, and nitrate nitrogen + nitrite nitrogen in the mixed nitrogen source medium were all 200 mg / L; in ammonia nitrogen + nitrate nitrogen + nitrite nitrogen, the ammonia nitrogen concentration was 200 mg / L, the nitrate nitrogen concentration was 100 mg / L, and the nitrite nitrogen concentration was mg / L. The ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, and total nitrogen in them were measured every 6 h, and the results are as Figure 11 shown.

[0037] Example 5: Halomonas Halomonas sp . MY-11's denitrification efficiency in different types and concentrations of salts (1)KCl: The effects of different salinities of KCl (0%, 2%, 4%, 6%, 8%, 10%, 12%) on the denitrification performance of this strain were investigated.

[0038] The heterotrophic nitrifying and aerobic denitrifying bacterium obtained in Example 1 Halomonas sp. MY-11 was activated and cultured in a nitrification medium at 34 °C and 180 rpm / min to obtain a bacterial liquid with an OD 600 of 1.0. The bacterial liquid was inoculated into different rotational speed media (g / L) at an inoculation amount of 1% (v / v). The conditions were sodium succinate, C / N = 14, pH = 7, 34 °C, and 220 rpm / min. After culturing for 60 h, the concentrations of ammonia nitrogen and nitrate nitrogen in them were measured respectively, and the results were as Figure 12 shown.

[0039] (2) Na2SO4: The effects of different salinities of Na2SO4 (0%, 2%, 4%, 6%, 8%, 10%, 12%) on the denitrification performance of this strain were investigated.

[0040] The heterotrophic nitrifying-aerobic denitrifying bacteria obtained in Example 1 Halomonas sp . MY-11 was activated and cultured in a nitrification medium at 34 °C and 180 rpm / min to obtain a bacterial liquid with an OD 600 of 1.0. The bacterial liquid was inoculated into different rotational speed media (g / L) at an inoculation amount of 1% (v / v). The conditions were sodium succinate, C / N = 14, pH = 7, 34 °C, and 220 rpm / min. After culturing for 60 h, the concentrations of ammonia nitrogen and nitrate nitrogen in them were measured respectively, and the results were as Figure 13 shown.

[0041] (3) K2SO4: The effects of different salinities of K2SO4 (0%, 2%, 4%, 6%, 8%, 10%, 12%) on the denitrification performance of this strain were investigated.

[0042] The heterotrophic nitrifying-aerobic denitrifying bacteria obtained in Example 1 Halomonas sp . MY-11 was activated and cultured in a nitrification medium at 34 °C and 180 rpm / min to obtain a bacterial liquid with an OD 600 of 1.0. The bacterial liquid was inoculated into different rotational speed media (g / L) at an inoculation amount of 1% (v / v). The conditions were sodium succinate, C / N = 14, pH = 7, 34 °C, and 220 rpm / min. After culturing for 60 h, the concentrations of ammonia nitrogen and nitrate nitrogen in them were measured respectively, and the results were as Figure 14 shown.

Claims

1. A method for treating high-salt and high-ammonia-nitrogen sewage using heterotrophic nitrifying and aerobic denitrifying bacteria, characterized in that, The method includes the following steps: (1) Enrichment, isolation and screening of heterotrophic nitrifying and aerobic denitrifying Halomonas S1. The mud-water mixture sample taken from the aerobic tank is stored at 2-5°C. 1-6 mL of the sample is taken and inoculated into a conical flask containing enrichment medium on a sterile operating table. After shaking well, it is cultured in a constant temperature shaker at 30-40°C and 120-160 rpm / min for 1-3 days; after the culture is completed, 1-6 mL of the bacterial liquid is taken and transferred into a conical flask containing enrichment medium, and the above operation is repeated three times; S2. Dilute the seed solution obtained by the above enrichment using the gradient dilution method to obtain cell dilutions with concentration gradients of 10 -1 ~10 -9 . (Dilution steps: Take 1 mL of the seed solution and add 9 mL of sterile water, and so on). Take 0.1 - 0.3 mL of the bacterial solutions with each concentration gradient and evenly spread them on the aerobic denitrification medium, and culture them in a constant temperature incubator at 30 - 40 °C for 2 - 3 days; After culturing, select the colonies whose medium near the colonies changes from yellow - green to blue and inoculate them into the heterotrophic nitrification medium for multiple three - zone streak cultures to improve their purity; Finally, inoculate the purified strain into the heterotrophic nitrification medium and store it at 2 - 5 °C; S3. 1-3 loops of the single colonies obtained from the culture are picked with an inoculation loop and cultured in a heterotrophic nitrification liquid medium with a salinity of 4% for 2-20 h to obtain a seed liquid. 1 mL of the seed liquid is taken and cultured in a heterotrophic nitrification liquid medium at 30-40°C and 120-160 rpm / min for 12-48 h, and its ammonia nitrogen removal efficiency is measured. Finally, the strains with an ammonia nitrogen removal efficiency greater than 90% are further screened; S4. The seed liquid of the strains with a removal efficiency greater than 90% is taken and cultured in an aerobic denitrification medium with a salinity of 4% at 30-40°C and 120-160 rpm / min for 12-48 h, and its nitrate nitrogen removal efficiency is measured. Finally, the strains with a nitrate nitrogen removal efficiency greater than 90% are further screened; S5. Based on the above removal efficiencies of ammonia nitrogen and nitrate nitrogen under high-salt conditions, the halomonas with the strongest tolerance under high-salt conditions was obtained, Halomonas sp . MY-11, and it was cryopreserved; (2) Nitrogen removal by Halomonas in high-salt wastewater For the nitrogen removal of this bacterium in wastewater with a salinity of 4%, the above-mentioned heterotrophic nitrification liquid medium is taken respectively, and 0%, 2%, 4%, 6%, 8%, 10%, 12% of NaCl, KCl, Na2SO4, and K2SO4 are added in turn. 1%-3% of the bacterial strain seed liquid is inoculated, and the concentrations of ammonia nitrogen and nitrate nitrogen in the heterotrophic nitrification medium and aerobic denitrification medium are measured at 0-48 h. This bacterium maintains the removal of ammonia nitrogen and nitrate nitrogen under the influence of high salt.

2. A method for treating high-salt and high-ammonia-nitrogen sewage using heterotrophic nitrifying and aerobic denitrifying bacteria according to claim 1, characterized in that, The composition of the enrichment medium is as follows: ammonium chloride 0.2-0.6 g, sodium succinate 4-8 g, vitamin solution 30-50 mL, sodium chloride 30-40 g, made up to 1-1.5 L, vitamin 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, made up to 1-2 L, pH 7.0-8.

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3. A method for treating high-salt and high-ammonia-nitrogen sewage using heterotrophic nitrifying and aerobic denitrifying bacteria according to claim 1, characterized in that, The composition of the medium and trace elements includes: 1) The composition of the aerobic denitrification solid medium includes: NaNO3 0.5-3 g, sodium succinate 5-40 g, NaCl 30-40 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; 2) The composition of the aerobic denitrifying medium includes: 0.5 - 3 g of NaNO3, 5 - 40 g of succinate, 30 - 40 g of NaCl, 0.5 - 1 g of K2HPO4, 1 - 2 g of KH2PO4, 0.1 - 0.2 g of MgSO4·7H2O, 0.05 - 0.1 g of FeSO4·7H2O, 1 - 2 mL of 1% bromothymol blue ethanol solution (BTB), pH 7.0 - 7.8, 1 - 2 mL of trace element solution, and 1 - 2 L of distilled water; 3) The solid composition of the heterotrophic nitrification medium includes: 0.5 - 2 g of ammonium chloride, 5 - 40 g of succinate, 30 - 40 g of NaCl, 0.5 - 1 g of K2HPO, 1 - 2 g of KH2PO4, 0.1 - 0.2 g of MgSO·7H2O, 0.05 - 0.1 g of FeSO4·7H2O, pH 7.0 - 7.8, 20 - 50 g of agar, and 1 - 2 L of distilled water; 4) The composition of the heterotrophic nitrification medium includes: 0.5 - 2 g of ammonium chloride, 5 - 40 g of succinate, 30 - 40 g of NaCl, 0.5 - 1 g of K2HPO, 1 - 2 g of KH2PO4, 0.1 - 0.2 g of MgSO·7H2O, 0.05 - 0.1 g of FeSO4·7H2O, pH 7.0 - 7.8, and 1 - 2 L of distilled water; 5) Trace element solution: 40 - 70 mg / L of EDTA - 2Na, 2 - 6 mg / L of ZnSO·7H2O, 5 - 9 mg / L of CaCl2·2H20, 6 - 8 mg / L of MnCl2·4H2O, 6 - 8 mg / L of FeSO·7H20, 1 - 5 mg / L of CuSO·5H2O, 1 - 5 mg / L of COCl.

4. A method for treating high-salt and high-ammonia-nitrogen sewage using heterotrophic nitrifying and aerobic denitrifying bacteria according to claim 1, characterized in that, The heterotrophic nitrifying and aerobic denitrifying bacterium is classified and named as Halomonas ( Halomonas sp. Halomonas sp. ), named Halomonas MY11, belonging to the genus Halomonas ( Halomonas Halomonas ), and is deposited in the China Center for Type Culture Collection with the deposit number CCTCC M 20242923.

5. A method for treating high-salt and high-ammonia-nitrogen sewage using heterotrophic nitrifying and aerobic denitrifying bacteria according to claim 1, characterized in that, The heterotrophic nitrifying and aerobic denitrifying Halomonas sp. removes nitrogen using ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen as the sole nitrogen source, as well as in a mixed nitrogen source.

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

7. A method for treating high-salt and high-ammonia-nitrogen sewage using heterotrophic nitrifying and aerobic denitrifying bacteria according to claim 1, characterized in that, The nitrogen removal by the heterotrophic nitrifying and aerobic denitrifying Halomonas sp. in high - salinity wastewater: Take the above - mentioned heterotrophic nitrification liquid medium, and sequentially add 4%, 6%, 8%, 10%, and 12% of NaCl, KCl, Na2SO4, and K2SO4. Inoculate 1% - 3% of the strain seed liquid, and measure the concentrations of ammonia nitrogen and nitrate nitrogen in the heterotrophic nitrification medium and the aerobic denitrification medium at 0 - 48 h, and maintain the removal of ammonia nitrogen and nitrate nitrogen under the influence of high salinity.

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