Method for denitrifying wastewater by using low-temperature-resistant heterotrophic nitrification and aerobic denitrification bacteria

Abstract

The invention discloses a method for denitrifying wastewater by using a low-temperature-resistant heterotrophic nitrification and aerobic denitrification bacterium, and relates to a method for denitrifying wastewater by using a biological bacterium, the bacterium is Pseudomonas putida, is named as Pseudomonas umsongensis BK17, belongs to Pseudomonas, and is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC M 20242924, and the preservation date is 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 low-temperature stress, and is high in low-temperature tolerance. The strain can efficiently treat nitrogen-containing wastewater under the condition of 10 DEG C, and the removal efficiency can reach 99% or above. The method has great potential in the application of inorganic nitrogen pollution treatment of domestic sewage and other water bodies in cold regions in China.

Description

Technical Field

[0001] The present invention belongs to an environmental microorganism and a method for nitrogen removal from wastewater, and particularly relates to a method for using a low-temperature-tolerant heterotrophic nitrifying and aerobic denitrifying bacterium for nitrogen removal from wastewater. Background Art

[0002] In recent years, although the phenomenon of water pollution has improved, the discharge of nitrogen into water bodies caused by human activities is still very serious. Therefore, removing nitrogen from wastewater remains an important issue in solving water pollution at present. Currently, the methods for solving water pollution include physical methods, chemical methods, and biological methods. Among them, biological methods can effectively treat nutrients in wastewater. Traditional biological nitrogen removal methods are generally divided into two types. Conventional biological methods rely on autotrophic nitrifying bacteria (i.e., AOB and NOB) to convert ammonia into nitrate through nitrification in an aerobic environment, and heterotrophic nitrifying bacteria to reduce nitrate to gas through denitrification in an anoxic / anaerobic environment. Conventional biological ecological nitrogen control technologies usually separate nitrification and denitrification into two stages to maintain suitable growth conditions for autotrophic and heterotrophic microorganisms, autotrophic nitrification and heterotrophic denitrification. This requires separate operations in two reactors, wasting material and financial resources and having complex operations and low efficiency. With the discovery of heterotrophic nitrifying and aerobic denitrifying bacteria, biological nitrogen removal technology has taken a big step forward. Heterotrophic nitrification-aerobic denitrification can simultaneously remove ammonia, nitrate, and organic matter in a single process and is currently the most concerned technology.

[0003] In northern China, the winter is long and the climate is cold. It is difficult to achieve efficient removal of nitrogen nutrients in cold lakes in winter using biological methods. Currently, the effect of sewage biological nitrogen removal technology is poor at low temperatures and the resources of low-temperature bacterial strains are insufficient. Research has found that low-temperature environments (<15°C) will inhibit the growth and metabolism of autotrophic nitrifying bacteria. To overcome this limitation, research scholars have continuously innovated and improved, and isolated and screened cold-tolerant nitrogen-removing microorganisms. Heterotrophic nitrifying-aerobic denitrifying bacteria have gradually attracted wide attention due to their strong adaptability to low-temperature environments. More and more cold-tolerant heterotrophic nitrifying-aerobic denitrifying functional bacteria have been screened and isolated from various environments, and their low-temperature tolerance characteristics have gradually been discovered, becoming a feasible alternative for treating nitrogen-containing pollutants and improving nitrogen removal efficiency in cold regions. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for using a low-temperature-tolerant heterotrophic nitrifying and aerobic denitrifying bacterium for nitrogen removal from wastewater. This method provides Pseudomonas yinchengensis ( Pseudomonas umsongensis ), which can efficiently treat nitrogen-containing wastewater under low-temperature conditions of 10°C. This strain can achieve rapid degradation of ammonia nitrogen and nitrate nitrogen within 48 hours and has a significant effect on removing inorganic nitrogen pollution in water bodies under low-temperature conditions.

[0005] The present invention provides the following technical solutions: A method for wastewater denitrification using a low-temperature-tolerant heterotrophic nitrifying and aerobic denitrifying bacterium. The heterotrophic nitrifying and aerobic denitrifying bacterium is Pseudomonas guazhouensis, named Pseudomonas yinchengensis BK17, belonging to the genus Pseudomonas. The strain is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC M 20242924, and the preservation date is December 27, 2024; (1)Strain source: The strains screened in the experiment are from the water samples collected from the activated sludge in the aerobic tank of a sewage treatment plant; (2)Culture medium formula: Enrichment medium: NH4Cl 0.5 g / L, C4H4Na2O4 3.5 g / L, MgSO4·7H2O 0.05 g / L, K2HPO4 0.2 g / L, NaCl 0.12 g / L, MnSO4·H2O 0.01 g / L, FeSO4·7H2O 0.01 g / L; Nitrifying liquid medium: C4H4Na2O4 4.5025 g / L, NH4Cl 0.3821 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml; Denitrifying liquid medium: C4H4Na2O4 4.5025 g / L, KNO3 0.72 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml, 1% bromothymol blue ethanol solution 1 ml; Nitrifying solid medium: C4H4Na2O4 4.5025 g / L, NH4Cl 0.3821 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml, agar 25 g / L; Denitrifying solid medium: C4H4Na2O4 4.5025 g / L, KNO3 0.72 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml, 1% bromothymol blue ethanol solution 1 ml, agar 25 g / L; Mixed culture medium: 4.5025 g / L of C4H4Na2O4, 0.2675 g / L of NH4Cl, 0.2164 g / L of KNO3, 1 g / L of K2HPO4, 0.5 g / L of KH2PO4, 0.1 g / L of MgSO4·7H2O, 0.05 g / L of FeSO4·7H2O, 2 ml of trace element solution; (3)Specific process: 1) Prepare the enrichment medium. Add the sample to a 250 ml conical flask containing 100 mL of enrichment medium and culture for 48 h. Set the parameters of the constant temperature shaker to 10 °C and 120 r / min. When the bacteria grow until the medium becomes significantly turbid, pipette 10 mL of the cell suspension in the turbid medium and transfer it to 100 ml of fresh enrichment medium, and continue to culture at 10 °C and 120 r / min for 48 h. Repeat the above process 3 times.

[0006] 2) Dilute the enriched sample by the gradient dilution method into bacterial suspensions of various gradients such as 10 -1, 10 -2,..., 10 -8. Select three gradients of 10 -6, 10 -7, and 10 -8, and take 100 μL each and spread them evenly on the heterotrophic nitrification solid medium. Culture in a constant temperature incubator at 10 °C for 2 - 3 days. Single colonies will form on the medium. Pick the single colonies and perform three-zone streak culture on the heterotrophic nitrification solid medium multiple times to ensure the purity of the strains in the medium; 3) Inoculate the obtained pure strains onto the denitrification medium and place them in a biochemical incubator at 10 ºC for 72 h; preliminarily screen out the strains with denitrification ability based on the change of the color of the denitrification medium, and select the strains with blue halos for further screening; 4) Inoculate the screened strains into the nitrification liquid medium respectively, and shake and culture under the conditions of 10 ºC and 120 rpm. After 60 h, measure the ammonia nitrogen removal efficiency, and select the strain BK17 with the highest efficiency for subsequent experiments.

[0007] (4)Method for applying wastewater denitrification: It includes the following steps: Set different culture conditions for heterotrophic nitrifying aerobic denitrifying bacteria under low-temperature conditions to culture them, and test their ammonia nitrogen removal effects; the carbon sources of the nitrogen-containing wastewater are set as sucrose, glucose, sodium acetate, sodium succinate, and sodium citrate respectively, the C / N is 4 - 20, the PH is set at 5 - 10, the rotation speed is set at 40 - 200 rpm, the inoculation amount is set at 0.5% - 4%, and the salinity is set at 0 - 4%.

[0008] The described method for using a low-temperature-tolerant heterotrophic nitrifying aerobic denitrifying bacterium for wastewater denitrification, wherein the Pseudomonas sp. BK17 removes one or a combination of ammonia nitrogen and nitrate nitrogen under low-temperature conditions.

[0009] The technical advantages of the present invention are as follows: 1. The low-temperature heterotrophic nitrifying and aerobic denitrifying bacterium of the present invention is Pseudomonas yinchengensis, named Pseudomonas yinchengensis BK17, belonging to the genus Pseudomonas, and its preservation number is CCTCC M 20242924.

[0010] 2. The application of the bacterium of the present invention in efficiently treating nitrogen-containing wastewater under the condition of 10°C. Under the low-temperature condition of 10°C, the strain can rapidly degrade ammonia nitrogen and nitrate nitrogen within 48 h.

[0011] 3. The strain of the present invention still maintains good denitrification activity at low temperature and is used for sewage denitrification treatment in geographical regions with large annual temperature differences in northern and northwestern China.

[0012] 4. The application of the low-temperature heterotrophic nitrifying and aerobic denitrifying bacterium of the present invention under different environmental conditions when treating nitrogen-containing wastewater. When the C / N of the nitrogen-containing wastewater is 8 - 16, the pH value is 6 - 8, the rotation speed is 120 - 160 rpm, and the inoculation amount is 3% - 5%, rapid degradation of the nitrogen-containing wastewater can be achieved. Brief Description of the Drawings

[0013] Figure 1 It is the 16S-rRNA phylogenetic tree of the heterotrophic nitrifying and aerobic denitrifying bacterium BK17 of the present invention; Figure 2 It is a schematic diagram of the denitrification performance of BK17 at low temperature (10°C) with ammonia nitrogen as the sole nitrogen source; Figure 3 It is a schematic diagram of the denitrification performance of BK17 at low temperature (10°C) with nitrate nitrogen as the sole nitrogen source; Figure 4 It is a schematic diagram of the denitrification performance of BK17 at low temperature (10°C) with ammonia nitrogen and nitrate nitrogen as the mixed nitrogen source; Figure 5 It is the influence of different carbon sources on the growth and ammonia nitrogen removal rate of the present heterotrophic nitrifying and aerobic denitrifying strain; Figure 6 It is the influence of different C / N ratios on the growth and ammonia nitrogen removal rate of the present heterotrophic nitrifying and aerobic denitrifying strain; Figure 7 It is the influence of different pH values on the growth and ammonia nitrogen removal rate of the present heterotrophic nitrifying and aerobic denitrifying strain; Figure 8 It is the influence of different rotation speeds on the growth and ammonia nitrogen removal rate of the present heterotrophic nitrifying and aerobic denitrifying strain; Figure 9 It is the influence of different inoculation amounts on the growth and ammonia nitrogen removal rate of the present heterotrophic nitrifying and aerobic denitrifying strain. Detailed Embodiments

[0014] The present invention will be described in detail below in conjunction with the accompanying drawings and specific examples. The examples described herein are only used to explain and illustrate the present invention, but the protection scope of the present invention is not limited to the following examples. Example 1

[0015] A heterotrophic nitrifying and aerobic denitrifying bacterium of the present invention, which is Pseudomonas melonis, named Pseudomonas yinchengensis BK17, belongs to the genus Pseudomonas. The strain is deposited in the China Center for Type Culture Collection, Wuhan, Hubei Province, with the deposit number CCTCC M 20242924 and the deposit date of December 27, 2024.

[0016] (1) Strain source: The strain screened in the experiment is derived from the water sample collected from the activated sludge in the aerobic tank of Zhenxing Sewage Treatment Plant, Shenyang City, Liaoning Province.

[0017] (2) Medium formula: Enrichment medium: NH4Cl 0.5 g / L, C4H4Na2O4 3.5 g / L, MgSO4·7H2O 0.05 g / L, K2HPO4 0.2 g / L, NaCl 0.12 g / L, MnSO4·H2O 0.01 g / L, FeSO4·7H2O 0.01 g / L; Nitrifying liquid medium: C4H4Na2O4 4.5025 g / L, NH4Cl 0.3821 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml; Denitrifying liquid medium: C4H4Na2O4 4.5025 g / L, KNO3 0.72 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml, 1% bromothymol blue ethanol solution 1 ml; Nitrifying solid medium: C4H4Na2O4 4.5025 g / L, NH4Cl 0.3821 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml, agar 25 g / L; Denitrifying solid medium: C4H4Na2O4 4.5025 g / L, KNO3 0.72 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml, 1% bromothymol blue ethanol solution 1 ml, agar 25 g / L; Mixed medium: C4H4Na2O4 4.5025 g / L, NH4Cl 0.2675 g / L, KNO3 0.2164 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml; (3) Specific process: 1. Prepare the enrichment medium, add the sample to a 250 ml conical flask containing 100 mL of enrichment medium and culture for 48 h. Set the parameters of the constant temperature shaker to 10 °C and 120 r / min. When the bacteria grow to make the medium significantly turbid, aspirate 10 mL of the cell suspension from the turbid medium and transfer it to 100 ml of fresh enrichment medium, and continue to culture at 10 °C and 120 r / min for 48 h. Repeat the above process 3 times.

[0018] 2. Dilute the enriched sample by gradient dilution method into bacterial suspensions of various gradients such as 10 -1, 10 -2,..., 10 -8. Select three gradients of 10 -6, 10 -7, and 10 -8, and take 100 μL each and evenly spread them on the heterotrophic nitrification solid medium. Culture in a constant temperature incubator at 10 °C for 2 - 3 days. Single colonies will form on the medium. Pick the single colonies and perform multiple three-zone streak cultures on the heterotrophic nitrification solid medium to ensure the purity of the strains in the medium.

[0019] 3. Inoculate the obtained pure strains onto the denitrifying medium and culture in a biochemical incubator at 10 ºC for 72 h. Preliminarily and qualitatively screen out the strains with denitrifying ability according to the color change of the denitrifying medium, and select the strains with blue halos for further screening.

[0020] 4. Inoculate the screened strains into the nitrification liquid medium respectively, and culture them by shaking at 10 ºC and 120 rpm. After 60 h, measure the ammonia nitrogen removal efficiency, and select the strain BK17 with the highest efficiency for subsequent experiments.

[0021] Example 2: Strain identification: The selected strains were inoculated into the nitrification medium and cultured at 10°C and 120 rpm for 60 h. The cultured bacterial liquid was sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. The test sequences were analyzed and homology comparison was carried out by online alignment using the NCBI database. A phylogenetic tree was constructed based on the 16S rDNA gene sequence of this strain and some phylogenetically related strains. The results are as Figure 1 .

[0022] Example 3: Denitrification performance test of Pseudomonas yinchengensis BK17 at low temperature (10°C) with ammonia nitrogen as the sole nitrogen source The BK17 strain in Example 1 was inoculated into the nitrification medium and then placed in a shaker at 10°C for cultivation. After it grew to the logarithmic phase, 1% of the cultured bacterial liquid by volume was inoculated into 100 mL of nitrification medium with NH4 + -N as the sole nitrogen source (concentration 100 mg / L) and cultured for 60 h. The culture conditions were 10°C and 120 r / min. Samples were taken at 0, 12, 24, 36, 48, and 60 h respectively. After centrifugation and filtration, the heterotrophic nitrification performance of the strain was tested. At the same time, the possible products NO3 - -N and NO2 - -N contents during the degradation process were also detected. Among them, the method used to test the NH4 + -N content was the Nessler reagent spectrophotometry method, the method used to test the NO3 - -N content was the ultraviolet spectrophotometer method, and the method used to test the NO2 - -N content was the N-(1-naphthyl)-ethylenediamine spectrophotometry method.

[0023] As Figure 2 shown, at 36 h, the removal efficiency of this strain for NH4 + -N reached 99%, and the average removal rate from 0 to 36 h was 2.78 mg / L / h. There was no accumulation of NO3 - -N and NO2 - -N in the middle.

[0024] When NH4Cl was the sole nitrogen source in the medium, the strain could well reduce the concentration of ammonia nitrogen, and there was basically no accumulation of nitrate nitrogen and nitrite nitrogen.

[0025] Example 4: Denitrification performance test of Pseudomonas yinchengensis BK17 at low temperature (10°C) with nitrate nitrogen as the sole nitrogen source The BK17 strain in Example 1 was inoculated into the denitrification medium and then placed in a shaker at 10°C for cultivation. After it grew to the logarithmic phase, 1% of the cultured bacterial liquid by volume was inoculated into 100 mL of medium with NO3- Cultured for 60 h in a nitrification medium with -N as the sole nitrogen source (concentration 100 mg / L), the culture conditions were 10 °C and 120 r / min. Samples were taken at 0, 12, 24, 36, 48, and 60 h respectively. After centrifugation and filtration, the heterotrophic nitrification performance of the strain was tested. Meanwhile, the possible products NH4 + -N and NO2 - -N content during the degradation process were also detected. The method used to test the NH4 + -N content was the Nessler's reagent spectrophotometry. The method used to test the NO3 - -N content was the ultraviolet spectrophotometry. The method used to test the NO2 - -N content was the N-(1-naphthyl)-ethylenediamine spectrophotometry.

[0026] As Figure 3 shown, the removal efficiency of NO3--N by the strain reached 75.68% at 42 h. By 60 h, the removal efficiency of NH4 + -N reached 83.64%, and the average removal rate from 0 to 242 h was 1.81 mg / L / h.

[0027] When KNO3 was used as the sole nitrogen source in the medium, although the degradation of NO3 - -N by the strain was not as fast as that of NH4 + -N, it could still effectively degrade NO3 - -N, and there was basically no accumulation of ammonia nitrogen and nitrite nitrogen.

[0028] Example 5: Denitrification performance test of Pseudomonas yinchengensis BK17 at low temperature (10 °C) with ammonia nitrogen and nitrate nitrogen as mixed nitrogen sources Inoculate the BK17 strain in Example 1 into the denitrification medium, and then place it in a shaker at 10 °C for cultivation. After it grows to the logarithmic phase, inoculate 1% of the cultured bacterial liquid by volume into 100 mL of a nitrification-denitrification medium with NH4 + -N (concentration 70 mg / L) and NO3 - -N (concentration 30 mg / L) as nitrogen sources and culture for 60 h. The culture conditions were 10 °C and 120 r / min. Samples were taken at 0, 12, 24, 36, 48, and 60 h respectively. After centrifugation and filtration, measure the concentrations of NH4 + -N, NO3 - -N, and NO2 - -N in the supernatant. The method used to test the NH4 + -N content was the Nessler's reagent spectrophotometry. The method used to test the NO3 -The method for measuring the -N content is the ultraviolet spectrophotometer method for testing NO2 - The method for measuring the -N content is the N-(1-naphthyl)-ethylenediamine spectrophotometry method.

[0029] As Figure 4 shown, when ammonia nitrogen and nitrate nitrogen coexist, the strain preferentially utilizes ammonia nitrogen. At 36 h, the removal efficiency of the strain for NH4 + -N reaches 99.51%, and NH4 + -N is basically completely removed. At this time, the strain begins to degrade NO3 - -N. By 60 h, the removal efficiency of NO3 - -N reaches 99.12%, and almost no NO2 - -N accumulates during the process.

[0030] When using a mixed nitrogen source of NH4Cl and KNO3 as the culture medium, the strain preferentially utilizes ammonia nitrogen and then nitrate nitrogen, and almost no nitrite nitrogen accumulates during the degradation process.

[0031] Example 6: Factors affecting the heterotrophic nitrification of strain BK17: Effect of carbon source on heterotrophic nitrification: Fix the C / N ratio of the culture medium at 8, and adjust the carbon sources to sucrose, glucose, sodium acetate, sodium succinate, and sodium citrate respectively. Use a sterile pipette to aspirate 1% of the seed solution and place it into 100 ml of the above liquid culture media with different carbon sources respectively. Incubate at 10 °C and 120 rpm for 60 h. Sampling is carried out at 0, 12, 24, 36, 48, and 60 h respectively. After centrifugation and filtration, measure the NH4 + -N content and OD 600 value, and repeat three parallel experiments. The results are as Figure 5 .

[0032] Effect of C / N ratio on heterotrophic nitrification: Adjust the C / N in the culture medium to 4, 8, 12, 16, and 20 respectively. Use a sterile pipette to aspirate 1% of the seed solution and place it into 100 ml of the above liquid culture media with different C / N ratios respectively. Incubate at 10 °C and 120 rpm for 60 h. Sampling is carried out at 0, 12, 24, 36, 48, and 60 h respectively. After centrifugation and filtration, measure the NH4 + -N content and OD 600 value, and repeat three parallel experiments. The results are as Figure 6 .

[0033] Effect of pH on heterotrophic nitrification: Adjust the pH values of the culture medium to 5, 6, 7, 8, and 9 respectively. Use a sterile pipette to aspirate 1% of the seed liquid and place it into 100 ml of the above-mentioned liquid culture media with different pH values. Incubate at 10 °C and 120 rpm for 60 h. Take samples at 0, 12, 24, 36, 48, and 60 h respectively. After centrifugation and filtration, measure the NH4 + -N content and OD 600 value. Repeat the experiment in three parallel sets. The results are as Figure 7 .

[0034] Effect of rotation speed on heterotrophic nitrification: Use a sterile pipette to aspirate 1% of the seed liquid and place it into 100 ml of the liquid culture medium. Incubate at 10 °C and place it in an incubator at 40, 80, 120, 160, and 200 rpm respectively for 60 h of shaking culture. Take samples at 0, 12, 24, 36, 48, and 60 h respectively. After centrifugation and filtration, measure the NH4 + -N content and OD 600 value. Repeat the experiment in three parallel sets. The results are as Figure 8 .

[0035] Effect of inoculum size on heterotrophic nitrification: Use a sterile pipette to aspirate 0.5%, 1%, 3%, 5%, and 7% of the seed liquid respectively and place them into 100 ml of the liquid culture medium. Incubate at 10 °C and shake culture at 120 rpm for 60 h. Take samples at 0, 12, 24, 36, 48, and 60 h respectively. After centrifugation and filtration, measure the NH4 + -N content and OD 600 value. Repeat the experiment in three parallel sets. The results are as Figure 9 .

[0036] The results show that ( Figures 5 - 9 ) the optimal carbon source for this strain is sodium citrate, and the removal efficiency is the best within the range of C / N ratio of 8 - 16, with the best reaching 100%. The efficiency is the best within the pH range of 6 - 8, the appropriate rotation speed is 80 - 160 rpm, and the optimal inoculum size is 3% - 5%.

[0037] Finally, it should be noted that the above are only the preferred examples of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for wastewater denitrification using low-temperature-tolerant heterotrophic nitrifying and aerobic denitrifying bacteria, characterized in that, The heterotrophic nitrifying and aerobic denitrifying bacterium is Pseudomonas gua, named Pseudomonas yinchengensis BK17, belonging to the genus Pseudomonas. The strain is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC M 20242924 and the preservation date of December 27, 2024; (1) Strain source: The strain screened in the experiment was derived from the water sample collected from the activated sludge in the aerobic tank of a sewage treatment plant; (2) Medium formula: Enrichment medium: NH4Cl 0.5 g / L, C4H4Na2O4 3.5 g / L, MgSO4·7H2O 0.05 g / L, K2HPO4 0.2 g / L, NaCl 0.12 g / L, MnSO4·H2O 0.01 g / L, FeSO4·7H2O 0.01 g / L; Nitrification liquid medium: C4H4Na2O4 4.5025 g / L, NH4Cl 0.3821 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml; Denitrification liquid medium: C4H4Na2O4 4.5025 g / L, KNO3 0.72 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml, 1% bromothymol blue ethanol solution 1 ml; Nitrification solid medium: C4H4Na2O4 4.5025 g / L, NH4Cl 0.3821 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml, agar 25 g / L; Denitrification solid medium: C4H4Na2O4 4.5025 g / L, KNO3 0.72 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml, 1% bromothymol blue ethanol solution 1 ml, agar 25 g / L; Mixed medium: C4H4Na2O4 4.5025 g / L, NH4Cl 0.2675 g / L, KNO3 0.2164 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.05 g / L, trace element solution 2 ml; (3) Specific process: 1) Prepare an enrichment medium. Add the sample to a 250 ml conical flask containing 100 mL of the enrichment medium and culture for 48 h. Set the parameters of the constant temperature shaker to 10 °C and 120 r / min. When the bacteria grow to make the medium significantly turbid, pipette 10 mL of the cell suspension from the turbid medium and transfer it to 100 ml of fresh enrichment medium. Continue to culture at 10 °C and 120 r / min for 48 h. Repeat the above process 3 times; 2) After enrichment, dilute the sample by the gradient dilution method to obtain bacterial suspensions of various gradients such as 10 -1, 10 -2, …, 10 -8. Select three gradients of 10 -6, 10 -7, and 10 -8. Take 100 μL of each and evenly spread them on the heterotrophic nitrification solid medium. Culture in a constant temperature incubator at 10 °C for 2 - 3 days. Single colonies will form on the medium. Pick the single colonies and perform three-zone streak culture on the heterotrophic nitrification solid medium multiple times to ensure the purity of the strains in the medium; 3) Inoculate the obtained pure strains onto the denitrification medium and culture in a biochemical incubator at 10 ºC for 72 h; Initially screen out the strains with denitrification ability based on the change in the color of the denitrification medium. Select the strains with a blue halo for further screening; 4) Inoculate the screened strains into the nitrification liquid medium respectively and culture them by shaking at 10 ºC and 120 rpm. After 60 h, measure the ammonia nitrogen removal efficiency and select the strain BK17 with the highest efficiency for subsequent experiments; (4) Method for applying wastewater denitrification: It includes the following steps: Set different culture conditions for heterotrophic nitrifying aerobic denitrifying bacteria under low-temperature conditions to culture them and test their ammonia nitrogen removal effect; The carbon sources of the nitrogen-containing wastewater are set as sucrose, glucose, sodium acetate, sodium succinate, and sodium citrate respectively, C / N is 4 - 20, PH is set at 5 - 10, the rotation speed is set at 40 - 200 rpm, the inoculation amount is set at 0.5% - 4%, and the salinity is set at 0 - 4%.

2. A method for wastewater denitrification using a low-temperature tolerant heterotrophic nitrifying and aerobic denitrifying bacterium according to claim 1, characterized in that, The Pseudomonas sp. BK17 removes one or a combination of ammonia nitrogen and nitrate nitrogen under low-temperature conditions.

Images