Strain capable of anaerobic degradation of nitrite and application thereof in denitrification of sewage

Abstract

The present application relates to the field of microorganism and water treatment, and discloses a strain capable of anaerobically degrading nitrite and application thereof in sewage denitrification. Leuconostoc falkenbergense The strain capable of anaerobically degrading nitrite is named KS-A1, and the microbial classification is named Falkenstenia sp. Leuconostoc falkenbergense , which has been preserved in China Center for Type Culture Collection on April 15, 2026, and the preservation number is CCTCC NO: M 2026684. A new type of Falkenstenia sp. Leuconostoc falkenbergense is screened in the present application, which has excellent water environment tolerance, can maintain high activity in a higher nitrite nitrogen concentration and a wider temperature and pH range, and can degrade nitrite (nitrite nitrogen) into nitrogen under anaerobic conditions, thus having a good application prospect in sewage biological denitrification.

Description

Technical Field

[0001] This invention relates to the fields of microorganisms and water treatment, and more particularly to a strain of microorganisms capable of anaerobic degradation of nitrite and its application in wastewater denitrification. Background Technology

[0002] High concentrations of nitrogen are commonly found in various types of wastewater, especially domestic sewage, industrial wastewater, and agricultural wastewater. Direct discharge of such high-nitrogen wastewater can have a negative impact on the ecological environment, so it must undergo denitrification treatment before discharge.

[0003] Currently, common wastewater denitrification methods include physical denitrification, chemical denitrification, and biological denitrification. Among these, biological denitrification has the advantages of being environmentally friendly and having high denitrification efficiency, and therefore has gradually become a research focus in recent years. A more traditional biological denitrification method utilizes nitrifying bacteria to remove ammonia nitrogen (NH3-N or NH4) from wastewater. + -N) is oxidized to nitrite nitrogen (NO2). - Nitrogen (NO3-) is further oxidized to nitrate nitrogen (NO3). - Nitrate nitrogen is converted into nitrogen gas by microorganisms. The disadvantages of this method are that the denitrification pathway is relatively long and requires a large amount of oxygen and carbon to be added to the water body during the treatment process.

[0004] To shorten the denitrification pathway, short-cut nitrification technology has been developed in recent years. The key feature of this technology is that it first uses short-cut nitrifying bacteria, which oxidize ammonia nitrogen to nitrite (nitrite nitrogen), to treat the water. This stops the oxidation of ammonia nitrogen in the water at the nitrite stage. Then, in conjunction with other denitrifying microorganisms, the nitrite is directly converted into nitrogen gas. Compared with traditional biological denitrification methods, this technology omits the step of further oxidizing nitrite to nitrate (nitrate nitrogen), resulting in a shorter denitrification pathway, lower consumption, significantly improved denitrification efficiency, and reduced costs.

[0005] The core of the aforementioned short-cut nitrification technology lies in the screening of microbial strains, including strains that oxidize ammonia nitrogen to nitrite (short-cut nitrifying bacteria) and strains that convert nitrite into nitrogen gas (denitrifying strains). Since wastewater composition is typically complex, and different types of wastewater have varying treatment temperatures, pH levels, salinity, and other parameters, suitable strains capable of tolerating the target wastewater environment need to be screened separately for different types of wastewater. In the early stages of research and development, the applicant has screened several pairs of short-cut nitrifying strains with good tolerance to high-nitrogen water environments. However, for denitrifying strains capable of converting nitrite into nitrogen gas, the selection remains limited due to the scarcity of existing strain resources. Therefore, there is an urgent need to obtain more denitrifying strains capable of tolerating high-nitrogen water environments and converting nitrite into nitrogen gas.

[0006] Leuconostoc falkenberghei ( Leuconostoc falkenbergense This strain belongs to the genus *Leuconostoc*. Currently, it is mainly used for food fermentation, such as in the fermentation of yogurt, kimchi, and pickled beans. The applicant's search revealed no reports regarding this strain's ability to convert nitrite to nitrogen under anaerobic conditions or its use for wastewater denitrification. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a strain capable of anaerobic nitrite degradation and its application in wastewater denitrification. This invention screened and obtained a novel *Leuconostoc falkenberghei* strain (…). Leuconostoc falkenbergense This strain exhibits excellent tolerance to aquatic environments, maintaining high activity at high nitrite nitrogen concentrations and over a wide range of temperatures and pH levels. It can degrade nitrite (nitrite nitrogen) into nitrogen gas under anaerobic conditions, thus showing promising application prospects in biological denitrification of wastewater.

[0008] The specific technical solution of this invention is as follows: First, this invention provides a strain capable of anaerobic degradation of nitrite, the strain being named... Leuconostoc falkenbergense KS-A1, microbiologically classified as Leuconostoc falkenberghemin ( Leuconostoc falkenbergense This strain was deposited at the China Center for Type Culture Collection on April 15, 2026, with accession number CCTCC NO: M 2026684, and the deposit address is Wuhan University, Wuhan, China.

[0009] This invention screened and obtained a novel strain of Leuconostoc falkenberghei (Falkenberghei). Leuconostoc falkenbergense During testing, this invention unexpectedly discovered that the strain possesses the activity of degrading nitrite (nitrite nitrogen) into nitrogen gas under anaerobic conditions. Even more remarkably, further testing revealed that this strain exhibits excellent tolerance to aquatic environments, maintaining high nitrite degradation activity across high nitrite nitrogen concentrations and a wide range of temperature and pH. Therefore, this strain shows promising application potential in biological denitrification of wastewater.

[0010] Secondly, the present invention provides activated sludge and microbial agents containing the above-mentioned strains capable of anaerobically degrading nitrite.

[0011] Preferably, the microbial agent is freeze-dried bacterial powder or bacterial liquid.

[0012] Furthermore, this invention provides the application of the above-mentioned anaerobic nitrite-degrading strains in wastewater denitrification: Leuconostoc falkenbergenseKS-A1 converts nitrite in wastewater into nitrogen gas under anaerobic conditions.

[0013] Preferably, the wastewater denitrification process includes: 1) Short-range nitrifying bacteria convert ammonia nitrogen in wastewater into nitrite; 2) Through Leuconostoc falkenbergense KS-A1 converts nitrite in wastewater into nitrogen gas.

[0014] Based on the present invention Leuconostoc falkenbergense KS-A1's anaerobic nitrite-degrading property allows it to be used in conjunction with existing short-cut nitrifying bacteria for biological denitrification of wastewater. In this process, the short-cut nitrifying bacteria first convert ammonia nitrogen in the wastewater into nitrite, and then... Leuconostoc falkenbergense KS-A1 then converts nitrite in wastewater into nitrogen. Compared to traditional biological denitrification methods, this denitrification pathway skips the step of further oxidizing nitrite (nitrite nitrogen) into nitrate (nitrate nitrogen), resulting in a shorter denitrification pathway, lower consumption, and significantly improved denitrification efficiency while reducing costs.

[0015] Preferably, in step 2), the dissolved oxygen concentration of the wastewater is <0.2 mg / L, the water temperature is 10~50℃, the initial pH of the wastewater is 5~9, and the nitrite nitrogen concentration is 5~2000 mg / L.

[0016] The strain of this invention has aquatic environmental tolerance and can maintain high nitrite degradation activity in a wide range of nitrite nitrogen concentrations and temperatures and pH.

[0017] Finally, the present invention provides a biological denitrification method for wastewater, which includes the following steps: A) After the sewage has settled, it is introduced into the inlet tank.

[0018] B) Pass the wastewater from the inlet tank into the inoculation tank. Leuconostoc falkenbergense In the anoxic pool of KS-A1.

[0019] C) The wastewater in the anoxic tank is introduced into an aerobic tank inoculated with short-range nitrifying bacteria, so that the ammonia nitrogen in the wastewater is converted into nitrite (nitrite nitrogen).

[0020] D) Wastewater in the aerobic tank is returned to the anoxic tank, where nitrite in the wastewater is converted into nitrogen gas. After the total nitrogen in the water reaches the standard, the wastewater in the aerobic tank is fed into the secondary sedimentation tank for secondary settling, thus completing the denitrification of the wastewater.

[0021] In the above method, the reason why the anoxic tank is located upstream of the aerobic tank in this invention is that the influent contains abundant carbon sources, so this design can ensure a stable oxygen supply without adding external carbon sources. Leuconostoc falkenbergense Proliferation of KS-A1.

[0022] Preferably, in step B), the treatment in the anoxic tank is carried out under stirring conditions.

[0023] Preferably, in step C), the treatment in the aerobic tank is carried out under aeration conditions.

[0024] Preferably, in step D), the sludge obtained after the secondary sedimentation tank is refluxed into the anoxic tank.

[0025] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention provides a novel strain of Leuconostoc falkenberghemin ( Leuconostoc falkenbergense This strain exhibits the activity of degrading nitrite (nitrite nitrogen) into nitrogen gas under anaerobic conditions; it also demonstrates excellent tolerance to aquatic environments, maintaining high nitrite degradation activity at high nitrite nitrogen concentrations and over a wide range of temperatures and pH.

[0026] (2) The present invention will Leuconostoc falkenbergense KS-A1, used in conjunction with short-cut nitrifying bacteria, is employed for biological nitrogen removal from wastewater. The short-cut nitrifying bacteria first convert ammonia nitrogen in the wastewater into nitrite, and then... Leuconostoc falkenbergense KS-A1 then converts nitrite in wastewater into nitrogen. Compared to traditional biological denitrification methods, this denitrification pathway skips the step of further oxidizing nitrite (nitrite nitrogen) into nitrate (nitrate nitrogen), resulting in a shorter denitrification pathway, lower consumption, and significantly improved denitrification efficiency while reducing costs. Attached Figure Description

[0027] Figure 1 This is a comparison diagram of the Griss reagent in Example 1 of the present invention; Figure 2 This is a graph showing the change in nitrogen concentration in headspace gas detected by gas chromatography in Example 2 of the present invention; Figure 3 In Embodiment 2 of the present invention 15 Figure showing the results of nitrogen generation detection using N isotope tracing method; Figure 4 This is a nitrogen balance analysis diagram of KS-A1 degradation of nitrite under anaerobic conditions in Example 2 of the present invention; Figure 5 The variation of COD concentration in the influent and effluent of the pilot test in Example 5 of this invention (the horizontal axis represents the number of days). Figure 6 The influent and effluent NH4 in Example 5 of this invention + -N concentration change (horizontal axis is number of days); Figure 7The total nitrogen (TN) concentration changes in the influent and effluent of Example 5 of this invention are shown on the horizontal axis (number of days). Detailed Implementation

[0028] First, this invention provides a strain capable of anaerobically degrading nitrite, which is named... Leuconostoc falkenbergense KS-A1, microbiologically classified as Leuconostoc falkenberghemin ( Leuconostoc falkenbergense This strain was deposited at the China Center for Type Culture Collection on April 15, 2026, with accession number CCTCC NO: M 2026684.

[0029] Second, the present invention provides an activated sludge containing the above-mentioned strains capable of anaerobically degrading nitrite.

[0030] Third, the present invention provides a microbial agent containing the above-mentioned strains capable of anaerobically degrading nitrite.

[0031] In some specific embodiments, the microbial agent is freeze-dried bacterial powder or bacterial liquid.

[0032] Fourth, this invention provides the application of the above-mentioned anaerobic nitrite-degrading strains, activated sludge, or microbial agents in wastewater denitrification. This invention... Leuconostoc falkenbergense KS-A1 can convert nitrite in wastewater into nitrogen gas under anaerobic conditions.

[0033] In some specific implementations, the wastewater denitrification process includes: 1) Short-range nitrifying bacteria convert ammonia nitrogen in wastewater into nitrite; 2) Through Leuconostoc falkenbergense KS-A1 converts nitrite in wastewater into nitrogen gas.

[0034] In some more specific implementations, in step 2), the dissolved oxygen concentration of the wastewater is <0.2 mg / L, the water temperature is 10~50℃, the initial pH of the wastewater is 5~9, and the nitrite nitrogen concentration is 5~2000 mg / L.

[0035] Fifth, the present invention provides a biological denitrification method for wastewater, which includes the following steps: A) After the sewage has settled, it is introduced into the inlet tank.

[0036] B) Pass the wastewater from the inlet tank into the inoculation tank. Leuconostoc falkenbergense In the anoxic pool of KS-A1.

[0037] In some more specific embodiments, in step B), the treatment in the anoxic tank is carried out under stirring conditions.

[0038] C) The wastewater in the anoxic tank is passed into an aerobic tank inoculated with short-range nitrifying bacteria, so that the ammonia nitrogen in the wastewater is converted into nitrite.

[0039] In some more specific implementations, in step C), the treatment in the aerobic tank is carried out under aeration conditions.

[0040] D) Wastewater in the aerobic tank is returned to the anoxic tank, where nitrite in the wastewater is converted into nitrogen gas. After the total nitrogen in the water reaches the standard, the wastewater in the aerobic tank is fed into the secondary sedimentation tank for secondary settling, thus completing the denitrification of the wastewater.

[0041] In some more specific implementations, in step D), the sludge obtained after the secondary sedimentation tank is refluxed into the anoxic tank. Specific Implementation The present invention will be further described below with reference to embodiments.

[0043] (1) Anaerobic ZPM02 agar medium: The formula is shown in Table 1. The preparation method is as follows: Weigh the corresponding amount of reagent according to the formula in Table 1 into an Erlenmeyer flask, add the corresponding amount of boiled pure water, stir with a glass rod to dissolve, and adjust the pH to 6.5-7.0; after boiling, add the second-boil reagent, add about 200 mL of purified water, turn on the Hengate device, and boil the medium again under N2 protection, continuously purging N2 for about 20 min; under N2 protection, dispense into anaerobic bottles that have been deoxygenated by purging with N2, and the liquid dispensing height should not exceed 2 / 3 of the height of the anaerobic bottle; stopper and attach the medium label, and sterilize at 121℃ for 15 min. Note: Before use, confirm that the pH of the medium is 6.5-7.0.

[0044] Table 1: Formula for Anaerobic ZPM02 Agar Medium (2) Anaerobic ZPM42 liquid culture medium: The formula is shown in Table 2. The preparation method is as follows: Weigh the corresponding amount of reagent into an Erlenmeyer flask according to the formula in Table 2, boil, and then add about 200 mL of pure water. Turn on the Hengate device to boil the culture medium again under N2 protection, and continue to pass N2 for about 30 min. Cool to room temperature, and dispense into deoxygenated anaerobic bottles under N2 protection. The dispensing height should not exceed 2 / 3 of the container height. Stopper the bottles and label them. Sterilize at 121℃ for 20 min. Note: Before using the culture medium, confirm that the pH of the culture medium is about 7.0-7.2. Because colorimetric reaction detection is required, resazurin and cysteine ​​salts are not added to the culture medium.

[0045] Table 2: Formulation of Anaerobic ZPM42 Liquid Culture Medium Example 1:Leuconostoc falkenbergense Identification of KS-A1 and verification of its anaerobic nitrite degradation function Sample processing: Take approximately 5 mL inside the glove box. Leuconostoc falkenbergense KS-A1 bacterial culture samples were treated with anaerobic sterile protectant at a ratio of 1:1 (v / v) and vortexed for 2 min. The samples were then dispensed into sterile screw cap tubes, 1 mL per tube.

[0046] Separation and plating: The treated sample was serially diluted 10-fold. 100 μL of the diluted stock solution (treated sample) was plated onto anaerobic ZPM02 agar medium and incubated anaerobically at 30°C for 10 days. Single colonies were picked and transferred to 200 μL of ZPM42 liquid medium and incubated anaerobically at 30°C for 4 days. 50 μL of the cultured bacterial suspension from a 96-well plate was taken, and 20 μL of Gliese reagent A (Feijing Biotechnology; PLM053) was added. After mixing, 20 μL of Gliese reagent B (Feijing Biotechnology; PLM053) was added, and the color reaction of the bacterial suspension was observed. A change in the color from purplish-red to lighter shades indicated a positive result. The remaining bacterial suspension from the positive wells in the 96-well plate was then streaked onto anaerobic ZPM02 agar medium for purification and incubated anaerobically at 30°C for 4 days. After single colonies have grown, a single colony from the third region of the plate was picked and streaked a second time onto anaerobic ZPM02 agar medium and incubated anaerobically at 30°C for 4 days. After single colonies have grown, a single colony from the third region was picked and incubated anaerobically at 30°C for 2 days before being submitted for preservation. 2 mL of bacterial suspension was resuspended in 2 mL of anaerobic sterile cryoprotectant, mixed thoroughly, transferred to sterile screw-cap tubes, and stored at -80°C. The preserved strain was identified by PCR sequencing, including forward and reverse sequencing of 16S-27F and 16S-1492R, and the full-length sequence was assembled. BLAST alignment was used to annotate species information.

[0047] The sample strain was identified as Leuconostoc falkenberghemin (Falkenberghemin). Leuconostoc falkenbergense Therefore, the strain was named Leuconostoc falkenbergense KS-A1. The preservation information for this strain is as follows: preservation date: April 15, 2026; preservation institution: China Center for Type Culture Collection; preservation number: CCTCC NO: M2026684.

[0048] Griess reagent re-screening verification: Take 1.5 mL of anaerobic ZPM42 liquid culture medium (cultured for 10 days), add 20 μL of Griess reagent A, mix well, then add 20 μL of Griess reagent B, and observe the color reaction of the bacterial solution. The principle of the above test is as follows: anaerobic ZPM42 liquid culture medium is the positive control group; anaerobic ZPM42 liquid culture medium without sodium nitrite is the negative control group. The higher the nitrite concentration in the bacterial solution, the darker the color of the bacterial solution, mainly showing a red or even purple color. When the nitrite concentration decreases, the color of the bacterial solution becomes lighter, thus corroborating the concentration of nitrite.

[0049] like Figure 1 As shown, the negative bacterial solution is transparent and colorless ( Figure 1 The leftmost test tube in the middle), the test bacterial solution is purple-red ( Figure 1 The rightmost test tube in the picture turned light, indicating a positive result. Figure 1 (the middle test tube), the result proves Leuconostoc falkenbergense KS-A1 has the ability to degrade nitrite under anaerobic conditions.

[0050] Example 2: Verification of degradation products obtained from the anaerobic degradation of nitrite by KS-A1 To further verify the degradation products of KS-A1 after degrading nitrite, the following experiments were conducted.

[0051] (1) Gas chromatography test Test method: KS-A1 bacterial culture was inoculated at a rate of 3 wt% into ZPM42 liquid medium containing 100 mg / L nitrite nitrogen, with a blank control group not inoculated. Anaerobic culture was performed at 37℃. Samples were taken at 0, 6, 12, 24, 36, 48, and 72 hours. 1 mL of headspace gas was drawn using a gas-tight needle and injected into the chromatograph for detection. Triple replicates were performed for each time point. A gas chromatograph with a thermal conductivity detector (TCD) was used, with a 5 Å molecular sieve packed column, a column temperature of 60℃, a detector temperature of 120℃, argon as the carrier gas, and a flow rate of 30 mL / min. Results are shown in Table 3 and [Table data missing]. Figure 2 .

[0052] Table 3: Changes in nitrogen concentration in headspace gas detected by gas chromatography The data above show that in the KS-A1 inoculated group, the headspace nitrogen concentration continuously increased over time, rising from 78.5% at 0h to 89.7% at 72h, an increase of 11.2%. In contrast, the nitrogen concentration in the blank control group remained almost unchanged, increasing by only 0.4%, possibly due to the release of a small amount of nitrogen dissolved in the culture medium. These results indicate that KS-A1 produces nitrogen during the degradation of nitrite.

[0053] (2) 15 Nitrogen isotope tracing experiment.

[0054] Using Na 15 NO2, 15 The nitrogen atom percentage exceeds 99%. KS-A1 bacterial culture was inoculated at a 3% inoculum into a solution containing 100 mg / L nitrite (Na₂O₃). 15 The culture was anaerobic at 37°C in ZPM42 liquid medium containing NO2. Samples were taken at 0, 12, 24, 36, 48, and 72 hours, and the concentration of NO2 in the headspace gas was measured using GC-IRMS (Gas Chromatography-Isotope Ratio Mass Spectrometry). 15 The atomic percentage of N2 was measured, and the remaining sodium nitrite in the culture medium was also measured. 15 N abundance. Results are shown in Table 4 and... Figure 3 .

[0055] Table 4: 15 Results of nitrogen generation detection by N isotope tracer method The data above shows that, over time, the top of the sky... 15 The atomic percentage of N2 continued to rise, from 0 to 5.50%; meanwhile, the percentage of sodium nitrite... 15 The remaining proportion of N continued to decline, from 100% to 8.8%. This indicates... 15 The nitrogen in N-labeled sodium nitrite was converted into... 15 N2. This indicates that KS-A1 has denitrification capabilities.

[0056] (3) Nitrogen balance analysis KS-A1 bacterial culture was inoculated at a rate of 3 wt% into ZPM42 liquid medium containing 100 mg / L nitrite nitrogen and cultured anaerobically at 37℃. Samples were taken at 0, 12, 24, 36, 48, and 72 hours, and the following parameters were measured: Nitrite nitrogen: N-(1-naphthyl)ethylenediamine hydrochloride spectrophotometry; Ammonium nitrogen: Nessler's reagent spectrophotometry; Nitrate nitrogen: UV spectrophotometry; Cell-assimilated nitrogen: Kjeldahl method for total nitrogen in the cells; Nitrogen gas: Gas chromatography combined with headspace analysis. The results are shown in Table 5. Figure 4 .

[0057] Table 5: Nitrogen balance analysis of KS-A1 degradation of nitrite under anaerobic conditions The data above shows that the total nitrogen level remained relatively constant at around 100 mg / L over different time periods, which is consistent with the law of conservation of mass (fluctuations are due to error); nitrite nitrogen decreased continuously over time, from 100 mg / L to 9.7 mg / L; nitrogen levels continued to rise, eventually dropping to 86.5 mg / L, accounting for about 95.8% of the degraded nitrite nitrogen.

[0058] The concentration of ammonium nitrogen remained consistently low, fluctuating between 0.5 mg / L and 2.2 mg / L, accounting for approximately 2% of the initial total nitrogen. This indicates that KS-A1 primarily degrades nitrite through the denitrification pathway, with the DNRA pathway contributing very little and essentially negligible. Furthermore, the concentration of nitrogen assimilated by the bacteria was also low, reaching a maximum of only 3.2 mg / L, suggesting that most of the nitrite nitrogen was converted into gaseous nitrogen and escaped rather than being absorbed by the bacteria.

[0059] The above three experiments confirm that KS-A1 can efficiently convert nitrite into nitrogen under anaerobic conditions, and therefore can be used for biological denitrification of wastewater.

[0060] Example 3: Effects of different initial nitrite nitrogen concentrations on Leuconostoc falkenbergense Effect of KS-A1 on the ability of nitrite degradation under anaerobic conditions Anaerobic liquid culture media with different nitrite nitrogen concentrations were prepared according to the following formula: NaNO2 concentrations of 24.64 mg / L, 147.86 mg / L, 295.71 mg / L, 492.86 mg / L, 985.71 mg / L, and 9857.14 mg / L (equivalent to nitrite nitrogen concentrations of 5, 30, 60, 100, 200, and 2000 mg / L in the culture medium), K2HPO4 1.0 g / L, MgSO4 0.5 g / L, CaCO3 1.0 g / L, NaCl 10.0 g / L, and purified water as the solvent; the pH was adjusted to 7.0; and the medium was sterilized by high-temperature moist heat at 121℃ for 15 min.

[0061] Pick Leuconostoc falkenbergense Single colonies of KS-A1 were inoculated into TSB medium and cultured anaerobically at 37°C until the logarithmic growth phase. Leuconostoc falkenbergense KS-A1 bacterial culture. Take... Leuconostoc falkenbergense KS-A1 bacterial suspension was inoculated into anaerobic liquid culture media with different nitrite nitrogen concentrations at an inoculum size of 3 wt%. After incubation at 37°C under anaerobic conditions for 3 days, the nitrite nitrogen content in the culture media was measured, and the results are shown in Table 6.

[0062] Table 6: Results of the detection of the effect of initial nitrite nitrogen concentration on the nitrite degradation capacity of strains under anaerobic conditions As can be seen from the data in Table 6, the present invention Leuconostoc falkenbergense KS-A1 can tolerate and effectively treat high concentrations of nitrite nitrogen. When the initial nitrite nitrogen concentration reaches 2000 mg / L, this strain can still play a good degradation role, efficiently converting nitrite into nitrogen gas under anaerobic conditions. The ammonia nitrogen removal rate can reach 97.26% within 3 days.

[0063] Example 4: Different initial pH values Leuconostoc falkenbergense Effect of KS-A1 on the ability of nitrite degradation under anaerobic conditions Prepare anaerobic liquid culture media with different pH values ​​according to the following formula: NaNO2 9857.14 mg / L (equivalent to 2000 mg / L nitrite nitrogen concentration in the culture medium), K2HPO4 1.0 g / L, MgSO4 0.5 g / L, CaCO3 1.0 g / L, NaCl 10.0 g / L, with purified water as the solvent; adjust the pH to 5.0, 6.0, 7.0, 8.0, and 9.0 respectively; sterilize at 121℃ for 15 min using moist heat.

[0064] Picking grid Leuconostoc falkenbergense Single colonies of KS-A1 were inoculated into TSB medium and cultured anaerobically at 37°C until the logarithmic growth phase. Leuconostoc falkenbergense KS-A1 bacterial culture. Take... Leuconostoc falkenbergense KS-A1 bacterial suspension was inoculated into anaerobic liquid culture media with different pH values ​​at an inoculum of 3 wt%. After incubation at 37°C under anaerobic conditions for 3 days, the nitrite nitrogen in the culture media was measured, and the results are shown in Table 7.

[0065] Table 7: Results of the test on the effect of pH on the nitrite degradation ability of strains under anaerobic conditions As can be seen from the data in Table 7, the present invention Leuconostoc falkenbergense KS-A1 can effectively degrade nitrite in anaerobic environments with pH 5.0~9.0, converting nitrite nitrogen into nitrogen gas. Its nitrite nitrogen removal rate can reach more than 67% within 3 days. Especially under the strongly alkaline condition of pH=9.0, the nitrite nitrogen removal rate can still reach 85.62% within 3 days.

[0066] Example 5: Effects of different temperatures on Leuconostoc falkenbergense Effect of KS-A1 on the ability of nitrite degradation under anaerobic conditions Prepare anaerobic liquid culture medium according to the following formula: NaNO2 9857.14 mg / L (equivalent to 2000 mg / L nitrite nitrogen in the culture medium), K2HPO4 1.0 g / L, MgSO4 0.5 g / L, CaCO3 1.0 g / L, NaCl 10.0 g / L, with purified water as the solvent; adjust the pH to 7.0; sterilize at 121℃ for 15 min using moist heat.

[0067] Pick Leuconostoc falkenbergense Single colonies of KS-A1 were inoculated into TSB medium and cultured anaerobically at 37°C until the logarithmic growth phase. Leuconostoc falkenbergense KS-A1 bacterial culture. Take... Leuconostoc falkenbergense KS-A1 bacterial suspension was inoculated into anaerobic liquid culture medium at an inoculum of 3 wt%. After incubation at 10℃, 20℃, 37℃, 40℃ and 50℃ for 3 days under anaerobic conditions, the nitrite nitrogen in the culture medium was measured, and the results are shown in Table 8.

[0068] Table 8: Results of the test on the effect of temperature on the nitrite degradation ability of the strains under anaerobic conditions As can be seen from the data comparison in Table 8, the present invention Leuconostoc falkenbergense KS-A1 can survive well in anaerobic environments within a temperature window of 10~50℃ and exhibit excellent nitrite degradation, with the optimal temperature around 37℃. Remarkably, Leuconostoc falkenbergense Even at a relatively high temperature of 50℃, KS-A1 can still maintain high nitrite degradation activity, with a nitrite nitrogen degradation rate of up to 65.08%. Therefore, it is suitable for the treatment of some wastewater with high treatment temperatures and has a wide range of applications.

[0069] Example 6: Leuconostoc falkenbergense Application of KS-A1 in biological nitrogen removal from wastewater (pilot-scale experiment) In this embodiment, the influent wastewater comes from a water treatment plant. The wastewater treatment test system consists of a primary sedimentation tank, an influent tank, an anoxic tank, an aerobic tank, and a secondary sedimentation tank connected in sequence. Wastewater in the aerobic tank can be returned to the anoxic tank, and sludge from the secondary sedimentation tank can be returned to the anoxic tank. The anoxic tank is equipped with a stirring device, and the aerobic tank has an aeration device at the bottom. The anoxic tank is filled with 0.5 wt% inoculum. Leuconostoc falkenbergense The activated sludge of KS-A1 is filled with 0.5 wt% Klebsiella pneumoniae in the aerobic tank. Klebsiella grimontii A5 (accession number CCTCC No: M 2025642, published strain) activated sludge.

[0070] The testing method is as follows: A) After the wastewater from the water plant has settled in the sedimentation tank, it is then introduced into the inlet tank.

[0071] B) The wastewater in the inlet tank is introduced into the anoxic tank; the water temperature in the anoxic tank is controlled at 30℃, the dissolved oxygen (DO) content is less than 0.2 mg / L, the pH is 7.0, and the hydraulic retention time is 12h.

[0072] C) The wastewater in the anoxic tank is introduced into the aerobic tank to convert the ammonia nitrogen in the wastewater into nitrite; the water temperature in the aerobic tank is controlled at 30℃, the dissolved oxygen (DO) content is controlled at 0.3-0.6 mg / L, the pH is 7.5, and the hydraulic retention time is 16h through aeration.

[0073] D) Wastewater in the aerobic tank is recirculated to the anoxic tank, where nitrite in the wastewater is converted into nitrogen gas. The effluent quality is monitored. Once the total nitrogen level in the water meets the standard, the wastewater in the aerobic tank is introduced into the secondary sedimentation tank for secondary settling. During stable operation of the device, the effluent quality is monitored, and the results are as follows: Figures 5-7 As shown.

[0074] Figure 5 The graph shows the COD concentration changes in the influent and effluent during the pilot-scale test (x-axis represents days). The average influent COD concentration was 68.71 mg / L, and the average effluent concentration was 30.68 mg / L. The effluent COD concentration remained relatively stable throughout the pilot-scale test. Furthermore, the relatively high influent COD concentration in the pilot-scale test was related to COD desorption. No external carbon source was added to the pilot-scale unit during the entire test.

[0075] Figure 6 For pilot-scale influent and effluent ammonia nitrogen (NH4) + -N) concentration changes (x-axis: days). Average influent NH4 concentration during the pilot test. + The -N concentration was 8.404 mg / L, with an average effluent concentration of 0.644 mg / L. Throughout the pilot-scale test, the effluent NH4 concentration was... + -N concentration decreased significantly.

[0076] Figure 7 The graph shows the changes in total nitrogen (TN) concentration in the influent and effluent of the pilot test (x-axis represents days). During the pilot test, the average influent TN concentration was 9.313 mg / L, and the average effluent concentration was 6.047 mg / L. It is evident that after the above biological denitrification treatment, the denitrification effect of the system was significantly improved, and the average effluent TN concentration of the pilot unit was reduced by approximately 40% compared to the water plant (before the modification) during the same period.

[0077] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of this invention should be considered within the scope of protection of this invention.

Claims

1. A bacterial strain capable of anaerobic degradation of nitrite, characterized in that: Strain naming Leuconostoc falkenbergense KS-A1, microbiologically classified as Leuconostoc falkenberghemin ( Leuconostoc falkenbergense This strain was deposited at the China Center for Type Culture Collection on April 15, 2026, with accession number CCTCC NO: M 2026684.

2. An activated sludge containing the anaerobic nitrite-degrading strain of claim 1.

3. A microbial agent containing the strain of claim 1 that can anaerobically degrade nitrite.

4. The microbial agent as described in claim 3, characterized in that: The microbial agent is a freeze-dried bacterial powder or bacterial liquid.

5. The application of the anaerobic nitrite-degrading strain as described in claim 1, the activated sludge as described in claim 2, or the microbial agent as described in claim 3 or 4 in wastewater denitrification, characterized in that: Leuconostoc falkenbergense KS-A1 converts nitrite in wastewater into nitrogen gas under anaerobic conditions.

6. The application as described in claim 5, characterized in that: The wastewater denitrification process includes: 1) Short-range nitrifying bacteria convert ammonia nitrogen in wastewater into nitrite; 2) Through Leuconostoc falkenbergense KS-A1 converts nitrite in wastewater into nitrogen gas.

7. The application as described in claim 6, characterized in that: In step 2), the dissolved oxygen concentration of the wastewater is <0.2 mg / L, the water temperature is 10~50℃, the initial pH of the wastewater is 5~9, and the nitrite nitrogen concentration is 5~2000 mg / L.

8. The application as described in claim 5, characterized in that: Includes the following steps: A) After the wastewater has settled, it is introduced into the inlet tank; B) Pass the wastewater from the inlet tank into the inoculation tank. Leuconostoc falkenbergense In the anoxic pool of KS-A1; C) The wastewater in the anoxic tank is passed into an aerobic tank inoculated with short-range nitrifying bacteria, so that the ammonia nitrogen in the wastewater is converted into nitrite; D) Wastewater in the aerobic tank is returned to the anoxic tank, where nitrite in the wastewater is converted into nitrogen gas. After the total nitrogen in the water reaches the standard, the wastewater in the aerobic tank is fed into the secondary sedimentation tank for secondary settling, thus completing the denitrification of the wastewater.

9. The application as described in claim 8, characterized in that: In step B), the treatment in the anoxic tank is carried out under stirring conditions; In step C), the treatment in the aerobic tank is carried out under aeration conditions.

10. The application as described in claim 9, characterized in that: In step D), the sludge obtained after the secondary sedimentation tank is refluxed into the anoxic tank.

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