Low-temperature-resistant heterotrophic nitrification-aerobic denitrification strain and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and provides a low-temperature-resistant heterotrophic nitrification-aerobic denitrification bacterial strain and application thereof, the low-temperature-resistant heterotrophic nitrification-aerobic denitrification bacterial strain is paracoccus versutus YQ18 and pseudomonas mendocina YQ25, the two bacterial strains are both preserved in the China General Microbiological Culture Collection Center, the preservation number of the two bacterial strains is CGMCC (China General Microbiological Culture Collection Center), and the preservation number of the two bacterial strains is CGMCC NO. The preservation numbers of the two strains are CGMCC No.34641 and CGMCC No.34642 respectively, the two strains both have a heterotrophic nitrification-aerobic denitrification function, have wide temperature adaptability and have efficient removal capacity on ammonia nitrogen and total nitrogen in sewage at normal temperature, and the mixed microbial agent also has efficient denitrification capacity at the low temperature of 8 DEG C, can be applied to sewage treatment in medium and high latitude areas in winter, and has wide application prospects. The sewage treatment efficiency under the low-temperature condition is effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to low-temperature resistant heterotrophic nitrification-aerobic denitrification strains and their applications, specifically the variable Paracoccus strain (… Paracoccus versutus YQ18 and Mendoza strain ( Pseudomonas mendocina YQ25 and its applications. Background Technology

[0002] Nitrogen pollution in aquatic ecosystems is a significant environmental issue. A major source of nitrogen pollution in water bodies is domestic sewage. Due to accelerated urbanization, the sources of nitrogen in domestic sewage have become more complex, increasing the difficulty of water treatment. Wastewater nitrogen removal primarily relies on microbial ammonia oxidation and denitrification processes, generally through aerobic nitrification by ammonia-oxidizing bacteria and anaerobic / anoxic denitrification by denitrifying bacteria. This necessitates the establishment of strict aerobic / anaerobic processes. Heterotrophic nitrification-aerobic denitrification bacteria, however, can simultaneously perform ammonia oxidation and denitrification in an aerobic environment, requiring simpler water treatment processes and consuming less energy, thus being considered a sustainable direction for wastewater treatment. For northern cities, low temperatures inhibit microbial metabolic processes, significantly impacting the efficiency of wastewater treatment in winter. This makes it difficult for the effluent quality of most urban wastewater treatment plants and constructed wetlands used for effluent treatment to meet national standards. Therefore, finding microbial strains capable of efficiently removing nitrogen under low-temperature conditions is of great significance for the sustainable development of wastewater treatment and water resource utilization in northern cities. Summary of the Invention

[0003] This invention provides low-temperature resistant heterotrophic nitrification-aerobic denitrification strains and their applications, specifically the *Paragonimus* strain (…). Paracoccus versutus YQ18 and Mendoza strain ( Pseudomonas mendocina YQ25 and its applications.

[0004] This invention is achieved by the following technical solution: a low-temperature resistant heterotrophic nitrification-aerobic denitrification strain, wherein the low-temperature resistant heterotrophic nitrification-aerobic denitrification bacteria is a variable paracoccus strain ( Paracoccus versutus YQ18 and Mendoza strain ( Pseudomonas mendocina YQ25, both strains are deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession numbers CGMCC No. 34641 and CGMCC No. 34642, respectively, and possess heterotrophic nitrification-aerobic denitrification functions.

[0005] Both strains are selected from the winter sediment of tail water artificial wetland of sewage treatment plant in Yangqu County, Shanxi Province, and both strains are rod-shaped gram-negative bacteria. The colony of YQ18 is orange, opaque, small, moist and viscous, and facultative anaerobic; the colony of YQ25 is light yellow, irregular, with umbrella-shaped edge, and easy to pick up. Both strains are heterotrophic nitrification-aerobic denitrification strains, and have high-efficiency denitrification function under low temperature conditions.

[0006] The screening method adopted by the two strains is as follows: 1. Preparation of culture medium: the heterotrophic nitrification screening culture medium involved in the present application comprises: NH4SO40.5 g / L, C4H4Na2O46 g / L, and 50 ml / L of Wilkins salt solution; the aerobic denitrification culture medium comprises: KNO30.36 g / L, C4H4Na2O46 g / L, and 50 ml / L of Wilkins salt solution; wherein the Wilkins salt solution comprises: NaCl 2.5 g / L, MnSO4·4H2O 0.05 g / L, K2HPO4·3H2O 6.5 g / L, MgSO4·7H2O 0.2 g / L, and FeSO4·7H2O 0.05 g / L; the aerobic denitrification identification culture medium comprises: KNO31.0 g / L, C4H4Na2O48.5 g / L, K2HPO41.0 g / L, FeSO4·7H2O 0.25 g / L, MgSO4·7H2O 1.2 g / L, and 1% bromothymol blue ethanol solution 1 ml / L.

[0007] 2. Strain screening: the winter sediment of tail water artificial wetland of sewage treatment plant in Yangqu County, Shanxi Province is collected, and the preliminary screening is performed by using the heterotrophic nitrification culture medium, and the secondary screening and identification are performed on the screened strains by using the aerobic denitrification culture medium and the aerobic denitrification identification culture medium.

[0008] 3. Denitrification capacity detection: the screened strains are cultured by using NO3 - -N as a nitrogen source, and the denitrification capacity of the strains is determined by detecting NO3 - -N in the bacterial solution.

[0009] 4. Nitrogen removal capacity test: the screened strains are cultured by using different forms of nitrogen sources at different temperatures, and the low-temperature nitrogen removal capacity of the screened strains is detected.

[0010] 5. Strain identification: the obtained purified strains are subjected to strain identification by Shanghai Meiji Biomedicine Technology Co., Ltd. The 16S rDNA of the Parachlamydiales strain (Parachlamydiales strain) Paracoccus versutus ) YQ18 is a gene sequence as shown in SEQ ID No: 1, and the 16S rDNA of the Pseudomonas mendozae strain (Pseudomonas mendozae strain) Pseudomonas mendocina ) YQ25 is a gene sequence as shown in SEQ ID No: 2.

[0011] 6. Strain preservation: The screened Paracoccus versutus (P. versutus) YQ18 and Pseudomonas mendocina (P. mendocina) YQ25 are preserved in the China General Microbiological Culture Collection Center, and the preservation numbers are CGMCC No. 34641 and CGMCC No. 34642, respectively. Paracoccus versutus Pseudomonas mendocina The application also provides application of the low-temperature-tolerant heterotrophic nitrification-aerobic denitrification strain in removal of TN in sewage at normal temperature, YQ18 and YQ25 are inoculated according to a volume inoculation amount of 1%, and TN in the sewage is removed at normal temperature.

[0012] The application also provides application of the low-temperature-tolerant heterotrophic nitrification-aerobic denitrification strain in removal of NO3 - -N in sewage at normal temperature, the YQ18 and YQ25 are inoculated according to a volume inoculation amount of 1%, and the 48h removal rates of NO3 - -N at normal temperature reach 98.89% and 96.83%, respectively.

[0013] The application also provides application of the low-temperature-tolerant heterotrophic nitrification-aerobic denitrification strain in nitrogen removal in sewage at low temperature, under the condition of 8℃, inoculation is performed according to a ratio of 1:1 and a volume ratio of 1%, and the removal rates of NH4 + -N and TN in the sewage are significantly improved.

[0014] The application also provides application of the low-temperature-tolerant heterotrophic nitrification-aerobic denitrification strain in nitrogen removal in tail water of a domestic sewage treatment plant, a DO value of sewage treated by the strain YQ18 or YQ25 is 0.5-8 mg / L, and a C / N ratio is not less than 1.

[0015] The nitrogen in the sewage is one or more of NH4 + -N, nitrate NO3 - -N and nitrite NO2 - -N.

[0016] The strain of the application has the following advantages:

[0017] The temperature tolerance range is wide, and the strain has good nitrogen removal ability under the condition of 8-30℃; the nitrogen removal range is wide, and the strain also has good nitrogen removal ability in tail water of a sewage treatment plant with low nitrogen content; mixed inoculation of the two strains can effectively improve the nitrogen removal ability in sewage; under the condition of 8℃, the 48h removal efficiencies of NH4 + -N and TN reach 73.44% and 99.74%, respectively.

[0018] ​​The two strains in the bacterial combination disclosed in this invention were both screened from winter sediments of the artificial wetland in the effluent of a sewage treatment plant in Yangqu County, Shanxi Province, and are deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession numbers CGMCC No. 34641 and CGMCC No. 34642, respectively. This combination can effectively address the problem of reduced nitrogen removal efficiency in the effluent wetlands of sewage treatment plants in northern cities during winter, providing a new solution for the sustainable development of water treatment.

[0019] The bacterial strain described in this invention is: *Paragonimus westermani* (…). Paracoccus versutus YQ18 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 22, 2025, with accession number CGMCC No. 34641; Mendoza Pseudomonas ( Pseudomonas mendocina YQ25 was deposited on May 22, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 34642. Attached Figure Description

[0020] Figure 1 These are heterotrophic nitrifying-aerobic denitrifying bacteria; in the image: the left image shows a variable strain of *Paragonimus* (…). Paracoccus versutus YQ18; the right image shows a strain of Pseudomonas mendoza ( Pseudomonas mendocina YQ25; Figure 2 The results show the denitrification identification; in the figure: the left image shows the variable paracoccus strain ( Paracoccus versutus YQ18; the right image shows a strain of Pseudomonas mendoza ( Pseudomonas mendocina YQ25; Figure 3 The results show the denitrification efficiency of the strain at 30℃ with nitrate nitrogen as the sole nitrogen source. Figure 4 Nitrogen removal rate for different inoculum amounts; Figure 5 The changes in OD values ​​for different inoculation methods; Figure 6 The nitrogen removal rate of the mixed bacterial solution in the constructed wetland of tailwater is given. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0023] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0025] Example 1: Screening of cold-tolerant heterotrophic nitrification-aerobic denitrification strains 1. Culture medium preparation: Vickers salt solution: NaCl 2.5 g / L, MnSO4·4H2O 0.05 g / L, K2HPO4·3H2O 6.5 g / L, MgSO4·7H2O 0.2 g / L, FeSO4·7H2O 0.05 g / L; Heterotrophic nitrifying bacteria selection medium: NH4SO4 0.5 g / L, C4H4Na2O4 6 g / L, Vickers salt solution 50 ml / L; Selection medium for aerobic denitrifying bacteria: KNO3 0.36 g / L, C4H4Na2O4 6 g / L, Vickers salt solution 50 ml / L; Identification medium for aerobic denitrifying bacteria: KNO3 1.0 g / L, C4H4Na2O4 8.5 g / L, K2HPO4 1.0 g / L, FeSO4·7H2O 0.25 g / L, MgSO4·7H2O 1.2 g / L, 1% bromothymol blue ethanol solution 1 ml / L; The pH of each culture medium was adjusted to 7.0-7.2. For solid culture medium, 15-20g of agar was added to the above formula.

[0026] 2. Preparation of bacterial suspension: Weigh 5g of sediment sample from the artificial wetland and place it in an Erlenmeyer flask containing 45ml of sterile water. Set the shaker temperature to 30℃ and the rotation speed to 125rpm. Shake for 30min and then remove and let stand for 2h to obtain bacterial suspension.

[0027] 3. Strain screening: Add 10 ml of bacterial suspension to 90 ml of heterotrophic nitrifying bacteria screening medium, incubate in the dark at 30°C and 125 rpm. After the medium becomes turbid, dilute to 10% with sterile water. -4, take 0.1 ml of the diluent evenly coated in solid heterotrophic nitrifying bacteria screening medium plate, 30℃ dark culture, select the colony morphology, well-grown colonies, line separation on solid medium until the screening of the same single colony colony characteristics; the above single colony inoculated in aerobic denitrifying bacteria screening medium, 30℃ dark culture, normal growth of colonies may be aerobic denitrifying bacteria ( Figure 1 ), with inoculation needle inoculation in aerobic denitrifying bacteria identification medium, inverted culture, observe the changes around the colony, the appearance of blue halo around the colony is the primary screening strain, further identify its denitrification ability.

[0028] 3, cold acclimation: using the way of gradually reducing temperature to the strains screened for acclimation, set 25℃, 20℃, 15℃, 13℃, 10℃, 8℃, 6 temperature gradient, each gradient under the strain culture to OD600≈0.7, take 10 ml of bacterial suspension inoculated in 90 ml aerobic denitrifying bacteria culture medium for the next temperature gradient culture, ultimately with the selection of 8℃ under the condition of 36h culture OD value greater than 0.6 strain as the target strain.

[0029] By the above method, 2 strains of cold-tolerant heterotrophic nitrification-aerobic denitrification bacteria ( Figure 2 ) were screened, which were Paracoccus versutus ( Paracoccus versutus ) YQ18 and Pseudomonas mendocina ( Pseudomonas mendocina ) YQ25, both strains had good aerobic denitrification capacity at room temperature; in the NO3 - -N concentration of 50mg / L denitrification medium, the removal rate of NO3 - -N of the two strains could reach about 80% in 30h, and the removal rate of NO3 - -N could reach 98.89% and 96.83% ( Figure 3 ) in 48h.

[0030] Example 2: The effect of different inoculation amount of strains on the removal efficiency of NH4 + -N and TN This example studied the effect of different inoculation amount of two strains on the removal efficiency of NH4 + -N and TN in wastewater. According to the method of constrained orthogonal test design, the nitrogen removal capacity of different inoculation amount of strains was tested, NH4SO4, KNO3 and NaNO2 were used as nitrogen source, the nitrogen content in aerobic denitrifying bacteria screening medium was adjusted to 100mg / L, the content of C4H4Na2O4 was adjusted to make the carbon-nitrogen ratio in the medium be 10; the two strains were cultured to OD600≈0.7, and then inoculated according to the volume percentage of 0.5%, 1% and 3%, respectively, and cultured at 30℃, 125rpm in the dark, and the NH4 + ​​​​​-N and TN content. The orthogonal test grouping of two strains and 48h nitrogen removal rate are shown in Table 1 and Table 2, respectively.

[0031] Removal effect of different inoculation amount of YQ18: three inoculation amounts of NH4 + -N and TN content. The orthogonal test grouping of two strains and 48h nitrogen removal rate are shown in Table 1 and Table 2, respectively. Figure 4 ).

[0032] Table 1: Orthogonal test grouping of different inoculation amount of YQ18 and 48h nitrogen removal rate Removal effect of different inoculation amount of YQ25: 0.5% inoculation amount of NH4 + -N has the lowest 24h removal rate, 1% and 3% inoculation amount of NH4 + -N can reach more than 90%, and there is no significant difference between them; the 24h removal rate of TN of three inoculation amounts is higher than that of YQ18, the 24h removal rate of TN of 0.5% inoculation amount is the lowest, and there is no significant difference between 1% and 3% inoculation amount of TN, from the economic point of view, 1% inoculation amount is the best inoculation amount ( Figure 4 ).

[0033] Table 2: Orthogonal test grouping of different inoculation amount of YQ25 and 48h nitrogen removal rate Example 3: verification of nitrogen removal efficiency of strains under low temperature conditions According to 1% inoculation amount, by inoculating YQ18, YQ25 alone and mixing YQ18 and YQ25 according to 1:1 volume ratio, three inoculation methods, using the experimental conditions in Example 2 for grouping, setting the culture temperature to 8℃, regularly measuring the NH4 + -N and TN content. The orthogonal grouping of different inoculation methods and 48h nitrogen removal rate are shown in Table 3, and the OD600 change is shown in Figure 5 The removal rate of NH4 + -N can reach more than 99%, the removal rate of TN of YQ18 alone is 45.39%-51.13%, the removal rate of TN of YQ25 alone is 44.69%-59.53%, the removal rate of TN of mixed inoculation can reach 67.69%-73.44%, which is significantly higher than that of single inoculation. Under low temperature conditions, mixed inoculation can effectively improve the removal rate of TN.

[0034] Table 3: Different inoculation mode orthogonal test grouping and 48h nitrogen removal rate Example 4: Application test of strains in tail water constructed wetland of domestic sewage treatment plant Compared with other wetlands, tail water constructed wetland of domestic sewage treatment plant has the characteristics of low content of TN and NH4 + -N, and the strains obtained by the application can be used for nitrogen removal in tail water wetland. Tail water was collected at the inlet of a tail water wetland of a domestic sewage treatment plant in Shanxi Province, and the water quality parameters were as follows: NH4 + -N 6.28 mg / L, TN 13.55 mg / L, DO 6.52, C / N 6.42, and pH 7.67. The tail water was divided into 1000 ml triangular bottles, with 300 ml in each bottle, and a total of 9 bottles. YQ18 and YQ25 were cultured to OD≈0.7, and mixed bacterial solution was prepared at a ratio of 1:1. Mixed bacterial solution was added to 6 bottles of tail water at a volume ratio of 1% (about 3 ml), and the remaining three bottles were used as controls. The culture temperature was set to 8℃, and the shaking speed was 125 rpm. The 48h nitrogen removal capacity of the strains was tested, and the results are shown in Table 4. Figure 6 As shown in Table 4, the NH4 + -N removal rate of the inoculated group was 98.79%, and the NH4 + -N removal rate of the control group was 39.87%. The TN removal rate of the inoculated group was 69.83%, and the TN removal rate of the control group was 48.37%. The NH4 + -N and TN removal rates of the inoculated group were significantly higher than those of the control group. This result shows that the mixed bacterial solution has good nitrogen removal capacity for tail water of domestic sewage treatment plant under low temperature conditions.

[0035] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the application, and are not limited thereto. Although the application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the application.

Claims

1. A psychrophilic heterotrophic nitrifying-aerobic denitrifying bacterial strain, characterized in that: The low-temperature resistant heterotrophic nitrifying-aerobic denitrifying bacteria are variable paracoccal strains ( Paracoccus versutus YQ18 and Mendoza strain ( Pseudomonas mendocina YQ25, both strains are deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession numbers CGMCC No. 34641 and CGMCC No. 34642, respectively, and the deposit date is May 22, 2025. They possess heterotrophic nitrification-aerobic denitrification functions.

2. The psychrophilic heterotrophic nitrifying-aerobic denitrifying bacterial strain according to claim 1, characterized in that: the 16S rDNA of the Paracoccus zeaxanthinifaciens strain (P. Paracoccus versutus ) YQ18 is a gene sequence as shown in SEQ ID No: 1, the 16S rDNA of the Pseudomonas mendocina strain (P. Pseudomonas mendocina ) YQ25 is a gene sequence as shown in SEQ ID No:

2.

3. Use of the psychrophilic heterotrophic nitrifying-aerobic denitrifying strain according to claim 1 or 2 for the removal of TN from wastewater at room temperature, characterized in that: The YQ18 and YQ25 are inoculated at a volume inoculation of 1%, and the TN in the sewage is removed at normal temperature.

4. Use of the psychrophilic heterotrophic nitrifying-aerobic denitrifying strain according to claim 1 or 2 for the removal of NO3 - - N at room temperature from sewage, characterized in that: The YQ18 and YQ25 are inoculated at a volume inoculation amount of 1%, and the 48h removal rates of NO3 - The 48h removal rates of N are 98.89% and 96.83% respectively.

5. Use of the psychrophilic heterotrophic nitrifying-aerobic denitrifying strain according to claim 1 or 2 for the removal of nitrogen from wastewater at low temperatures, characterized in that: At 8℃, according to the inoculation mode of 1:1, 1% of the volume ratio mixed inoculation, significantly improve the NH4 + The removal rates of N and TN.

6. Use of the psychrophilic heterotrophic nitrifying-aerobic denitrifying strain according to claim 1 or 2 for nitrogen removal from effluent of a domestic wastewater treatment plant, characterized in that: The DO value of the sewage treated by the strains YQ18 and YQ25 is 0.5-8 mg / L, and the C / N is not less than 1.

7. Use according to claim 6, characterized in that: The nitrogen in the wastewater is NH4 + -N, nitrate NO3 - -N and nitrite NO2 - one or more of -N.

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