A heterotrophic nitrification-aerobic denitrification bacterium with high tolerance to ammonia nitrogen and organic matter and its application in landfill leachate treatment

Abstract

The application discloses a heterotrophic nitrification-aerobic denitrification bacterium resistant to high ammonia nitrogen and high organic matter and application of the bacterium in landfill leachate treatment. The heterotrophic nitrification-aerobic denitrification bacterium provided by the application is Paracoccus pantotrophus HNAD-9, the bacterium is preserved in the China General Microbiological Culture Collection Center on August 22, 2024, and the preservation number is CGMCC No.31698. The application can remove ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in the same heterotrophic and aerobic system, and there is no accumulation of nitrate nitrogen and nitrite nitrogen in the ammonia nitrogen removal process, and the application has an important application prospect in the field of high ammonia nitrogen and high organic matter concentration landfill leachate treatment.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology technology, specifically to the application of a salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacterium in landfill leachate treatment. Background Technology

[0002] Landfill leachate is a complex, high-concentration organic wastewater produced during waste treatment (landfill, incineration, etc.) and landfilling processes. It is generated due to the decomposition of the waste itself, as well as the effects of precipitation and surface runoff. It is characterized by high ammonia nitrogen content, high organic matter concentration, a wide variety of pollutants, and an imbalance of nutrients for microorganisms. According to the *China Statistical Yearbook*, the amount of urban domestic waste collected in my country increased from 215.209 million tons in 2017 to 248.692 million tons in 2021, and is projected to reach 3.4 billion tons by 2050. With the continuous increase in waste treatment volume, the amount of landfill leachate generated is also showing a year-on-year upward trend. Studies have shown that every ton of waste landfilled (landfilling more than 60% of urban domestic waste) generates 0.05–0.2 tons of leachate. Because landfill leachate contains a large number of toxic and harmful substances and pathogenic microorganisms, direct discharge without treatment will not only seriously pollute the surrounding ecological environment but also threaten human health.

[0003] To ensure ecological security and human health, the Ministry of Ecology and Environment issued the "Pollution Control Standard for Municipal Solid Waste Landfills" (GB16889-2024), which stipulates the COD (Chemical Oxygen Demand) of directly discharged water pollutants. Cr The general emission limits for total nitrogen, ammonia nitrogen, and total phosphorus are less than 100 mg / L, 40 mg / L, 25 mg / L, and 3 mg / L, respectively, while the special emission limits are less than 60 mg / L, 20 mg / L, 8 mg / L, and 1.5 mg / L, respectively. This sets forth specific requirements for the efficient treatment of landfill leachate.

[0004] "Pretreatment + biological treatment + advanced treatment" is the main treatment process for landfill leachate. This process improves the biodegradability of the leachate through pretreatment, then uses biological treatment to remove ammonia nitrogen and simultaneously remove organic matter, and finally achieves compliant discharge of the leachate through advanced treatment. Biological denitrification is the core step of this process, typically accomplished through aerobic nitrification and anoxic / anaerobic denitrification. Under aerobic conditions, autotrophic nitrite-oxidizing bacteria and nitrifying bacteria convert ammonia nitrogen into nitrate via nitrite, and then under anaerobic / anoxic conditions, heterotrophic denitrifying bacteria convert nitrate into nitrogen gas, ultimately achieving total nitrogen removal.

[0005] However, the aforementioned biological denitrification processes have several shortcomings in treating landfill leachate, making it difficult for the effluent from biological treatment to meet the water quality requirements for advanced treatment, thus increasing the difficulty of advanced treatment. First, nitrifying bacteria are autotrophic bacteria with poor tolerance to organic matter. When high concentrations of organic matter enter the system, their growth is inhibited. Furthermore, the long cultivation period and slow growth of nitrifying bacteria result in a long treatment cycle and a weak ability to withstand system loads and shocks. Second, nitrifying and denitrifying bacteria have different oxygen and nutrient requirements. To ensure abundant biomass within the system, nitrification liquid and excess sludge need to be recycled. Dissolved oxygen enters the denitrification unit along with the recycled liquid from the aerobic nitrification unit, inhibiting the denitrification activity of heterotrophic denitrifying microorganisms. Third, traditional denitrification microorganisms have weak tolerance to high concentrations of ammonia nitrogen, and are prone to inactivation and death when treating landfill leachate. Therefore, improving the denitrification efficiency of existing biological processes is crucial to ensuring that landfill leachate meets discharge standards and reducing ecological impact.

[0006] The use of heterotrophic nitrifying-aerobic denitrifying bacteria (HNAD) to treat landfill leachate has significant advantages. On the one hand, HN-AD can simultaneously remove ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, and organic matter, shortening the denitrification cycle and reducing the toxic effects of nitrite accumulation on the bacteria. On the other hand, HN-AD is a heterotrophic aerobic microorganism; the supply of oxygen and organic matter can accelerate cell proliferation and differentiation, rapidly initiating biological denitrification and enhancing the tolerance of denitrifying bacteria to environmental shocks. Currently, more than 20 genera and over 100 strains of HNAD bacteria have been isolated and purified, mainly from sludge, water bodies, and certain extreme environments (low pH, low temperature, high salinity, etc.). HN-AD from landfill leachate has been rarely reported. Therefore, screening for highly efficient heterotrophic nitrifying-aerobic denitrifying bacteria from landfill leachate and studying their effects on landfill leachate is crucial. Summary of the Invention

[0007] The purpose of this invention is to provide a heterotrophic nitrifying-aerobic denitrifying bacterium tolerant to high ammonia nitrogen and high organic matter content, and its application in landfill leachate treatment. This heterotrophic nitrifying-aerobic denitrifying bacterium can remove ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen, and has important application prospects in the field of landfill leachate treatment with high ammonia nitrogen and high organic matter concentration.

[0008] In a first aspect, the present invention provides a heterotrophic nitrifying-aerobic denitrifying bacterium, wherein the heterotrophic nitrifying-aerobic denitrifying bacterium is Paracoccus pantotrophus HNAD-9, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 31698.

[0009] Among the heterotrophic nitrifying-aerobic denitrifying bacteria mentioned above, Paracoccus pantotrophus HNAD-9 is a Gram-negative bacterium with a spherical or short rod-shaped cell morphology, arranged singly, in pairs, or in clusters. The 16S rDNA sequence of this strain is shown in SEQ ID NO:1 of the sequence listing.

[0010] In the aforementioned heterotrophic nitrifying-aerobic denitrifying bacteria, the pantrophic Paracoccus pantotrophus HNAD-9 is derived from landfill leachate, wherein the COD concentration in the landfill leachate is greater than 10000 mg / L and the ammonia nitrogen concentration is greater than 400 mg / L. (The last sentence appears to be incomplete and possibly refers to a different topic.) Cr TN and NH4 + Landfill leachate with high ammonia nitrogen and high COD concentrations of -N was 19300 mg / L, 2320 mg / L and 2250 mg / L, respectively.

[0011] The method for isolating the aforementioned heterotrophic nitrifying-aerobic denitrifying bacteria includes the following steps:

[0012] 1) Using fresh landfill leachate as the inoculum source and LB liquid medium as the enrichment medium, the culture was carried out at 30℃ and 150rpm for 36h with constant temperature shaking; the bacterial suspension obtained by enrichment on LB medium was transferred to fresh heterotrophic nitrification liquid medium and cultured at 30℃ and 150rpm for 36h with constant temperature shaking.

[0013] 2) Take the bacterial suspension obtained in step 1) and cultured in heterotrophic nitrification liquid medium, and serially dilute it to 10⁻⁶ with sterile physiological saline. -1 10 -2 10 -3 10 -4 The bacteria were screened by spreading on heterotrophic nitrification solid medium; the plates were inverted in a constant temperature incubator and cultured at 30°C for several days. Colonies with different appearance characteristics were picked and isolated and purified on heterotrophic nitrification solid medium; the heterotrophic nitrification performance of the strains was analyzed to obtain strains with heterotrophic denitrification ability.

[0014] 3) Inoculate the heterotrophic nitrifying bacteria obtained in step 2) into LB liquid medium and culture at 30℃ and 150 r / min for 24 h. Then, take the bacterial suspension and drop it into bromothymol blue solid medium and culture it in a constant temperature incubator at 30℃ for 24-48 h. Select the strain clones that show a blue halo on the medium and inoculate them into aerobic denitrification medium for streak culture. Analyze the denitrification performance of the strains to obtain strains with aerobic denitrification ability.

[0015] In an embodiment of the present invention, the LB liquid culture medium has the following composition: 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl, and pH 7.0–7.2;

[0016] The heterotrophic nitrification medium is composed of the following: sodium succinate 15.34 g / L, Na2HPO4·12H2O 8.0 g / L, KH2PO4 1.5 g / L, (NH4)2SO4 1.0 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.01 g / L, trace elements 2 mL / L, and pH value of 7.0–7.2.

[0017] The composition of the aerobic denitrification medium is as follows: sodium succinate 15.04 g / L, Na2HPO4·12H2O 8.0 g / L, KH2PO4 1.5 g / L, KNO3 1.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.01 g / L, trace elements 2 mL / L, pH 7.0–7.2;

[0018] The trace element composition is as follows: MnSO4·4H2O 0.10 g / L, ZnSO4·7H2O 0.12 g / L, H3BO3 0.07 g / L, Na2MoO4·2H2O 0.04 g / L, CuSO4·5H2O 0.02 g / L, CoCl2·6H2O 0.04 g / L.

[0019] Secondly, the present invention provides a culture of the paracoccus pantotrophus HNAD-9, which is a substance obtained by culturing the paracoccus pantotrophus HNAD-9 in a bacterial culture medium.

[0020] The substances in the above-mentioned culture include the Paracoccus pantotrophus HNAD-9 (the bacterial cell itself) and the metabolites of the Paracoccus pantotrophus HNAD-9.

[0021] In the above-mentioned cultures, the bacterial culture medium can be a solid culture medium or a liquid culture medium.

[0022] The term "culture" refers to any liquid or solid culture medium that has grown a microbial community after artificial inoculation and cultivation. It is the product obtained by growing and / or amplifying microorganisms; it can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process. The term "culture" also includes passaged cultures obtained by subculturing microorganisms; these can be cultures of a single generation or mixtures of several generations.

[0023] Thirdly, the present invention provides metabolites of the paracoccus pantotrophus HNAD-9.

[0024] The term "metabolite" refers to the primary and / or secondary metabolites produced during microbial metabolism. Primary metabolism refers to the process by which microorganisms absorb various nutrients from the external environment and, through catabolism and anabolism, generate substances and energy to sustain life activities. The products of primary metabolism are called primary metabolites, such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids, and various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, and lipids. Secondary metabolism refers to the process by which microorganisms, at a certain growth stage, use primary metabolites as precursors to synthesize substances that have no clearly defined function for their life activities. The products of secondary metabolism are called secondary metabolites, and are mostly compounds with relatively complex molecular structures. Based on their functions, they can be classified into types such as antibiotics, hormones, alkaloids, and toxins.

[0025] Fourthly, the present invention provides a microbial agent comprising the aforementioned Paracoccus pantotrophus HNAD-9 or the aforementioned culture or the aforementioned metabolite.

[0026] The aforementioned microbial agents are those that remove or degrade ammonia nitrogen, nitrate nitrogen, and / or nitrite.

[0027] In the above-mentioned microbial agents, in addition to the active ingredients, a carrier is also included. The carrier can be a commonly used and biologically inert carrier in microbial agents. The carrier can be a solid or liquid carrier; the solid carrier can be a mineral material, plant material, or polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material can be at least one of corn flour, soybean flour, and starch; the polymer compound can be polyvinyl alcohol and / or polyethylene glycol; the liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent can be decane and / or dodecane.

[0028] The above-mentioned microbial agents can be in various formulations, such as liquids, emulsions, suspensions, powders, granules, wettable powders, or water-dispersible granules. Depending on the requirements, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc., may also be added to the microbial agents.

[0029] Fifthly, the present invention provides the use of the aforementioned Paracoccus pantotrophus HNAD-9, or the aforementioned culture, or the aforementioned metabolite, or the aforementioned bacterial agent in any of the following:

[0030] (A1) Removes ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen;

[0031] (A2) Prepare products that remove ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen;

[0032] (A3) Remove organic matter, ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen from landfill leachate;

[0033] (A4) Prepare products for removing organic matter, ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen from landfill leachate.

[0034] In one embodiment of the present invention, the nitrogen removal performance of heterotrophic nitrifying-aerobic denitrifying bacteria for ammonia nitrogen is evaluated by the following steps: the purified strain is inoculated into fresh LB liquid medium and cultured overnight at 30°C and 150 rpm; the activated strain HNAD-9 is transferred at an inoculum of 3% to a medium containing NH4+. + In a heterotrophic nitrification medium with -N as the sole nitrogen source, the culture was carried out under aerobic conditions at 30℃ and 150 rpm for 96 h, and the NH4+ was measured. + -N concentration. The results showed that when NH4+ + When -N is used as the sole nitrogen source, after 60 hours of culture of strain HNAD-9, NH4+ + The removal rate of -N was 88.93% after 72 hours of incubation. + The removal rate of -N reached its maximum at 98.73%.

[0035] In another embodiment of the present invention, the nitrogen removal performance of heterotrophic nitrifying-aerobic denitrifying bacteria for nitrate nitrogen was evaluated by the following steps: the purified strain was inoculated into fresh LB liquid medium and cultured overnight at 30°C and 150 rpm; the activated strain HNAD-9 was transferred at an inoculum of 3% to a medium containing NO3-. - In an aerobic denitrification medium with -N as the sole nitrogen source, the culture was carried out under aerobic conditions at 30℃ and 150 rpm for 96 h, and NO3 was measured. —N concentration. The results showed that when NO3... - When -N is used as the sole nitrogen source, NO3- was reduced after 72 hours of culture of strain HNAD-9. - The removal rate of -N reaches its maximum, at 100%.

[0036] In another embodiment of the present invention, the nitrogen removal performance of heterotrophic nitrifying-aerobic denitrifying bacteria for ammonia nitrogen and nitrate nitrogen was evaluated by the following steps: The purified paracoccus pantotrophus HNAD-9 strain was inoculated into fresh LB liquid medium and cultured overnight at 30°C and 150 rpm; the activated strain HNAD-9 was inoculated at a 3% inoculum into NH4+. + -N and NO3 — In a liquid culture medium with N as a mixed nitrogen source, the culture was carried out under aerobic conditions at 30°C and 150 rpm for 96 h, and the NH4 content was measured. + -N, NO3 - The results showed that after 36 h of culture of strain HNAD-9, the concentration of NH4+ changed. + -N was almost completely removed, NO3 - -N was also almost completely removed after 60 hours of culture of strain HNAD-9. The present invention's strain, Paracoccus pantotrophus HNAD-9, was cultured with NH4+. + -N and NO3 - When -N is a mixed nitrogen source, for NH4 + -N and NO3 - The removal efficiency of -N is 100%.

[0037] In a sixth aspect, the present invention provides a method for removing or degrading ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen, comprising the following steps: adding the Paracoccus pantotrophus HNAD-9 or the culture or the metabolite or the bacterial agent to the sample to be treated to react, so as to remove or degrade ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen.

[0038] In the above method, the reaction is carried out under aerobic conditions, with sodium succinate as the carbon source and ammonia nitrogen, nitrate nitrogen or nitrite nitrogen as the sole nitrogen source, or a mixture of any two or three of them as the nitrogen source.

[0039] In the above method, the sample to be tested can specifically be a body of water.

[0040] In a seventh aspect, the present invention provides a method for treating landfill leachate, characterized by comprising the following steps: adding the aforementioned Paracoccus pantotrophus HNAD-9 or the aforementioned culture or the aforementioned metabolite or the aforementioned bacterial agent to the landfill leachate for reaction, so as to remove or degrade organic matter, ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen in the landfill leachate; wherein the concentration of COD in the landfill leachate is greater than 1000 mg / L and the concentration of ammonium nitrogen is greater than 400 mg / L.

[0041] The term "landfill leachate" refers to a high-concentration organic wastewater originating from the moisture contained in the landfill itself, rainwater and snowmelt entering the landfill, and other moisture, after deducting the saturated water-holding capacity of the landfill and the cover soil layer, and passing through the landfill and cover soil layers. As an example, the COD in the landfill leachate is... Cr TN and NH4 + The concentrations of -N were 19300 mg / L, 2320 mg / L, and 2250 mg / L, respectively. In a specific embodiment of the present invention, the inoculum size of the Paracoccus pantotrophus HNAD-9 was 3%, and the reaction was carried out by culturing at 30°C and 150 rpm for 96 h. The results showed that the strain had an effect on COD in landfill leachate. Cr TN and NH4 + The removal efficiencies of -N were 91.14%, 94.87%, and 99.31%, respectively.

[0042] In an embodiment of the present invention, the heterotrophic nitrifying-aerobic denitrifying bacterial denitrification pathway is evaluated through the following steps: The purified bacterial strain is inoculated into fresh LB liquid medium and cultured overnight at 30°C and 150 rpm; 3% of the overnight culture is inoculated into sterilized heterotrophic nitrification liquid medium and cultured in a constant-temperature shaking incubator at 30°C and 150 rpm for 96 h, and the NH4+ ions are measured during the reaction. + -N, NH2OH, NO2 - -N, NO3 - Based on -N, TN, and bacterial growth, it is inferred that the ammonia nitrogen metabolism pathway of this strain is NH4+. + -N→NH2-OH→NO2 - -N→NO→N2O→N2, the metabolic pathway of nitrate nitrogen is NO3. - -N→NO2 - -N→NO→N2O→N2. This invention can simultaneously remove ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in the same heterotrophic and aerobic system, and there is no accumulation of nitrate nitrogen and nitrite nitrogen during the ammonia nitrogen removal process.

[0043] Eighthly, the present invention provides a product for removing ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen, wherein the active ingredient is the aforementioned Paracoccus pantotrophus HNAD-9 or the aforementioned culture or the aforementioned metabolite or the aforementioned bacterial agent.

[0044] The present invention has the following beneficial effects:

[0045] The pantotrophic paracoccus (Paracoccus pantotrophus) HNAD-9 provided by this invention exhibits highly efficient heterotrophic nitrification and aerobic denitrification performance under both aerobic and heterotrophic conditions, and is effective against NH4+. + -N and NO3 - The removal efficiencies of -N were 98.73% and 100%, respectively. Specifically, under aerobic conditions, when strain HNAD-9 grew and metabolized using ammonia nitrogen as the sole nitrogen source, the ammonia nitrogen removal rate was 98.73% within 72 hours. Strain HNAD-9 also grew and metabolized using nitrate nitrogen as the sole nitrogen source, achieving a 100% nitrate nitrogen removal rate within 72 hours. When strain HNAD-9 grew and metabolized using a mixed nitrogen source of ammonia nitrogen and nitrate nitrogen, both ammonia nitrogen and nitrate nitrogen were completely removed after 30 hours and 48 hours of reaction. The pantrophic Paracoccus pantotrophus HNAD-9 provided by this invention can also efficiently treat landfill leachate, significantly reducing COD in the leachate. Cr TN and NH4 + The removal efficiencies of -N were 91.14%, 94.87%, and 99.31%, respectively. Specifically, when the strain provided by this invention was inoculated into landfill leachate, after 124 hours of degradation, the COD in the landfill leachate was reduced. Cr TN and NH4 + The concentrations of -N decreased from 19300 mg / L, 2320 mg / L, and 2250 mg / L to 1710 mg / L, 119 mg / L, and 15.6 mg / L, respectively. This biological nitrogen removal method belongs to the field of novel biological nitrogen removal technology. It can simultaneously remove ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in the same heterotrophic and aerobic system, and there is no accumulation of nitrate nitrogen and nitrite nitrogen during the ammonia nitrogen removal process. It has good application prospects in practical applications.

[0046] Instructions for the Preservation of Biological Materials

[0047] Classification and nomenclature: Paracoccus pantotrophus;

[0048] Strain number: HNAD-9;

[0049] Abbreviation of depositary institution: CGMCC;

[0050] Name of the depository: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0051] Address of the depository: No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101;

[0052] Deposit date: August 22, 2024;

[0053] Registration number at the Preservation Center: CGMCC No. 31698. Attached Figure Description

[0054] Figure 1 This shows the growth of heterotrophic nitrifying bacteria in heterotrophic nitrifying liquid culture medium in Example 1.

[0055] Figure 2 This shows the growth of the dominant heterotrophic nitrifying bacteria in Example 1 in an aerobic denitrification liquid culture medium.

[0056] Figure 3 The results are Gram staining results for strain HNAD-9 in Example 1.

[0057] Figure 4 This is a scanning electron microscope image of strain HNAD-9 from Example 1.

[0058] Figure 5 The strain HNAD-9 in Example 3 of this invention degrades ammonia nitrogen (NH4). + The process changes of -N).

[0059] Figure 6 The strain HNAD-9 in Example 4 of this invention degrades nitrate nitrogen (NO3). - The process changes of -N).

[0060] Figure 7 The strain HNAD-9 in Example 5 of this invention degrades ammonia nitrogen (NH4). + -N) and nitrate nitrogen (NO3) - The process changes of -N).

[0061] Figure 8 The strain HNAD-9 in Example 6 of this invention degrades ammonia nitrogen (NH4). + Changes in metabolites during the -N process.

[0062] Figure 9 This describes the denitrification pathway of strain HNAD-9 in Example 6 of this invention.

[0063] Figure 10 The effects of strain HNAD-9 in Example 7 of this invention on CODCr, TN and NH4 in landfill leachate +-N removal efficiency. Detailed Implementation

[0064] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0065] Unless otherwise specified, the methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0066] The compositions of the culture media and trace elements in the following examples are as follows:

[0067] LB medium: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, pH 7.0–7.2.

[0068] Heterotrophic nitrification liquid culture medium: sodium succinate 15.34 g / L, Na2HPO4·12H2O 8.0 g / L, KH2PO4 1.5 g / L, (NH4)2SO4 1.0 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.01 g / L, trace elements 2 mL / L, pH 7.0–7.2.

[0069] Heterotrophic nitrification solid medium: sodium succinate 15.34 g / L, Na2HPO4·12H2O 8.0 g / L, KH2PO4 1.5 g / L, (NH4)2SO4 1.0 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.01 g / L, trace elements 2 mL / L, agar 20 g / L, pH 7.0–7.2.

[0070] Aerobic denitrification liquid culture medium: sodium succinate 15.04 g / L, Na2HPO4·12H2O 8.0 g / L, KH2PO4 1.5 g / L, KNO3 1.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.01 g / L, trace elements 2 mL / L, pH 7.0–7.2.

[0071] Aerobic denitrification solid medium: sodium succinate 15.04 g / L, Na2HPO4·12H2O 8.0 g / L, KH2PO4 1.5 g / L, KNO3 1.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.01 g / L, trace elements 2 mL / L, agar 20 g / L, pH 7.0–7.2.

[0072] Mixed nitrogen source liquid culture medium: sodium succinate 14.466 g / L, Na2HPO4·12H2O 8.0 g / L, KH2PO4 1.5 g / L, (NH4)2SO4 0.472 g / L, KNO3 0.722 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 0.01 g / L, NaCl 150 g / L, trace elements 2 mL / L, pH 7.0–7.2.

[0073] Trace elements: MnSO4·4H2O 0.10g / L, ZnSO4·7H2O 0.12g / L, H3BO3 0.07g / L, Na2MoO4·2H2O 0.04g / L, CuSO4·5H2O 0.02g / L, CoCl2·6H2O 0.04g / L.

[0074] Example 1: Screening of heterotrophic nitrifying-aerobic denitrifying bacteria

[0075] Take 2 mL of fresh landfill leachate (COD) Cr TN and NH4 + The concentrations of -N (19300 mg / L, 2320 mg / L, and 2250 mg / L, respectively) were inoculated into 50 mL of fresh LB liquid medium and cultured at 30 °C with constant temperature shaking at 150 rpm for 36 h. 2 mL of the bacterial suspension enriched in LB medium was then added to 50 mL of fresh heterotrophic nitrification liquid medium and cultured at 30 °C with constant temperature shaking at 150 rpm for 36 h.

[0076] Take 2 mL of bacterial suspension cultured in heterotrophic nitrification liquid medium and serially dilute it to 10⁻⁶ with sterile physiological saline. -1 10 -2 10 -3 10 -4 The strains were screened by plating on heterotrophic nitrification solid medium. The plates were inverted and incubated at 30°C for several days. Colonies with different appearance characteristics were then picked for isolation and purification. The purified strains were inoculated into heterotrophic nitrification liquid medium at a 3% inoculum and incubated at 30°C and 150 rpm for 42 hours. The growth of the strains in the culture medium was then measured (OD). 600 Changes over time ( Figure 1This yielded four dominant heterotrophic nitrifying bacteria strains: HNAD-8, HNAD-9, HNAD-10, and HNAD-11.

[0077] The aforementioned dominant heterotrophic nitrifying bacteria were inoculated into LB liquid medium using an inoculation loop (a small amount of bacteria was directly inoculated using an inoculation loop). After incubation at 30°C and 150 rpm for 24 h, 0.2 mL of the bacterial suspension was dropwise onto bromothymol blue solid medium and incubated at 30°C for 24–48 h. Clones exhibiting a blue halo on the medium were selected and inoculated into aerobic denitrification solid medium for isolation and purification. The purified strains were inoculated into aerobic denitrification liquid medium at a 3% inoculation rate and incubated at 30°C and 150 rpm for 42 h. The growth of the strains (OD) was then measured. 600 Changes over time Figure 2 This yields the dominant heterotrophic nitrifying-aerobic denitrifying bacteria HNAD-9.

[0078] HNAD-9 was identified as a Gram-negative bacterium, with a morphological form of spherical or short rod-shaped cells, arranged singly, in pairs, or in clusters. Gram staining images of the bacteria are shown below. Figure 3 As shown, the scanning electron microscope image is as follows: Figure 4 As shown.

[0079] Example 2: Molecular biological identification of bacterial strains

[0080] Total HNAD-9 DNA was extracted using the Omega 200T bacterial DNA extraction kit, following the kit's instructions. The extracted genome was then subjected to 16S rDNA PCR amplification in a PCR instrument to determine the bacterial species. The primers used for the amplification experiment were P1-27 F (5'-AGAGTTTGATCCTGGCTCAG-3') and P2-1492 R (5'-GGTTACCTTGTTACGACTT-3').

[0081] The obtained PCR products were sequenced. After the sequencing results were returned, the sequences were submitted to NCBI for BLAST (Basic Local Alignment Search Tool) comparison. The bacterial species were determined by finding the same or similar strain sequences in the database.

[0082] Based on the sequencing results (nucleotide sequence of 16S rDNA of Paracoccus pantotrophus HNAD-9 SEQ ID NO:1), strain HNAD-9 was identified as Paracoccus pantotrophus and designated as Paracoccus pantotrophus strain HNAD-9. This strain was deposited on August 22, 2024, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCCNo. 31698.

[0083] Example 3: Utilization of ammonia nitrogen by strain HNAD-9

[0084] The purified strain was inoculated into fresh LB liquid medium and cultured overnight at 30°C and 150 rpm. The activated strain HNAD-9 was then transferred at a 3% inoculum to a medium containing NH4+. + In a heterotrophic nitrification liquid medium with -N as the nitrogen source, the culture was carried out under aerobic conditions at 30℃ and 150 rpm for 96 h, and the NH4+ was measured. + -N concentration and bacterial growth, experimental results are as follows: Figure 5 As shown. When NH4 + When NH4+ was used as the sole nitrogen source, strain HNAD-9 grew rapidly, reaching a maximum growth rate of 1.753 after 60 hours of culture. Simultaneously, NH4+ in the culture medium... + The concentration of NH4+ decreased significantly with the growth of strain HNAD-9. After 60 h of culture, the concentration of NH4+ decreased. + The removal rate of -N was 88.93% after 72 hours of incubation. + The removal rate of -N reached its maximum at 98.73%. Strain HNAD-9 can efficiently utilize NH4+ in water. + -N is involved in growth and metabolism.

[0085] Example 4: Utilization of nitrate nitrogen by strain HNAD-9

[0086] The purified strain was inoculated into fresh LB liquid medium and cultured overnight at 30°C and 150 rpm. The activated strain HNAD-9 was then transferred at a 3% inoculum to a medium containing NO3. - In an aerobic denitrification liquid medium with nitrogen (N-N) as the nitrogen source, the culture was carried out under aerobic conditions at 30℃ and 150 rpm for 96 h, and NO3 was measured. - -N concentration and bacterial growth are as follows Figure 6 As shown. When NO3 -When -N is used as the sole nitrogen source, strain HNAD-9 rapidly enters the logarithmic growth phase, reaching its maximum growth at 72 hours of culture, at which point the growth rate of strain HNAD-9 reaches 1.788. Simultaneously, NO3- in the culture medium... - The concentration of NO3- decreased significantly with the growth of strain HNAD-9. After 72 h of culture of strain HNAD-9, the concentration of NO3- decreased significantly. - The removal rate of -N reached its maximum, at 100%. The strain can efficiently utilize NO3- in the water. - -N is involved in growth and metabolism.

[0087] Example 5: Utilization of mixed nitrogen sources (ammonia nitrogen and nitrate nitrogen) by strain HNAD-9

[0088] The purified strain was inoculated into fresh LB liquid medium and cultured overnight at 30°C and 150 rpm. The activated strain HNAD-9 was then transferred at a 3% inoculum to a medium containing NH4+. + -N and NO3 - In a mixed nitrogen source liquid medium with -N as the mixed nitrogen source, the HNAD-9 strain was cultured under aerobic conditions at 30°C and 150 rpm for 96 h. The growth and nitrogen metabolism of the strain are as follows: Figure 7 As shown. When NH4 + -N and NO3 - When -N is used as a mixed nitrogen source, NH4 + -N and NO3 - -N is removed at the same time, but NH4 + The removal rate of -N is compared to NO3. - -N will be faster. When strain HNAD-9 was cultured for 36 hours, NH4+... + -N was almost completely removed, NO3 - -N was almost completely removed after 60 hours of culture in strain HNAD-9. The bacterial growth reached its maximum of 1.621 after 84 hours of culture. After this point, as strain HNAD-9 entered its death phase, NH4+ was released through autolysis. + -N, leading to NH4+ in the culture medium + The content of -N increased slightly.

[0089] Example 6: Analysis of the nitrogen removal pathway of heterotrophic nitrification-aerobic denitrification bacteria HNAD-9

[0090] Currently, there are three main nitrogen metabolism pathways in HNAD denitrifying bacteria: NO3-, NO2-, and NO3-. - / NO2 - The NO pathway, the NO pathway, and the N2O pathway. NO3 - / NO2 -The pathway refers to the process by which the strain produces NH4 under aerobic conditions. + -N is converted to NO2 via NH2OH. - -N and NO3 - -N, then NO2 - -N and NO3 - -N is converted to N2, thereby removing nitrogen from the water; the NO pathway involves the strain converting NH4+ into N2 under aerobic conditions. + -N is converted to NH2OH, and the resulting NH2OH does not pass through NO2. - -N and NO3 - -N is directly converted to NO, and then to N2 via N2O; the N2O pathway refers to the process by which the strain converts NH4+ into NO under aerobic conditions. + -N is converted to NH2OH, and the resulting NH2OH does not pass through NO2. - -N and NO3 - -N is directly converted to N2O, and then directly converted to N2 and completely removed.

[0091] When strain HNAD-9 reacts with NH4 + When -N is used as the sole nitrogen source, in NH4 + In the metabolism of -N, intermediate products NH2OH and NO2 were detected. - -N( Figure 8 According to strain HNAD-9, NH4 + The degradation process of -N was investigated, and a heterotrophic nitrification-aerobic denitrification pathway for strain HNAD-9 was proposed, such as... Figure 9 As shown. NH4 + -N is first converted to NH2OH, and the generated NH2OH is immediately converted to NO2. - -N, NO2 - -N is then converted into gaseous nitrogen through denitrification, ultimately achieving nitrogen removal from the water. To further verify this denitrification pathway, the effects of strain HNAD-9 on the denitrification intermediates NH2OH and NO2 were analyzed. - -N and NO3 - The utilization of -N was investigated as follows: The purified bacterial strain was inoculated into fresh LB liquid medium and cultured overnight at 30°C and 150 rpm; 3% of the overnight culture was then inoculated into sterilized heterotrophic nitrification liquid medium and placed in a constant temperature shaking incubator at 30°C and 150 rpm for 96 h. NH4+ was measured during the reaction. + -N, NH2OH, NO2 - -N and NO3 - The changes in -N concentration indicated that strain HNAD-9 could utilize not only NH4+, but also... + -N is used for growth and metabolism, and can also utilize NH2OH and NO2.- -N and NO3 - -N, and can completely remove it, suggesting that the strain's ammonia nitrogen metabolism pathway is NH4+. + -N→NH2-OH→NO2 - -N→NO→N2O→N2, the metabolic pathway of nitrate nitrogen is NO3. - -N→NO2 - -N→NO→N2O→N2.

[0092] Example 7: Study on the effect of strain HNAD-9 on landfill leachate treatment

[0093] The landfill leachate used in the experiment came from a landfill in Beijing, and its basic water quality indicators are shown in Table 1. (COD of the landfill leachate) Cr TN, NH4 + -N, NO3 - -N and NO2 - The concentrations of nitrogen (N) were 19300 mg / L, 2320 mg / L, 2250 mg / L, 1.81 mg / L, and 2.66 mg / L, respectively. The landfill leachate had high concentrations of organic matter and ammonia nitrogen, with ammonia nitrogen contributing primarily to the total nitrogen. Treatment of landfill leachate using the heterotrophic nitrifying-aerobic denitrifying strain HNAD-9 is feasible.

[0094] Table 1. Changes in pollutant indicators in landfill leachate before and after treatment.

[0095]

[0096] The purified strain was inoculated into fresh LB liquid medium and cultured overnight at 30°C and 150 rpm. The activated strain HNAD-9 was then transferred to landfill leachate at a 3% inoculation rate and cultured aerobically at 30°C and 150 rpm for 124 h. The COD in the landfill leachate was then measured. Cr TN, NH4 + -N, NO3 - -N and NO2 - The changes in -N content were observed in the experimental results as follows: Figure 10 As shown in Table 1, each experiment was conducted in triplicate, with landfill leachate uninoculated with strain HNAD-9 serving as a blank control. Table 1 demonstrates that strain HNAD-9 can efficiently treat landfill leachate; after 124 hours of degradation, the COD in the leachate was significantly reduced. Cr TN and NH4 + The concentrations of -N decreased from 19300 mg / L, 2320 mg / L, and 2250 mg / L to 1710 mg / L, 119 mg / L, and 15.6 mg / L, respectively. Figure 10 As shown, strain HNAD-9 has an effect on CODCr TN and NH4 + The removal efficiencies of NO3- were 91.14%, 94.87%, and 99.31%, respectively. Furthermore, compared to the untreated landfill leachate, the NO3- removal efficiency was significantly reduced after treatment with strain HNAD-9. - The concentration of -N increased from 1.81 mg / L to 80.3 mg / L, NO2 — The nitrogen (N) remained essentially unchanged, indicating that strain HNAD-9 converted NH4+ into nitrogen through heterotrophic nitrification. + -N is converted to NO3 - -N, NO3 - -N is converted into N2 through aerobic denitrification.

[0097] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including modifications made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Paracoccus pantrophicus ( Paracoccus pantotrophus HNAD-9, its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 31698.

2. A microbial agent, characterized in that, Contains the pantrophic paracoccus as described in claim 1 ( Paracoccus pantotrophus HNAD-9.

3. The microbial agent according to claim 2, characterized in that: The bacterial agent is a bacterial agent that removes or degrades ammonia nitrogen, nitrate nitrogen, and / or nitrite.

4. The pantrophic paracoccus as described in claim 1 ( Paracoccus pantotrophus The use of HNAD-9 or the microbial agent according to claim 2 or 3 in any of the following: (A1) Removes ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen; (A2) Prepare products that remove ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen; (A3) Remove organic matter, ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen from landfill leachate; (A4) Prepare products for removing organic matter, ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen from landfill leachate.

5. A method for removing ammonia nitrogen, nitrate nitrogen, and / or nitrite nitrogen, comprising the following steps: adding the *Paragonimococcus pantrophicans* as described in claim 1 to the sample to be treated. Paracoccus pantotrophus The sample is reacted with HNAD-9 or the bacterial agent of claim 2 or 3 to remove ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen from the sample.

6. The method according to claim 5, characterized in that: The reaction is carried out under aerobic conditions, using sodium succinate as the carbon source and ammonia nitrogen, nitrate nitrogen or nitrite nitrogen as the sole nitrogen source, or a mixture of any two or three of them as the nitrogen source.

7. A method for treating landfill leachate, characterized in that, The steps include: adding the pantrophic paracoccus (as described in claim 1) to the landfill leachate. Paracoccus pantotrophus The leachate is reacted with HNAD-9 or the microbial agent of claim 2 or 3 to remove organic matter, ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen from the landfill leachate; the concentration of COD in the landfill leachate is ≥10000 mg / L and the concentration of ammonia nitrogen is ≥400 mg / L.

8. A product for removing ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen, wherein the active ingredient is the pantrophic paracoccus described in claim 1 (…). Paracoccus pantotrophus HNAD-9 or the bacterial agent as described in claim 2 or 3.

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