Heterotrophic nitrification-aerobic denitrification bacterium resistant to high ammonia nitrogen and high organic matters and application of heterotrophic nitrification-aerobic denitrification bacterium in landfill leachate treatment

Abstract

The invention discloses a heterotrophic nitrification-aerobic denitrification bacterium resistant to high ammonia nitrogen and high organic matters and application of the heterotrophic nitrification-aerobic denitrification bacterium in landfill leachate treatment. The heterotrophic nitrification-aerobic denitrification bacterium provided by the invention is Paracoccus pantotrophus HNAD-9, the strain is preserved in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms on August 22, 2024, and the preservation number of the strain is CGMCC No.31698. The invention further discloses a preparation method of the heterotrophic nitrification-aerobic denitrification bacterium. According to the method, ammonia nitrogen, nitrate nitrogen and nitrite nitrogen can be removed in the same heterotrophic and aerobic system at the same time, accumulation of nitrate nitrogen and nitrite nitrogen is avoided in the ammonia nitrogen removal process, and the method has important application prospects in the field of high-ammonia-nitrogen and high-organic-matter-concentration landfill leachate treatment.

Description

Technical Field

[0001] The invention relates to the technical field of environmental microorganisms, and in particular to application of a salt-tolerant heterotrophic nitrification-aerobic denitrification bacterium in the treatment of landfill leachate. Background Art

[0002] Garbage leachate is a kind of complex high-concentration organic wastewater produced during the garbage treatment (landfill, incineration, etc.) and stacking process due to the decomposition of the garbage itself, as well as the influence of precipitation and surface runoff. It has the characteristics of high ammonia nitrogen content, high organic matter concentration, many types of pollutants, and imbalance of microbial nutrient elements. According to the "China Statistical Yearbook", the amount of urban domestic garbage collected and transported in my country has increased from 215.209 million tons in 2017 to 248.692 million tons in 2021. It is estimated that by 2050, my country's garbage production will increase to 3.4 billion tons. With the continuous growth of garbage treatment, the amount of garbage leachate produced has also shown an increasing trend year by year. Studies have shown that every ton of garbage landfilled (landfill treatment of urban domestic garbage is greater than 60%) will produce 0.05 to 0.2 tons of garbage leachate. Since the garbage leachate contains a large amount 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] In order to ensure ecological safety and human health, the Ministry of Ecology and Environment has issued the "Pollution Control Standards for Municipal Waste Landfills" (GB16889-2024), which stipulates the direct discharge of water pollutants COD Cr The general emission limits of total nitrogen, ammonia nitrogen and total phosphorus are less than 100 mg / L, 40 mg / L, 25 mg / L and 3 mg / L respectively, and the special emission limits are less than 60 mg / L, 20 mg / L, 8 mg / L and 1.5 mg / L respectively, which puts forward specific requirements for the efficient treatment of leachate.

[0004] "Pretreatment + biochemical treatment + deep treatment" is the main treatment process for landfill leachate at present. This process improves the biodegradability of landfill leachate through pretreatment, then uses biochemical treatment to remove ammonia nitrogen in the landfill leachate, and simultaneously removes organic matter, and finally achieves the discharge of landfill leachate in compliance with standards through deep treatment. Biochemical denitrification is the core step of the above process, which is usually completed through aerobic nitrification and anoxic / anaerobic denitrification, that is, under aerobic conditions, autotrophic nitrite bacteria and nitrifying bacteria convert ammonia nitrogen into nitrate through nitrite, and then under anaerobic / anoxic conditions, heterotrophic denitrifying bacteria convert nitrate into nitrogen gas, and finally achieve total nitrogen removal.

[0005] However, the above-mentioned biological denitrification process has many shortcomings when treating landfill leachate, which makes it difficult for the biochemical treatment effluent to meet the water quality requirements of deep treatment, thereby increasing the difficulty of deep treatment. First, nitrifying bacteria are autotrophic bacteria with poor tolerance to organic matter. When high-concentration organic matter enters the system, its growth is inhibited. In addition, due to the long culture cycle and slow growth of nitrifying bacteria, the process treatment cycle is long, and the load and impact bearing capacity of the system are weak; second, nitrifying bacteria and denitrifying bacteria have different requirements for oxygen and nutrients. In order to ensure abundant biomass in the system, nitrification liquid and residual sludge need to be refluxed. Dissolved oxygen enters the denitrification unit with the reflux liquid of the aerobic nitrification unit, resulting in the inhibition of the denitrification activity of heterotrophic denitrifying microorganisms; third, traditional denitrifying microorganisms have weak tolerance to high concentrations of ammonia nitrogen, and are prone to inactivation and death when treating landfill leachate. Therefore, in order to ensure that landfill leachate meets the discharge standards and reduce the impact on the ecological environment, it is crucial to improve the denitrification efficiency of existing biochemical processes.

[0006] There are significant advantages in using heterotrophic nitrifying-aerobic denitrifying bacteria (HNAD) to treat landfill leachate. On the one hand, HN-AD can achieve the simultaneous removal of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and organic matter, which not only shortens the denitrification cycle, but also reduces the toxic effects of nitrite nitrogen accumulation on bacteria; on the other hand, HN-AD belongs to heterotrophic aerobic microorganisms. The supply of oxygen and organic matter can accelerate the proliferation and differentiation of cells, which can not only quickly start biological denitrification, but also enhance the tolerance of denitrifying bacteria to environmental shocks. At present, more than 20 genera and more than 100 strains of HNAD have been isolated and purified. They are mainly derived from sludge, water bodies and some extreme environments (low pH, low temperature, high salt, etc.). There are few reports on HN-AD from landfill leachate. Therefore, it is very important to screen efficient heterotrophic nitrifying-aerobic denitrifying bacteria from landfill leachate and study their effects on landfill leachate. Summary of the invention

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

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

[0009] Among the above heterotrophic nitrification-aerobic denitrification bacteria, the Paracoccus pantotrophus HNAD-9 is a Gram-negative bacterium with spherical or short rod-shaped cells arranged singly, in pairs or in piles. The 16S rDNA sequence of the strain is shown in the sequence table SEQ ID NO: 1.

[0010] In the above-mentioned heterotrophic nitrification-aerobic denitrification bacteria, the Paracoccus pantotrophus HNAD-9 is derived from landfill leachate, wherein the concentration of COD in the landfill leachate is greater than 10000 mg / L, and the concentration of ammonia nitrogen is greater than 400 mg / L. Cr TN and NH4 + The concentrations of -N in the landfill leachate with high ammonia nitrogen and high COD are 19300mg / L, 2320mg / L and 2250mg / L respectively.

[0011] In the above-mentioned heterotrophic nitrification-aerobic denitrification bacteria, the method for isolating the strain comprises the following steps:

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

[0013] 2) Take the bacterial suspension obtained in step 1) and culture it in heterotrophic nitrification liquid medium, and dilute it to 10% with sterile physiological saline. -1 , 10 -2 , 10 -3 , 10 -4 , and screened on a heterotrophic nitrification solid medium; invertedly placed the plate in a constant temperature incubator for culture at 30°C for several days, and then selected colonies with different appearance characteristics and separated and purified on a heterotrophic nitrification solid medium; analyzed the heterotrophic nitrification performance of the strain, and obtained a strain with heterotrophic denitrification ability;

[0014] 3) The heterotrophic nitrifying bacteria obtained in step 2) are inoculated into LB liquid culture medium, and cultured at 30° C. and 150 r / min for 24 h, then the bacterial suspension is taken and dripped into bromothymol blue solid culture medium, and cultured in a constant temperature incubator at 30° C. for 24 to 48 h, and the strain clone with a blue halo on the culture medium is selected and inoculated into an aerobic denitrification culture medium for streak culture; the denitrification performance of the strain is analyzed to obtain a strain with aerobic denitrification ability.

[0015] In an embodiment of the present invention, the LB liquid culture medium is composed of: 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl, and a pH of 7.0 to 7.2;

[0016] The heterotrophic nitrification medium is composed of: 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 value is 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] In a second aspect, 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] In the above culture, the substance includes the Paracoccus pantotrophus HNAD-9 (the bacteria itself) and metabolites of the Paracoccus pantotrophus HNAD-9.

[0021] In the above culture, the bacterial culture medium may be a solid culture medium or a liquid culture medium.

[0022] The term "culture" refers to a general term for liquid or solid culture media that have a microbial population after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying microorganisms, which 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 culture process. The term "culture" also includes a subculture obtained by subculturing microorganisms, which can be a culture of a certain generation or a mixture of several generations.

[0023] In a third aspect, the present invention provides metabolites of Paracoccus pantotrophus HNAD-9.

[0024] The term "metabolite" refers to the primary metabolites and / or secondary metabolites produced during the metabolism of microorganisms. Primary metabolism refers to the process in which microorganisms absorb various nutrients from the outside world and generate substances and energy to maintain life activities through catabolism and anabolism. The products of primary metabolism are primary metabolites, such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids and other monomers, as well as various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, lipids, etc. Secondary metabolism refers to the process in which microorganisms use primary metabolites as precursors to synthesize some substances that have no clear function in the life activities of microorganisms during a certain growth period. The products of secondary metabolism are secondary metabolites, most of which are compounds with relatively complex molecular structures. According to their functions, they can be divided into types such as antibiotics, hormones, alkaloids, toxins, etc.

[0025] In a fourth aspect, the present invention provides a bacterial agent comprising the Paracoccus pantotrophus HNAD-9 or the culture or the metabolite.

[0026] In the above-mentioned bacterial agent, the bacterial agent is a bacterial agent that removes or degrades ammonia nitrogen, nitrate nitrogen and / or nitrite.

[0027] In the above-mentioned microbial agent, in addition to the active ingredient, the microbial agent also contains a carrier. The carrier can be a carrier commonly used in microbial agents and is biologically inert. The carrier can be a solid carrier or a liquid carrier; the solid carrier can be a mineral material, a plant material or a 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 polyglycol; 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 bacterial agent can be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule. According to the needs, surfactant (such as Tween 20, Tween 80, etc.), binder, stabilizer (such as antioxidant), pH adjuster, etc. can also be added to the bacterial agent.

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

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

[0031] (A2) preparing a product for removing ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen;

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

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

[0034] In one embodiment of the present invention, the denitrification performance of heterotrophic nitrification-aerobic denitrification bacteria on ammonia nitrogen was evaluated by the following steps: the purified strain was inoculated into fresh LB liquid medium and cultured overnight at 30°C and 150rpm; the activated strain HNAD-9 was transferred to NH4 + -N was the only nitrogen source in a heterotrophic nitrification medium, and cultured at 30°C and 150 rpm for 96 h under aerobic conditions. NH4 + -N concentration. The results show that when NH4 + When -N was used as the only nitrogen source, NH4 + -N removal rate was 88.93%, and NH4 + The removal rate of -N reached the maximum, which was 98.73%.

[0035] In another embodiment of the present invention, the denitrification performance of heterotrophic nitrification-aerobic denitrification bacteria on 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 to NO3 with an inoculum of 3% - -N was the only nitrogen source in the aerobic denitrification medium, and the NO3 —N concentration. The results show that when NO3 - When -N was used as the only nitrogen source, NO3 - The removal rate of -N reached the maximum, which was 100%.

[0036] In another embodiment of the present invention, the denitrification performance of heterotrophic nitrification-aerobic denitrifiers on ammonia nitrogen and nitrate nitrogen was evaluated by the following steps: the purified Paracoccus pantotrophus HNAD-9 strain was inoculated into fresh LB liquid culture medium and cultured at 30°C and 150 rpm overnight; the activated strain HNAD-9 was inoculated at an inoculum of 3% into NH4 + -N and NO3 — The liquid medium with N as a mixed nitrogen source was cultured at 30°C and 150 rpm for 96 h under aerobic conditions to determine NH4 + -N, NO3 - -N concentration changes. The results showed that when strain HNAD-9 was cultured for 36 hours, NH4 + -N is almost completely removed, NO3 - -N was also completely removed after the strain HNAD-9 was cultured for 60 hours. + -N and NO3 - -N is a mixed nitrogen source, for NH4 + -N and NO3 - The removal efficiency of -N was 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 thereof as the nitrogen source.

[0039] In the above method, the sample to be tested may specifically be water.

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

[0041] The term "landfill leachate" refers to a high-concentration organic wastewater formed by the water contained in the garbage in the landfill, the rainwater and snow water entering the landfill, and other water, minus the saturated water holding capacity of the garbage and the covering soil layer, and passing through the garbage layer and the covering soil layer. As an example, the COD in the landfill leachate is Cr TN and NH4 + -N concentrations were 19300 mg / L, 2320 mg / L and 2250 mg / L respectively. In a specific embodiment of the present invention, the inoculation amount of the Paracoccus pantotrophus HNAD-9 was 3%, and the reaction was cultured at 30°C and 150 rpm for 96 hours. The results showed that the strain had an effect on COD 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 nitrification-aerobic denitrification bacteria denitrification pathway is evaluated by the following steps, including the following steps: inoculating the purified strain into a fresh LB liquid culture medium and culturing it at 30°C and 150rpm overnight; inoculating 3% of the overnight cultured bacterial solution into a sterilized heterotrophic nitrification liquid culture medium, and placing it in a constant temperature shaking incubator at 30°C and 150rpm for 96h, and measuring the NH4 + -N, NH2OH, NO2 - -N, NO3 - -N, TN and bacterial growth, it is inferred that the ammonia nitrogen metabolism pathway of the 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. The present invention can remove ammonia nitrogen, nitrate nitrogen and nitrite nitrogen simultaneously in the same heterotrophic and aerobic system, and there is no accumulation of nitrate nitrogen and nitrite nitrogen in the process of removing ammonia nitrogen.

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

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

[0045] The Paracoccus pantotrophus HNAD-9 provided by the present invention has high-efficiency heterotrophic nitrification and aerobic denitrification performance under aerobic and heterotrophic conditions, and has good denitrification performance for NH4 + -N and NO3 - -N removal efficiencies are 98.73% and 100% respectively. Specifically, under aerobic conditions, when strain HNAD-9 grows and metabolizes with ammonia nitrogen as the only nitrogen source, the removal rate of ammonia nitrogen is 98.73% within 72 hours. Strain HNAD-9 also grows and metabolizes with nitrate nitrogen as the only nitrogen source, and the removal rate of nitrate nitrogen is 100% within 72 hours. When strain HNAD-9 grows and metabolizes with a mixed nitrogen source of ammonia nitrogen and nitrate nitrogen, ammonia nitrogen and nitrate nitrogen can be completely removed after 30 hours and 48 hours of reaction. The Paracoccus pantotrophus HNAD-9 provided by the present invention can also efficiently treat landfill leachate, and can remove COD Cr TN and NH4 + The removal efficiencies of -N were 91.14%, 94.87% and 99.31% respectively. Specifically, the strain provided by the present invention was inoculated into the landfill leachate, and after 124 hours of degradation, the COD Cr TN and NH4 + The concentration of -N decreased from 19300mg / L, 2320mg / L and 2250mg / L to 1710mg / L, 119mg / L and 15.6mg / L respectively. This biological denitrification method belongs to the field of new biological denitrification technology. It can not only remove ammonia nitrogen, nitrate nitrogen and nitrite nitrogen at the same time in the same heterotrophic and aerobic system, but also there is no accumulation of nitrate nitrogen and nitrite nitrogen in the process of ammonia nitrogen removal, and has a good application prospect in practical applications.

[0046] Description of biological material deposit

[0047] Classification name: Paracoccus pantotrophus;

[0048] Strain ID: HNAD-9;

[0049] Abbreviation of depository unit: CGMCC;

[0050] Name of depository: General Microbiology Center of China Microbiological Culture Collection Administration;

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

[0052] Deposit date: August 22, 2024;

[0053] The registration number of the Collection Center is: CGMCC No.31698. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The growth conditions of the heterotrophic nitrifying bacteria in Example 1 in the heterotrophic nitrification liquid culture medium.

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

[0056] Figure 3 This is the Gram staining result of strain HNAD-9 in Example 1.

[0057] Figure 4 This is a scanning electron microscopic picture of the strain HNAD-9 in Example 1.

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

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

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

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

[0062] Fig. 9 This is the denitrification pathway of strain HNAD-9 in Example 6 of the present invention.

[0063] Fig.10 The strain HNAD-9 in Example 7 of the present invention is used to analyze the CODCr, TN and NH4 in the landfill leachate. +-N removal efficiency. DETAILED DESCRIPTION

[0064] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0065] The methods used in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0066] The compositions of each culture medium 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 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, 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 culture medium: sodium succinate 15.04g / L, Na2HPO4·12H2O 8.0g / L, KH2PO41.5g / L, KNO3 1.5g / L, MgSO4·7H2O 0.1g / L, FeSO4·7H2O 0.01g / L, trace elements 2mL / L, agar 20g / L, pH 7.0-7.2.

[0072] Mixed nitrogen source liquid culture medium: sodium succinate 14.466g / L, Na2HPO4·12H2O 8.0g / L, KH2PO4 1.5g / L, (NH4)2SO4 0.472g / L, KNO3 0.722g / L, MgSO4·7H2O 0.1g / L, FeSO4·7H2O 0.01g / L, NaCl150, trace elements 2mL / 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 nitrification-aerobic denitrifying bacteria

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

[0076] Take 2 mL of bacterial suspension cultured in heterotrophic nitrification liquid medium and dilute it to 10 -1 , 10 -2 , 10 -3 , 10 -4 , and screened on heterotrophic nitrification solid medium. The plate was inverted in a constant temperature incubator and cultured at 30°C for several days, and colonies with different appearance characteristics were picked for separation and purification. The isolated and purified strains were inoculated into heterotrophic nitrification liquid medium at a 3% inoculation rate, and cultured at 30°C and 150r / min for 42h, and the growth of the strains in the culture medium (OD 600 ) changes over time ( Figure 1), that is, 4 dominant heterotrophic nitrifying bacteria HNAD-8, HNAD-9, HNAD-10, and HNAD-11 were obtained.

[0077] Use an inoculation loop to inoculate the above-mentioned dominant heterotrophic nitrifying bacteria into LB liquid culture medium (use an inoculation loop to pick up a small amount of bacteria for direct inoculation), and after culturing at 30°C and 150r / min for 24h, take 0.2mL of bacterial suspension and drip it into bromothymol blue solid culture medium, and culture it in a 30°C constant temperature incubator for 24-48h. Select the strain clone with a blue halo on the culture medium and inoculate it into aerobic denitrification solid culture medium for separation and purification. The isolated and purified strain was inoculated into aerobic denitrification liquid culture medium at an inoculation rate of 3%, and cultured at 30°C and 150r / min for 42h, and the growth of the strain (OD 600 ) changes over time ( Figure 2 ), that is, the dominant heterotrophic nitrification-aerobic denitrification bacteria HNAD-9 was obtained.

[0078] It was identified that HNAD-9 is a Gram-negative bacterium. The bacterial morphology is spherical or short rod-shaped cells, arranged alone, in pairs or in piles. The Gram staining picture of the bacteria is as follows: Figure 3 As shown in the scanning electron microscope photos Figure 4 shown.

[0079] Example 2: Molecular Biological Identification of Bacteria

[0080] HNAD-9 total DNA was extracted using the Omega 200T bacterial DNA extraction kit, and the specific extraction steps were carried out according to the instructions of the kit; the extracted genome was subjected to a 16s rDNA PCR amplification experiment in a PCR instrument to determine the species of the bacteria. The primers used in the amplification experiment were P1-27 F (5'-AGAGTTTGATCCTGGCTCAG-3') and P2-1492 R (5'-GGTTACCTTGTTACGACTT-3').

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

[0082] According to the sequencing results (nucleotide sequence of Paracoccus pantotrophus HNAD-9 16s rDNA SEQ ID NO: 1), strain HNAD-9 was identified as Paracoccus pantotrophus and recorded as Paracoccus pantotrophus strain HNAD-9. The strain was deposited in the General Microbiology Center of China National Microbiological Culture Collection (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on ​​August 22, 2024, with the deposit number CGMCC No. 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 transferred to a 3% inoculum containing NH4 + -N was used as the nitrogen source in a heterotrophic nitrification liquid culture medium, and cultured at 30°C and 150 rpm for 96 h under aerobic conditions. NH4 + -N concentration and bacterial growth, the experimental results are as follows Figure 5 As shown. When NH4 + When -N was used as the only nitrogen source, strain HNAD-9 grew rapidly, and the bacterial growth reached a maximum value of 1.753 after 60 hours of cultivation. + The concentration of -N decreased significantly with the growth of strain HNAD-9. When strain HNAD-9 was cultured for 60 h, NH4 + -N removal rate was 88.93%, and NH4 + -N removal rate reached the maximum, which was 98.73%. Strain HNAD-9 can efficiently utilize NH4 in water + -N carries out growth 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 transferred to a 3% inoculum containing NO3 - -N was used as the nitrogen source in aerobic denitrification liquid culture medium, and cultured at 30°C and 150 rpm for 96 h under aerobic conditions to determine NO3 - -N concentration and bacterial growth Figure 6 As shown. When NO3 -When -N was used as the only nitrogen source, strain HNAD-9 quickly entered the logarithmic growth phase, and at 72h of culture, the growth of strain HNAD-9 reached its maximum, at which time the growth of strain HNAD-9 reached 1.788. - The concentration of -N decreased significantly with the growth of strain HNAD-9. When strain HNAD-9 was cultured for 72h, NO3 - -N removal rate reached the maximum, which was 100%. The strain can efficiently utilize NO3 in water - -N carries out growth 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 transferred to a 3% inoculum containing NH4 + -N and NO3 - -N was a mixed nitrogen source liquid medium, and cultured at 30°C and 150 rpm for 96 h under aerobic conditions. The growth and nitrogen metabolism of strain HNAD-9 were 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 + -N removal rate compared to NO3 - -N will be faster. When strain HNAD-9 was cultured for 36h, NH4 + -N is almost completely removed, NO3 - -N was also completely removed after the strain HNAD-9 was cultured for 60 hours. When the strain HNAD-9 was cultured for 84 hours, the bacterial growth reached a maximum of 1.621. After that, the growth of the strain HNAD-9 began to enter the decay phase, and the NH4 released by the autolysis of the bacteria + -N, resulting in NH4 + The -N content was slightly increased.

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

[0090] At present, there are three main nitrogen metabolism pathways of HNAD denitrifying bacteria, namely NO3 - / NO2 - pathway, NO pathway and N2O pathway. NO3 - / NO2 -The pathway refers to the process in which the strain converts NH4 + -N is converted into NO2 via NH2OH - -N and NO3 - -N, followed by NO2 - -N and NO3 - -N is converted into N2, thereby achieving the removal of nitrogen in the water body; the NO pathway is that under aerobic conditions, the strain converts NH4 + -N is converted into NH2OH, and the generated NH2OH does not pass through NO2 - -N and NO3 - -N, directly converted to NO, and then converted to N2 via N2O; the N2O pathway refers to the conversion of NH4 + -N is converted into NH2OH, and the generated NH2OH does not pass through NO2 - -N and NO3 - -N, directly converted into N2O, and then directly converted into N2 to be completely removed.

[0091] When strain HNAD-9 was treated with NH4 + -N as the sole nitrogen source, in NH4 + -N metabolism, the intermediate products NH2OH and NO2 were detected - -N( Figure 8 ). According to the strain HNAD-9 to NH4 + -N degradation process, and proposed the heterotrophic nitrification-aerobic denitrification pathway of strain HNAD-9, such as Fig. 9 NH4 + -N is first converted into NH2OH, and the generated NH2OH is immediately converted into NO2 - -N, NO2 - -N is then converted into gaseous nitrogen through denitrification, ultimately achieving nitrogen removal from the water body. To further verify this denitrification pathway, the effects of strain HNAD-9 on the denitrification intermediates NH2OH and NO2 - -N and NO3 - The experimental process was as follows: the purified strain was inoculated into fresh LB liquid medium and cultured overnight at 30°C and 150rpm; 3% of the overnight cultured bacterial solution was inoculated into sterilized heterotrophic nitrification liquid medium and cultured in a constant temperature shaking incubator at 30°C and 150rpm for 96h, and the NH4 + -N, NH2OH, NO2 - -N and NO3 - -N concentration changes, the results show that strain HNAD-9 can not only utilize NH4 + -N for growth and metabolism, and can also use NH2OH, NO2- -N and NO3 - -N, and it can completely remove it, and it is speculated that the ammonia nitrogen metabolism pathway of the 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.

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

[0093] The landfill leachate used in the experiment comes from a landfill in Beijing. The basic water quality indicators are shown in Table 1. COD of landfill leachate Cr TN, NH4 + -N, NO3 - -N and NO2 - The concentrations of -N were 19300mg / L, 2320mg / L, 2250mg / L, 1.81mg / L and 2.66mg / L respectively. The landfill leachate had a high concentration of organic matter and ammonia nitrogen, of which the total nitrogen was basically contributed by ammonia nitrogen. It is feasible to treat landfill leachate using heterotrophic nitrification-aerobic denitrification strain HNAD-9.

[0094] Table 1 Changes in pollution 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 150rpm. The activated strain HNAD-9 was transferred to the landfill leachate at a 3% inoculation rate and cultured for 124h at 30°C and 150rpm under aerobic conditions. The COD in the landfill leachate was determined. Cr TN, NH4 + -N, NO3 - -N and NO2 - -N content changes, experimental results such as Fig.10 Each experiment was set up in three parallel groups, and the landfill leachate without inoculation of strain HNAD-9 was used as a blank control. As shown in Table 1, strain HNAD-9 can efficiently treat landfill leachate. After 124 hours of degradation, the COD in the landfill leachate was Cr TN and NH4 + The concentration 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. Fig.10 As shown, strain HNAD-9 has a significant effect on CODCr TN and NH4 + -N removal efficiencies were 91.14%, 94.87% and 99.31% respectively. In addition, compared with the landfill leachate before treatment, the NO3 - -N concentration increased from 1.81mg / L to 80.3mg / L, NO2 — N remained basically unchanged, indicating that strain HNAD-9 converted NH4 + -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. For those skilled in the art, without departing from the purpose and scope of the present invention, can implement the present invention in a wider range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departing from the disclosed scope in the application and the changes made with conventional techniques known in the art.

Claims

1. Paracoccus pantotrophus HNAD-9, whose registration number in the General Microbiological Center of China Culture Collection Administration is CGMCC No.31698.

2. The culture of Paracoccus pantotrophus HNAD-9 according to claim 1, which is obtained by culturing the Paracoccus pantotrophus HNAD-9 according to claim 1 in a bacterial culture medium.

3. The metabolite of Paracoccus pantotrophus HNAD-9 according to claim 1.

4. A bacterial agent, characterized in that The method comprises the Paracoccus pantotrophus HNAD-9 of claim 1 or the culture of claim 2 or the metabolite of claim 3.

5. The bacterial agent according to claim 4, characterized in that: The bacterial agent is a bacterial agent that removes or degrades ammonia nitrogen, nitrate nitrogen and / or nitrite.

6. Use of the Paracoccus pantotrophus HNAD-9 according to claim 1, or the culture according to claim 2, or the metabolite according to claim 3, or the bacterial agent according to claim 4 or 5 in any of the following: (A1) removing ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen; (A2) preparing a product for removing ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen; (A3) removing organic matter, ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen from landfill leachate; (A4) preparing a product for removing organic matter, ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen from landfill leachate.

7. A method for removing ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen, comprising the steps of adding the Paracoccus pantotrophus HNAD-9 described in claim 1 or the culture described in claim 2 or the metabolite described in claim 3 or the bacterial agent described in claim 4 or 5 to a sample to be processed for reaction to remove ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen in the sample.

8. The method according to claim 7, characterized in that: 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 thereof as the nitrogen source.

9. A method for treating landfill leachate, characterized in that: The method comprises the following steps: adding the Paracoccus pantotrophus HNAD-9 described in claim 1 or the culture described in claim 2 or the metabolite described in claim 3 or the bacterial agent described in claim 4 or 5 to the landfill leachate for reaction to remove organic matter, ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen in the landfill leachate; the COD concentration in the landfill leachate is ≥10000 mg / L, and the ammonia nitrogen concentration is ≥400 mg / L.

10. A product for removing ammonia nitrogen, nitrate nitrogen and / or nitrite nitrogen, wherein the active ingredient is the Paracoccus pantotrophus HNAD-9 according to claim 1 or the culture according to claim 2 or the metabolite according to claim 3 or the bacterial agent according to claim 4 or 5.

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