Mycobacteria Based on Multiple Carbon and Nitrogen Metabolic Pathways, Their Isolation Methods, and Uses

Abstract

Disclosed is a mycobacterium sp., its isolation method, and its use. The deposit number of the mycobacterium sp. is CGMCC No. 21272 or CGMCC No. 21273. The mycobacterium has clear nitrogen fixation, carbon fixation, and ammonia oxidation abilities, and can directly oxidize ammonia nitrogen to nitrogen gas, exhibiting antagonistic effects against other microorganisms.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the rights and benefit of Chinese Patent Application No. 202210989249.4, filed on August 17, 2022, the contents of which are incorporated herein by reference. [Technical Field]

[0002] The present invention relates to the field of wastewater treatment and to mycobacteria used in the fermentation industry, specifically based on multiple carbon and nitrogen metabolic pathways, methods for their isolation and use. [Background technology]

[0003] Nitrification in nature is generally believed to be carried out by two types of nitrifying bacteria. In the first stage, nitrifying bacteria oxidize ammonia to nitrite, and in the second stage, denitrifying bacteria oxidize nitrite to nitrate. A 2015 study proposed that nitrification is carried out by a single type of bacteria. Due to their characteristics, nitrifying bacteria are widely used in the treatment of nitrogen-containing wastewater and the screening of nitrification inhibitors. However, conventional technology has assumed that nitrifying bacteria are "autotrophic" bacteria, and that the presence of organic matter is harmful to their growth. This perception severely limits the application of nitrifying bacteria. For example, given the recognition that nitrifying bacteria are autotrophic, the microbial basis and process principle of existing wastewater biochemical treatment technologies for removing carbon and nitrogen contaminants is a combination of the "ammonification" reaction by heterotrophic bacteria, the "nitrification" reaction by autotrophic bacteria oxidizing ammonia and nitrite, and the "denitrification" reaction by anaerobic (facultative) heterotrophic bacteria, which removes nitrogen. However, the above combination has problems such as a slow bacterial growth rate and a low total nitrogen removal rate.

[0004] Mycobacteria are elongated, slightly curved organisms that may be branched or filamentous, and are primarily characterized by their cell wall containing large amounts of lipids, primarily mycolic acids, lacking flagella and spores, lacking endotoxin and exotoxin production, and being aerobic saprophytes that grow on surfaces and adhere strongly to solid surfaces. Most research on mycobacteria has focused on the pathogenic bacteria Mycobacterium tuberculosis and Mycobacterium leprae. To date, no mycobacteria have been reported to have nitrifying activity.

[0005] Furthermore, existing methods for isolating nitrifying strains still have limitations. For example, Chinese Patent ZL 03118598.3 (Method for isolating, identifying, and purifying heterotrophic nitrifying microorganisms) discloses a method for isolating, identifying, and purifying heterotrophic nitrifying microorganisms from various soils through steps such as nutrient agar plate separation and Griess reagent color development. This method is versatile and can screen many heterotrophic strains with nitrifying activity, but it lacks specificity, making it difficult to find strains with high nitrifying activity. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to overcome the problems of the prior art and provide a mycobacterium based on multiple carbon and nitrogen metabolic pathways, a method for isolating the same, and its use. The strain or agent of the present invention is expected to be applied to sewage treatment, screening of nitrification inhibitors, etc. [Means for solving the problem]

[0007] To achieve the above object, a first aspect of the present invention provides a mycobacterium (Mycobacterium sp.) having deposit number CGMCC No. 21272 or CGMCC No. 21273.

[0008] A second aspect of the present invention provides a bacterial agent comprising a mycobacterium as described above.

[0009] Another aspect of the invention relates to the use of said mycobacterial or fungal agents.

[0010] The present invention also provides a method for isolating microorganisms having nitrification activity.

[0011] The mycobacteria of the present invention differ from existing mycobacteria in that they possess at least the following characteristics: 1) They have a clear ability to fix nitrogen; 2) They have a high ammonia oxidation activity, significant nitrification activity, and are capable of heterotrophy, achieving carbon capture (C) and removal of N-organic pollutants without the need for cooperation with other microorganisms; 3) They have the ability to dehydrogenate hydrazine sulfate to produce hydroxylamine; 4) They contain at least five dehydrogenases; 5) They possess a nitric oxide synthase gene and can oxidize arginine; Fe is an important element that affects their nitrification activity; and 6) They have a significant antagonistic effect against other microorganisms.

[0012] The separation method of the present invention makes it possible to stably, specifically, and reproducibly separate strains with high nitrification activity from samples (for example, soil).

[0013] biological deposit The mycobacterium (Mycobacterium sp.) according to the present invention was deposited on December 1, 2020, at the Center for Ordinary Microorganisms, China Microbial Species Depositary (address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, 100101). The abbreviation for the depository is CGMCC, and the deposit number of the strain is CGMCC No. 21272, with the abbreviation YT.

[0014] The mycobacterium (Mycobacterium sp.) according to the present invention was deposited on December 1, 2020, at the Center for Ordinary Microorganisms, China National Commission on the Depositary of Microorganisms (address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, postal code 100101). The abbreviation for the depository is CGMCC, and the deposit number of the strain is CGMCC No. 21273, abbreviation for JD. [Brief explanation of the drawings]

[0015] [Figure 1a-1b] 1 is a whole genome circosplot of mycobacteria of the present invention. [Figure 1c-1d] FIG. 1 is a schematic diagram of nitrogen metabolism pathways. [Figure 2a] Bacterial growth on LB-Malachite Green plates using a 10-7 dilution gradient. [Figure 2b] This is a transmission electron microscope photograph of the target bacteria. [Figure 2c-2e] FIG. 1 is a color diagram of the Griess reagent. [Figure 2f] This is a distribution diagram of target bacteria on a separation plate. [Figure 3a] 1 is a photograph of the mycobacteria of the present invention cultured in a liquid medium. [Figure 3b-3c] 1 is a photograph of the mycobacteria of the present invention cultured on a solid medium. [Figure 3d] FIG. 1 shows the morphology of the mycobacteria of the present invention under a Nikon stereomicroscope. [Figure 3e] 1 is a photograph of the mycobacteria of the present invention cultured on a solid medium. [Figure 3f] This is the result of PCR amplification of the nitrogenase iron protein gene. [Figure 3g] This shows the changes in culture OD600 and NH4 +-N concentration during the mycobacterial cultivation process using ammonium sulfate as a nitrogen source and pyruvate as a carbon source. [Figure 4a] FIG. 1 shows the changes in OD600 and pH during the culture process. [Figure 4b] FIG. 1 shows the change in pyruvate concentration during the culture process. [Figure 5] 1 shows a photograph of mycobacteria of the present invention cultured in a medium containing hydrazine sulfate as a nitrogen source. [Figure 6] 1 shows the results of transcriptome sequencing of YT cultures obtained with hydroxylamine as the nitrogen source. [Figure 7a]This is a photograph of the initial stage of pure culture of JD using trans-sodium hyponitrite as a nitrogen source and ammonium sulfate as a nitrogen source. [Figure 7b] 1 shows the morphology of the mycobacteria of the present invention under a Nikon stereomicroscope. [Figure 7c] FIG. 1 shows the results of qualitative measurement of the nitrification activity of a culture using trans-sodium hyponitrite as a nitrogen source. [Figure 8] 1 is a photograph of the mycobacteria of the present invention cultured in an iron-free liquid medium. [Figure 9a] FIG. 1 shows a comparison of 13-day YT pure culture and control. [Figure 9b] FIG. 1 shows the NOS gene amplification profile of mycobacteria of the present invention. [Figure 10a] 1 shows the changes in culture OD600 and pH during the process of culturing mycobacteria using sodium nitrite as a nitrogen source. [Figure 10b] FIG. 1 shows the change in total nitrogen concentration in a culture during the process of culturing the mycobacteria of the present invention using sodium nitrite as a nitrogen source. [Figure 11] This is a graph and photograph of the culture showing the change in NO2-N after JD was grown for 6 days in anaerobically cultured with sodium nitrite as the nitrogen source. [Figure 12a] FIG. 1 shows the changes in OD600 and pH of JD during the culture process. [Figure 12b] FIG. 1 shows the results of qualitative measurements of NO2 --N in JD cultures cultivated for 11 days. [Figure 12c] FIG. 1 shows changes in CO 2 and O 2 concentrations over time in the JD culture system. [Figure 12d] FIG. 1 shows the changes in N2 and N2O concentrations over the course of culture time in the JD culture system. [Figure 13a-13b] The effects of various nitrification inhibitors on YT growth are shown. [Figure 13c] FIG. 1 shows a comparison of TN concentrations in YT cultures grown with various nitrification inhibitors as nitrogen sources. [Figure 14a] FIG. 1 shows the antagonistic effect of YT on other strains. [Figure 14b] The inhibition zone formed by the antagonistic effect of YT against three standard strains and its electron micrographs. [Figure 14c] FIG. 1 shows the inhibition zones formed by the antagonistic action of YT against three standard strains, and their morphology observed under a stereomicroscope. DETAILED DESCRIPTION OF THE INVENTION

[0016] The range endpoints and values disclosed herein are not limited to the exact ranges or values, and should be understood to include values that are close to these ranges or values. In the case of numerical ranges, the range endpoints, the range endpoints and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which are considered to be specifically disclosed herein.

[0017] In the present invention, "mM" means "mmol / L", "M" means "mol / L", and "ammoniacal nitrogen" and "ammonium nitrogen" can be used interchangeably.

[0018] I. Strains and Agents A first aspect of the present invention provides a mycobacterium (Mycobacterium sp.) having the deposit number CGMCC No. 21272 or CGMCC No. 21273.

[0019] The inventors further confirmed through experiments that the CGMCC No. 21272 strain (abbreviated as YT) or the CGMCC No. 21273 strain (abbreviated as JD) of the present invention is different from all existing mycobacteria, and therefore the strain of the present invention is a new mycobacterial species and has been named Mycobacterium guanghaopengii sp. nov.

[0020] In the present invention, the cell morphology and physicochemical properties of the mycobacterium CGMCC No. 21272 are shown in Table 1a.

[0021] [Table 1a]

[0022] In the present invention, the 16S rRNA sequence of the mycobacterium CGMCC No. 21272 is shown in SEQ ID NO:1. SEQ ID NO:1: TIFF2025526926000003.tif72165

[0023] In the present invention, phylogenetic analysis based on the 16S rRNA gene sequence revealed that CGMCC No. 21272 is a Mycobacterium strain, and its whole genome circosplot is shown in Figure 1a.

[0024] In the present invention, the results of antibiotic susceptibility experiments of the mycobacterium CGMCC No. 21272 are shown in Table 1b.

[0025] [Table 1b]

[0026] In the present invention, the cell morphology and physicochemical properties of the mycobacterium CGMCC No. 21273 are shown in Table 2a.

[0027] [Table 2a]

[0028] In the present invention, the 16S rRNA sequence of the mycobacterium CGMCC No. 21273 is shown in SEQ ID NO:2. SEQ ID NO:2: TIFF2025526926000006.tif48107

[0029] In the present invention, phylogenetic analysis based on the 16S rRNA gene sequence showed that CGMCC No. 21273 is a Mycobacterium strain, and its whole genome circosplot is shown in Figure 1b.

[0030] In the present invention, the results of antibiotic susceptibility experiments of the mycobacterium CGMCC No. 21273 are shown in Table 2b.

[0031] [Table 2b]

[0032] The nitrogen metabolic pathways of the mycobacteria of the present invention are shown in Figure 1c (the upper diagram is a schematic diagram of the nitrogen metabolic pathways of the mycobacteria of the present invention, and the lower diagram is a schematic diagram of currently known nitrogen metabolic pathways) and Figure 1d. This shows that the mycobacteria of the present invention contain, in addition to conventional metabolic pathways, additional direct ammonia oxidation pathways and organic nitrogen (e.g., arginine) oxidation pathways.

[0033] A second aspect of the present invention provides a bacterial preparation characterized by containing the above-mentioned mycobacteria. The bacterial preparation may be a solid or liquid bacterial preparation, and may be in an appropriate form prepared using various auxiliary materials or carriers commonly used in the art. For example, to prepare a solid bacterial preparation, the auxiliary materials may include, but are not limited to, at least one of water-insoluble carbonates (e.g., calcium carbonate), rice bran, sawdust, cake powder, straw powder, etc., and the viable cell count of the mycobacteria in the solid preparation may be 10 5 ~10 18 It may be CFU / g.

[0034] II. Use in environmental protection Since the strain of the present invention has nitrogen fixation, nitrification, and denitrification abilities, a third aspect of the present invention provides a denitrification method comprising contacting the above-mentioned mycobacteria or the above-mentioned bacterial agent with a target system for denitrification. The target system for denitrification refers to various general systems in which N needs to be removed. In the target system for denitrification, N can be present in the form of general organic nitrogen and / or inorganic nitrogen, and preferably in the form of molecular nitrogen (N), ammoniacal nitrogen (NH and / or NH + ), nitrate nitrogen (NO3 - ), nitrite nitrogen (NO2 - Therefore, the denitrification target system can be various systems containing ammonia nitrogen (e.g., ammonia nitrogen (NH3 and / or ammonium salts (NH4 + )), waste materials containing nitrate-nitrogen, various systems (e.g., NO3 - waste materials containing nitrogen), nitrite nitrogen (e.g., NO2 -The denitrification target system may be a system containing nitrogen-containing pollutants, or any system requiring denitrification, such as wastewater, waste gas, or waste residue. The contact allows the mycobacteria to grow using the N in the denitrification target system as a nitrogen source, changing the form of N and facilitating its removal. Depending on the characteristics of the mycobacteria, an appropriate carbon source (e.g., at least one of glucose, pyruvate, and pyruvate) can also be added to the system. When the N in the denitrification system is molecular nitrogen, the contact converts the molecular nitrogen to ammonia nitrogen, nitrate nitrogen, and / or nitrite nitrogen (performing nitrogen fixation). In addition to the carbon and nitrogen sources, inorganic salts such as calcium chloride (0.1-1 g / L), magnesium sulfate (0.05-0.5 g / L), dipotassium hydrogen phosphate (0.5-5 g / L), sodium molybdate (0.005-0.05 g / L), and ferrous sulfate (0.01-0.1 g / L) are also added to the contact system. When the N in the denitrification system is ammonia nitrogen, the contact converts the ammonia nitrogen to nitrate nitrogen and / or nitrite nitrogen (performing nitrification). When the N in the denitrification system is nitrate nitrogen and / or nitrite nitrogen, the contact is carried out under anaerobic conditions, and the nitrate nitrogen and / or nitrite nitrogen are converted to N2 and / or NO (performing denitrification).

[0035] The present invention also provides the use of the above-mentioned mycobacteria or the above-mentioned bacterial agent in the treatment of nitrogen pollution (nitrogen-containing pollutants, particularly ammonia-nitrogen pollution). A fourth aspect of the present invention provides a method for treating nitrogen pollution, comprising contacting the above-mentioned mycobacteria or the above-mentioned bacterial agent with nitrogen-containing pollutants. The nitrogen-containing pollutants may be various common pollutants containing ammonia-nitrogen. Upon contact, the mycobacteria grow using the N in the nitrogen-containing pollutants as a nitrogen source, changing the form of the nitrogen and facilitating its removal. Upon contact, the ammonia-nitrogen is converted into nitrate-nitrogen, nitrite-nitrogen, and N2. In this method for treating nitrogen pollution, the contact system is first placed under conditions favorable for the mycobacteria to perform nitrification (converting ammonia-nitrogen to nitrate-nitrogen and / or nitrite-nitrogen), and then under conditions favorable for the mycobacteria to perform denitrification (converting nitrate-nitrogen and / or nitrite-nitrogen to N2), thereby converting the environmentally harmful ammonia-nitrogen into colorless and odorless nitrogen gas.

[0036] The mycobacteria of the present invention are capable of single-pass denitrification (direct oxidation of ammonia nitrogen (hydrazine and / or hydroxylamine) to nitrogen-containing gases (direct ammonia oxidation)). Specifically, under aerobic conditions in the presence of organic matter, the mycobacteria can oxidize ammonia nitrogen through the pathway of "ammonia nitrogen → hydrazine → hydroxylamine → nitrogen and nitrous oxide." This process can be completed before nitrification occurs, and the mycobacteria can complete the entire denitrification process under aerobic conditions. This solves the problem of conventional biological denitrification technologies, which require aerobic nitrification and anaerobic denitrification to be performed in stages. This significantly simplifies the denitrification process, saving processing space and costs. Therefore, the above-described method for ammonia oxidation using mycobacteria can be applied to various wastewaters containing ammonia nitrogen (especially wastewater containing hydroxylamine), including single-pass denitrification and nitrification. Single-pass denitrification involves directly oxidizing ammonia nitrogen to nitrogen-containing gases by controlling pH. In some embodiments of the present invention, the wastewater containing ammonia nitrogen is propellant wastewater and / or hydroxylamine wastewater. To enable the mycobacteria to more effectively achieve single-pass denitrification, it is preferable to use pyruvate as a carbon source.

[0037] Various types of hydrazine and / or its derivatives (e.g., hydrazine, hydrazine sulfate, unsymmetrical dimethylhydrazine) can be removed using mycobacteria as described above. Hydroxylamine and / or its derivatives (e.g., oximes) can also be removed using mycobacteria as described above.

[0038] Preferably, the nitrogen-containing contaminant is at least one of hydroxylamine, a hydroxylamine derivative, hydrazine, and a hydrazine derivative.

[0039] More preferably, the hydroxylamine derivative is an oxime.

[0040] More preferably, the hydrazine derivative is hydrazine sulfate and / or unsymmetrical dimethylhydrazine.

[0041] The mycobacteria of the present invention can convert CO2 into amino acids, sugars, and lipids through the reverse oxidative tricarboxylic acid cycle. Therefore, the present invention also provides the use of such mycobacteria or such bacterial agents in carbon capture. A fifth aspect of the present invention provides a method for capturing CO2, comprising contacting such mycobacteria or such bacterial agents with a system containing CO2. The contact allows the mycobacteria to grow using CO2 as a carbon source, thereby achieving carbon capture. Depending on the characteristics of the mycobacteria, an appropriate nitrogen source (e.g., ammonium salts) can also be added to the system. The CO2-containing system may be at least one of exhaust gas from a coal-fired power plant, exhaust gas from natural gas production, exhaust gas from a synthetic fuel plant, and exhaust gas from a fossil fuel-based hydrogen production plant.

[0042] In the above contact, unless otherwise specified, the amount of carbon source used is such that the carbon content in the contact system is preferably 0.5 to 5 g / L, and the amount of nitrogen source used is such that the nitrogen content in the contact system is preferably 0.2 to 2 g / L. Inorganic salts such as sodium dihydrogen phosphate (0.1 to 1 g / L), dipotassium hydrogen phosphate (0.5 to 5 g / L), magnesium sulfate (0.01 to 0.1 g / L), manganese sulfate (0.005 to 0.05 g / L), and ferrous sulfate (0.005 to 0.05 g / L) may also be added to the contact system. The above contact can be carried out at a temperature suitable for the growth of the mycobacteria (e.g., 15 to 35°C, preferably 25 to 32°C). The initial pH of the contact system can be adjusted to 6 to 9, preferably neutral (e.g., in the range of 7 to 7.2), using a pH adjuster. The contact time can be determined as needed, for example, 10 h to 50 d (e.g., 20 h, 1 d, 3 d, 4 d, 5 d, 6 d, 7 d, 8 d, 9 d, 10 d, 11 d, 13 d, 15 d, 17 d, 19 d, 21 d, 23 d, 25 d, 27 d, 29 d, 31 d, 35 d, 40 d, 45 d, 50 d, or any range thereof). Unless otherwise specified, the contact (cultivation / growth) is carried out under aerobic conditions.

[0043] WO2009018686A1 (Method for removing C and N pollutants from sewage using heterotrophic ammonia-oxidizing bacteria) still belongs to a mixed bacterial treatment process and is unable to maximize the ammonia oxidation rate, but the mycobacteria of the present invention can be applied in the form of a pure culture, which maximizes the absolute number of bacteria before addition, thereby maximizing the ammonia oxidation rate.

[0044] III. Use in Agriculture Based on conventional ammonia nitrogen metabolic pathways, the inventors have discovered that the ammonia nitrogen metabolic pathway of the bacterial strain of the present invention also includes the arginine oxidation pathway. Therefore, in addition to screening nitrification inhibitors using conventional pathways, any substance that inhibits the loss of ammonia nitrogen through the conversion of ammonia nitrogen to other forms by acting on any part of the pathway can be used as a target pesticide. Therefore, the present invention also provides the use of the above-mentioned mycobacteria or the above-mentioned fungicide in screening pesticides. A sixth aspect of the present invention provides a method for screening pesticides based on the above-mentioned mycobacteria, comprising inoculating and culturing the above-mentioned mycobacteria in an ammonia nitrogen medium containing the target pesticide, and determining whether the target pesticide has the function of inhibiting the loss of ammonia nitrogen based on changes in N or the growth status of the mycobacteria during the cultivation process. Monitoring changes in N during the cultivation process includes detecting the content of ammonia nitrogen in the culture, detecting the content of nitrate nitrogen and / or nitrite nitrogen in the culture, or detecting the production of N and / or NO in the culture system. If the ammoniacal nitrogen content remains almost constant after incubation, if nitrate, nitrite, N2, or N2O is not detected during incubation, or if mycobacterial growth is poor, the target pesticide is considered to be a product that inhibits the loss of ammoniacal nitrogen. The target pesticide concentration in the ammoniacal nitrogen medium containing the target pesticide may be 1-20 mM. This medium may be modified Stevenson's medium supplemented with the target pesticide. The specific composition may be 1-20 mM target pesticide, 0.5-5 g / L carbon source (C equivalent), 0.2-2 g / L nitrogen source (N equivalent), 0.1-1 g / L sodium dihydrogen phosphate, 0.5-5 g / L dipotassium hydrogen phosphate, 0.01-0.1 g / L magnesium sulfate, 0.005-0.05 g / L manganese sulfate, and 0.005-0.05 g / L ferrous sulfate. The carbon source is preferably at least one of glucose, pyruvic acid, and pyruvate. The nitrogen source is ammoniacal nitrogen (NH3 and / or NH4 +), preferably at least one of ammonia gas, ammonium salts, and amino acids, more preferably ammonium sulfate and / or arginine. The culture temperature is 15 to 35°C, preferably 25 to 32°C. The culture time may be 10 hours to 50 days (for example, 20 hours, 1 day, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 13 days, 15 days, 17 days, 19 days, 21 days, 23 days, 25 days, 27 days, 29 days, 31 days, 35 days, 40 days, 45 days, 50 days, or any range of the above times).

[0045] In the present invention, the term "agrochemical" refers primarily to products that can potentially inhibit the loss of ammoniacal nitrogen, particularly those that can reduce nitrogen fertilizer loss, improve nitrogen fertilizer utilization, and reduce the nitrite content in crops. These may also be called ammoniacal nitrogen loss inhibitors, including nitrification inhibitors and / or nitric oxide synthase inhibitors. As described above, the bacterial strains of the present invention possess a greater number of ammoniacal nitrogen metabolic pathways, and this can be used to select agrochemicals with more potent inhibitors. The nitrification inhibitors can inhibit the activity of enzymes in the nitrification process and the growth of nitrifying bacteria, thereby inhibiting nitrification. The ammoniacal nitrogen loss inhibitor may include, but is not limited to, non-heterocyclic inhibitors (e.g., aminoguanidines, acetamidines, cycloalkylamidines, thioureas, etc.) and heterocyclic inhibitors (e.g., pyridines, imidazoles, indazoles, triazoles, coumarins, piperazines, piperidines, pteridines, tetrahydropteridines, sulfur-nitrogen heterocycles, thiazoles, etc.), and specific examples thereof include ammonium sulfate, ammonium nitrate, potassium nitrate, calcium sulfate, copper sulfate, acetylene, ethynylpyridine, phenylacetylene, 4-amino-1,2,4-triazole, etc. The amines and amines used in the present invention include, but are not limited to, at least one of methyl thiourea (ATC), aminoguanidine, aminoguanidine hydrochloride, 2-amino-4-chloro-9-methylpyridine, potassium azide, sodium azide, 2-chloro-6-(trichloromethyl)pyridine (also known as nitrogen pyridine, CP), 3,4-dimethylpyrazole phosphate (DMPP), guanylthiourea (ASU), 2-methyl-4,6-bis(trichlorotoluene)-s-triazine (MDCT), thiourea-N-2,5-dichlorophenylsuccinamide, allylthiourea, 4-amino-1,2,3-triazole hydrochloride, and 2-sulfathiazole (ST).

[0046] The present inventors have discovered that non-amino acid nitrification inhibitors have potent inhibitory effects on nitric oxide synthase. Therefore, the present invention also provides a method for inhibiting nitric oxide synthase (particularly the nitric oxide synthase of the mycobacterium of the present invention) (in vivo, ex vivo, non-therapeutic), which comprises contacting nitric oxide synthase with at least one non-amino acid nitrification inhibitor. The non-amino acid nitrification inhibitor may be at least one selected from triazoles, aminoguanidines, and thioureas, preferably at least one of 4-amino-1,2,4-triazole, aminoguanidine, aminoguanidine hydrochloride, guanylthiourea, etc. When used to inhibit the nitric oxide synthase activity of the mycobacterium, the non-amino acid nitrification inhibitor can be directly added to the mycobacterial culture system at a concentration of 2 to 10 mM.

[0047] IV. Medicinal Use A seventh aspect of the present invention provides the use of a mycobacterium as described above or a fungicide as described above in the treatment and / or prevention of a skin disease, the treatment and / or prevention of a disease associated with low nitrite levels, the treatment and / or prevention of body odour, treatment by providing nitric oxide to a subject, or the inhibition of microbial growth.

[0048] In some embodiments, the mycobacterial or fungal agent can be used to treat at least one of HIV, dermatitis, ulcer (e.g., venous ulcer, e.g., leg ulcer, e.g., venous leg ulcer) infection (e.g., diabetic foot ulcer infection), allergic dermatitis, acne (e.g., acne vulgaris), eczema, contact dermatitis, allergic reactions, psoriasis, hives, rosacea-like dermatitis, skin infections, vascular disease, vaginal yeast infections, sexually transmitted diseases, heart disease, arteriosclerosis, alopecia, leg ulcers secondary to diabetes or being bedridden, angina pectoris (particularly chronic stable angina), ischemic disease, congestive heart failure, myocardial infarction, ischemia-reperfusion injury, laminitis, hypertension, hypertrophic organ degeneration, Raynaud's phenomenon, fibrosis, fibrotic organ degeneration, allergy, autoimmune sensitization, end-stage renal disease, obesity, impotence, pneumonia, primary immunodeficiency, epidermolysis bullosa, or cancer.

[0049] In some embodiments, the mycobacterial or fungal agent can be used to treat at least one of diabetic foot ulcer infections, allergic dermatitis, acne (e.g., acne vulgaris), eczema, psoriasis, hives, rosacea-like dermatitis, and skin infections.

[0050] In some embodiments of the present invention, the use is in the manufacture of a medicament or formulation for the treatment and / or prevention of skin disorders, treatment and / or prevention of disorders associated with low nitrite levels, treatment and / or prevention of body odor, treatment by providing nitric oxide to a subject, or inhibition of microbial growth.

[0051] In some embodiments of the invention, the inhibition is in vitro inhibition.

[0052] In some embodiments of the present invention, the microorganism is a Mycobacterium genus. The strain of the present invention is capable of producing nitric oxide and thus can inhibit various nitric oxide-sensitive microorganisms (including fungi, bacteria, and viruses), particularly various nitric oxide-sensitive microorganisms that cause skin diseases and / or various nitric oxide-sensitive microorganisms that grow well in vitro, more preferably at least one of Mycobacterium bambaareni, Mycobacterium vaccae, and Mycobacterium austroafricanum.

[0053] In the present invention, the drug or formulation may further contain common pharmaceutical auxiliary materials to be formulated into a specific dosage form for use, such as a tincture, ointment, cream, paste, aerosol, spray, powder, ear drop, lotion, rinse, smear, paint, film coating, gel, patch, etc.

[0054] V. Separation method An eighth aspect of the present invention provides a method for isolating mycobacteria having nitrifying activity, the method comprising the steps of: (1) enriching mycobacteria having nitrifying activity; and (2) selecting mycobacteria having high nitrifying activity using a medium supplemented with an antibiotic.

[0055] In some embodiments of the present invention, the medium used for the enrichment may be a medium commonly used in the art for isolating mycobacteria. In other embodiments of the present invention, the medium used for the enrichment is preferably a modified Stevenson's medium, more preferably containing 0.5 to 5 g / L (C equivalent) of any organic carbon source (e.g., glucose), 2 to 10 g / L (carbon equivalent) of a carbonate (e.g., calcium carbonate), 0.2 to 2 g / L (N equivalent) of ammonium sulfate, 0.1 to 1 g / L of sodium dihydrogen phosphate, 0.5 to 5 g / L of dipotassium hydrogen phosphate, 0.01 to 0.1 g / L of magnesium sulfate, 0.005 to 0.05 g / L of manganese sulfate, and 0.005 to 0.05 g / L of ferrous sulfate. In step (1), the temperature of the enrichment culture is 15 to 35°C, preferably 25 to 32°C. The enrichment culture time is 1 hour, 30 minutes, or 5 minutes ... + -N, NO2 - -N, and NO3 - The period can be determined based on the qualitative detection results of -N, and may be 4 to 30 days.

[0056] In the present invention, if the target strain is isolated by enrichment in a modified Stevenson medium, the strain can be selected for screening in step (2). If the target strain is not isolated by enrichment in a modified Stevenson medium, the procedure proceeds to step (2) after further acid treatment (removal of impurities). Therefore, in a preferred embodiment of the present invention, step (1) includes inoculating a sample into a modified Stevenson medium, culturing the culture, mixing the culture with an acid for acid treatment, and neutralizing the acid in the system after treatment. The amount of acid used for the acid treatment may be 0.2 to 2 mol (hydrogen ion equivalent), preferably 0.3 to 0.5 mol (hydrogen ion equivalent), per 1 L of culture. The acid used for the acid treatment may be any common inorganic acid, preferably HCl. The temperature for the acid treatment is preferably 3 to 8°C. The time for the acid treatment is preferably 1 to 5 minutes. The alkali used for neutralization is a common inorganic alkali, such as an alkali metal hydroxide (e.g., sodium hydroxide).

[0057] In some embodiments of the present invention, the antibiotic content in the medium supplemented with the antibiotic in step (2) is preferably 0.1 to 0.5 mg / L. The antibiotic may be any antibiotic commonly used in the art, but is preferably malachite green, penicillin, or the like. Preferably, the medium supplemented with the antibiotic is LB medium supplemented with the antibiotic, more preferably containing 5 to 15 g / L of peptone, 5 to 15 g / L of yeast extract, 5 to 15 g / L of sodium chloride, and 0.1 to 0.5 mg / L of antibiotic. The temperature of the enrichment culture is 15 to 35°C, preferably 25 to 32°C. The enrichment culture time is 4 to 30 days. After the enrichment in step (2), the dominant bacterium is the target bacterium with higher nitrification activity. The inventors have discovered that when the acid treatment time and antibiotic concentration are within the above-mentioned preferred ranges, the target strain can be better isolated, whereas isolation may fail otherwise. The method of the present invention is particularly advantageous for isolating the mycobacteria of the present invention.

[0058] The present invention will be described in detail below with reference to examples. In the following preparation examples and examples, some of the media and test methods used are as follows:

[0059] 1. Preparation of Basal Medium 1.1 Preparation of modified Stevenson's medium (ST medium) Weigh out 2.0 g of (NH4)2SO4, 0.25 g of NaH2PO4, 0.75 g of K2HPO4, 0.03 g of MgSO4·7H2O, 0.01 g of MnSO4·H2O, and 0.01 g of FeSO4·7H2O, dissolve them in 1000 ml of deionized water, adjust the pH to 7.0-7.2, sterilize with high-pressure steam, and prepare for use.

[0060] 1.2 Regular Nutrient Agar Plates (PM Plates) 3 g beef extract, 5 g peptone, 20 g agar, 1000 ml distilled water, pH 7.0-7.2. Before sterilization, adjust the pH value of each medium to the required pH range using 1 M NaOH or 25% (V / V) HCl solution.

[0061] 1.3 Medium with sodium pyruvate as a carbon source Prepare a sodium pyruvate solution of a specific concentration according to the concentration requirements of each experiment, sterilize it separately, and before use, take a certain volume (determine the volume according to the concentration required for each experiment) and add it to the modified Stevenson medium to prepare sodium pyruvate in the medium at a concentration that meets the needs of each experiment. Because sodium pyruvate partially decomposes during the preparation process, the actual concentration will be lower than the designed concentration. Fine-tune the required pH with 1 M NaOH solution.

[0062] 2 Nitrite nitrogen (NO2 - Qualitative measurement of -N 2.1 Preparation of Griess reagent (1) Sulfanilic acid reagent (Solution A): Dissolve 0.5 g of sulfanilic acid in 150 ml of 20% diluted acetic acid solution, place in a brown bottle, and store in the refrigerator until ready for use. (2) α-Naphthylamine Reagent (Solution B): Add 0.5 g of α-naphthylamine to 20 ml of distilled water and 150 ml of 20% diluted acetic acid solution, place in a brown bottle, and store in the refrigerator until ready for use.

[0063] 2.2 NO2 - Qualitative measurement of -N: A few drops of the culture medium were placed on a white porcelain colorimetric plate, and two drops each of solution A and solution B of Griess's reagent were added. If nitrite was present, the color turned red. Colors and corresponding concentration ranges: "+-" is a trace pink with a concentration less than 0.5 mg / L; "+" is a light pink with a concentration of approximately 0.5 mg / L; "++" is a pink with a concentration of approximately 1 mg / L; "+++" is a dark magenta with a concentration of approximately 5 mg / L; "++++" is a light brown with a concentration of approximately 10 mg / L; and ">++++" is a brown with a concentration of greater than 20 mg / L.

[0064] 3 Ammonium nitrogen (NH4 + Qualitative measurement of -N 3.1 Preparation of Nessler's Reagent

[0065] Weigh out 17 g of potassium mercury iodide and dissolve it in 10 ml of water. Dissolve 24.4 g of KOH in a 100 ml measuring flask containing 70 ml of water. Cool to room temperature, slowly pour the potassium mercury iodide into the measuring flask while shaking, add water up to the mark, and leave for 2 days before use. Store in a brown bottle in a dark place.

[0066] 3.2 NH4 + Qualitative determination of -N: A few drops of the culture medium were placed on a white porcelain colorimetric plate, and two drops of Nessler's reagent were added, which turned yellow if ammonium nitrogen was present. Colors and corresponding concentration ranges: "+" is slightly yellow and the concentration is less than 0.5 mg / L; "+++" indicates a concentration of approximately 5 mg / L; "++++" indicates a concentration of approximately 10 mg / L; ">++++" is dark yellow and the concentration is greater than 20 mg / L.

[0067] 4 Nitrate nitrogen (NO3 - Qualitative measurement of -N 4.1 Preparation of diphenylamine reagent Diphenylamine (0.5 g), concentrated sulfuric acid (100 ml), and distilled water (20 ml) are used. First, dissolve diphenylamine in distilled water, then slowly add concentrated sulfuric acid. The mixture is then placed in a brown bottle and prepared for use. For qualitative analysis, place a few drops of the culture medium on a white porcelain colorimeter and add two drops of diphenylamine reagent. If nitrate is present, a blue color will appear.

[0068] 4.2 NO3 - Qualitative determination of -N: Place a few drops of culture medium on a white porcelain colorimetric plate, add two drops of diphenylamine reagent, and a blue color will appear if nitrate is present. "-" indicates a negative result in the diphenylamine spot test, and "+" indicates a positive result in the diphenylamine spot test.

[0069] 5. Quantitative determination of various forms of nitrogen When quantitatively measuring various indices, the culture medium is centrifuged at 4°C for 15 minutes (5000 g) and the supernatant is collected for measurement. NH4 +Quantitative measurement of -N was performed using the indophenol blue colorimetric method, and NO2 - Quantitative measurement of -N was performed using the Griess reagent colorimetric method. TN was measured using a potassium peroxysulfate oxidation-flow analyzer. x - -N(NO2 - -N+NO3 - Quantitative measurements of NO (-N) were performed by flow analysis-spectroscopy using the hydrazine sulfate reduction method. x - -N(NO2 - -N+NO3 - -N), and NO2 - -N content was measured, and NO3 - -N content is NO x - -N content and NO2 - The basic principle is to use a peristaltic pump to compress pump tubes of different diameters, inject a reaction reagent and the test sample into a sealed continuous flow carrier at a predetermined rate, generate a color reaction in the chemical reaction unit, measure the signal value with a detector, and determine the concentration of the test sample according to the calibration curve method.

[0070] 6 Gas measurement in culture systems The contents of N2, CO2, O2, N2O, etc. in the culture gas are measured using a gas chromatograph (Agilent).

[0071] 7 Measurement of bacterial growth: Turbidimetric method, expressed as OD value at wavelength 600 nm.

[0072] 8 Measuring pH value: Measure using precision pH test paper and a pH meter.

[0073] 9. Measurement of pyruvate concentration: Collect the culture supernatant (4°C, 5000 rpm, 15 min high-speed centrifugation) and measure the pyruvate concentration using an Agilent 1260 liquid chromatograph (UV detector). Basic conditions: wavelength 214 nm, mobile phase: phosphate buffer:methanol = 98:2, flow rate 1.0 ml / min.

[0074] 10. Processing and analysis of cultures: When analyzing the cultured bacterial cells, first, collect the bacterial cells by centrifugation at 10,000 rpm for 5 minutes at 4°C using a high-speed centrifuge, and store them at 4°C until ready for use. To determine the dry weight and carbon and nitrogen content of the culture, the cells after centrifugation must be freeze-dried in a vacuum and weighed.

[0075] 11. Experimental Replicates: In each experiment, a medium without inoculation was used as a control (CK), and the experiment was set up so that there were three replicates for each detection.

[0076] Production Example 1: Isolation of Mycobacteria 1. Methods for isolation and identification of target bacteria

[0077] 1.1 Culture medium Media required: Modified Stevenson's medium, LB-Malachite Green solid medium, PM plates. Preparation of LB-Malachite Green solid medium (1) Preparation of malachite green mother liquor: 0.125 g of malachite green solid was weighed and dissolved in 500 ml of deionized water. After complete dissolution, the pH was adjusted to 7.0, and the solution was sterilized by high-pressure steam for use. (2) Preparation of LB solid medium: Weigh out 10 g of peptone, 10 g of yeast extract, and 10 g of sodium chloride, dissolve in 1,000 ml of deionized water, adjust the pH to 7-7.2, and sterilize under high pressure. Add 100 μl of 0.25 g / L malachite green to every 100 ml of LB solid medium, so that the concentration of malachite green in the LB solid medium becomes 0.25 mg / L.

[0078] 1.2 Separation method (1) Enrichment culture: 2 g of sample (original sample or sample after enrichment culture) is inoculated into 100 ml of modified Stevenson medium containing calcium carbonate, or a medium containing 0.5 g of calcium carbonate per 100 ml, or the sample is inoculated into modified Stevenson medium containing calcium carbonate supplemented with an organic carbon source (concentration 0.01 M). The culture is performed in a shaker at 28°C and 180 rpm to increase the abundance of the target bacteria in the medium. Generally, the amount of NH4 in the medium is increased. + -N concentration was negative in the Nessler reagent spot test, and NO2 - -N until a strongly positive Griess reagent spot test is obtained, or NH4 + -N and NO2 - -N were negative in spot tests, and NO3 - -N is isolated by enrichment culture until it is positive in the diphenylamine spot test. (2) Removal of impurities: Add 3 ml of cold concentrated hydrochloric acid (4°C, 3% of the volume of the culture solution) to the above culture system, shake to homogenize, and remove other contaminants. Then, quickly neutralize with cold sodium hydroxide solution (4°C) within 3 minutes to bring the pH of the system to approximately 7.0. (3) Isolation of target bacteria by selective (positive) plating: The bacterial solution that exhibits nitrification activity after impurity removal treatment is diluted 10-fold and smeared on a solid LB-Malachite Green plate to isolate the target bacteria.

[0079] 1.3 Purification After 7 days of incubation at 28°C, single colonies are picked onto PM plates, streaked and purified. Purity is confirmed by microscopy.

[0080] 1.4 Identification of activity The isolated and purified target bacteria are inoculated onto PM plates and cultured at 28°C for 10 days. To confirm nitrification activity, Griess reagent is dropped directly onto the plate, and a plate without bacteria is used as a blank control. Within 1 minute, the Griess reagent turns red, indicating the production of nitrite. Repeated testing again showed a positive color reaction, tentatively confirming that the bacteria are nitrifying. Analysis of the PM medium by high-pressure liquid chromatography revealed trace amounts of nitrite and nitrate, no nitrite was detected, and NO3 - The content of -N was less than 0.2 mg / L and did not interfere with the results.

[0081] 1.5 Verification of nitrification activity in liquid pure culture A loopful of pure bacterial lawn growing on a PM plate was taken, placed in 100 mL of PBS, and sonicated for 2 minutes to completely and uniformly disperse the bacteria, preparing a PBS-target bacterial suspension. One ml of the bacterial suspension was inoculated into 50 mL of modified Stevenson's medium (250 mL Erlenmeyer flask) containing various organic substances as carbon sources, with each group replicated three times. A blank control was used, with the medium not inoculated with bacteria. The culture was incubated at 28°C and 180 rpm for at least 14 days, after which the supernatant was collected and analyzed for NO2. - -Measure the content of N. NO2 - The -N content was 5 mg / L higher than that of the control group, indicating that there was clearly high nitrification activity.

[0082] 2. Isolation, purification, and identification of target bacteria in the soil of the waterside area of the Three Gorges Reservoir in Kaizhou District, Chongqing City The test soil samples were collected from the waterside area of the Three Gorges Reservoir in Kaizhou District, Chongqing City (latitude and longitude 31.07'56-57'', 108.28'34-35''). Five ml of 0.1 M glucose (concentration in the medium is 0.01 M) is added to 45 ml of modified Stevenson's medium (with calcium carbonate added), and 1 g of soil sample passed through a 20-mesh sieve is added, followed by incubation in a shaker at 28°C and 180 rpm for 14 days. Approximately 200 μl of sample was taken every 2 to 3 days, placed on a white porcelain colorimetric plate, and dropped with Griess's reagent (qualitative measurement). -After about 7 days of incubation, clear nitrification activity was observed. - The -N content was about 1.0 ppm, but exceeded 20 ppm after 13 days of cultivation. The results are shown in the table below.

[0083] [Table 3a]

[0084] After culturing for 14 days, concentrated HCl (3% of the volume of the culture solution) was added to the culture solution, and the mixture was shaken for 3 minutes. The mixture was then smeared on an LB-Malachite Green plate according to a 10-fold dilution gradient, and each dilution gradient was repeated three times. -7 The average number of target bacteria in the dilution gradient was 21 (Figure 2a, left and right are the results of two repeated experiments). At this point, the target bacteria were isolated.

[0085] Single colonies were picked and streaked onto PM plates. After incubation at 28°C, microscopic examination confirmed that the bacteria had a uniform external morphology and were pure bacteria (Figure 2b).

[0086] The isolated and purified bacteria were inoculated onto PM plates and cultured at 28°C for 10 days. When Griess reagent was added dropwise to the plate, the color immediately changed to brown (Fig. 2c). - The -N content was over 10 mg / L, and the nitrification activity was strong.

[0087] The pure bacterial colony growing on the PM plate was scraped to prepare a PBS-target bacterial suspension. 1 ml of the bacterial suspension was inoculated into 100 ml of modified Stevenso's medium containing 0.06 M pyruvate as a carbon source (container: 250 ml Erlenmeyer flask). This was repeated three times for each group. A medium without inoculation was used as a blank control. Culture was performed on a shaker at 28°C and 180 rpm for 24 days, and the supernatant of the culture medium was collected and analyzed for NO2. -The measurement of the -N content confirmed that it exceeded 100 mg / L. Figure 2d shows the color development with Griess's reagent after diluting the supernatant of the target bacteria culture 10-fold and 100-fold, indicating that the target bacteria have high nitrification activity.

[0088] 3. Isolation of target bacteria from moist soil in North China One gram of moist soil from the North China region, passed through a 100-mesh sieve, is inoculated into 50 ml of modified Stevenson's medium supplemented with calcium carbonate and cultured. - Changes in -N and the development of nitrification activity were periodically monitored. After 14 days of cultivation, concentrated HCl was added at a volume of 3% of the culture volume and shaken thoroughly. The culture was quickly neutralized with sodium hydroxide solution within 3 minutes until the pH reached approximately 7.0. 1 ml of the culture neutralized with alkali was added to 9 ml of sterile water, diluted 10-fold, and plated on LB-Malachite Green plates, with each dilution gradient repeated three times. Target colonies appeared on the plates. The statistics of the target bacteria are shown in the table below.

[0089] [Table 3b]

[0090] Single colonies were picked and streaked onto PM plates and verified by microscopy. After 14 days of incubation at 28°C, the Griess reagent spot test showed a brown color (Figure 2e), indicating strong nitrification activity.

[0091] The pure bacterial colony growing on the PM plate was scraped to prepare a bacterial suspension of the target bacteria in PBS. 1 ml of the bacterial suspension was inoculated into 100 ml of modified Stevenson's medium (250 ml Erlenmeyer flask) containing 0.06 M pyruvic acid as a carbon source, and the suspension was repeated three times for each group. A medium without inoculation was used as a blank control. The culture was incubated at 28°C and 180 rpm in a shaker for 28 days, and the supernatant of the culture was collected and analyzed for NO2. - The measured content of -N exceeded 100 mg / L. The target bacteria has high nitrification activity.

[0092] 4. Isolation of target bacteria from multiple other types of soil Using the target bacterium isolation scheme determined in this invention, target bacteria were also isolated from agricultural soil in Zhaogang Township, Fengqiu County, Xinxiang City, Henan Province (latitude and longitude 35.02', 114.05'), Wushan soil in Changshu, Jiangsu Province (latitude and longitude 31°32'58.98'', 120°41'55.37''), and loess soil (latitude and longitude 31°48.87'', 120°38'32.22''), and red soil in Yingtan, Jiangxi Province.

[0093] The results of the isolation revealed that, in general, contaminating bacteria usually appear within 24 hours, whereas target bacteria appear after 5 to 7 days. Figure 2f shows the appearance of target bacteria from soil or soil suspension (modified Stevenson's medium). The target bacteria are shown within a circle, clearly distinguishable from other contaminating bacteria.

[0094] Example 1 Indigenous nitrogen fixation ability of the mycobacteria of the present invention

[0095] 1. Materials and Methods 1.1 Preparation of experimental strains and PBS bacterial solution: The JD and YT strains were obtained according to Preparation Example 1 and deposited at CGMCC. The JD strain was isolated from soil in the waterside area of the Three Gorges Reservoir in Kaizhou District, Chongqing City (latitude and longitude 31°07'56-57'', 108°28'34-35''), and the YT strain was isolated from farmland soil in Zhaogang Township, Fengqiu County, Xinxiang City, Henan Province (latitude and longitude 35°02', 114°5'). One loopful of pure bacteria JD or YT was placed in 100 mL of PBS solution, sonicated for 2 minutes, and approximately 1 × 10 7 The bacteria were dispersed uniformly until the concentration reached CFU / mL, and a PBS-JD / YT bacterial solution was prepared for use.

[0096] 1.2 Spontaneous nitrogen fixation in the absence of bound nitrogen 1 ml of PBS-YT / JD bacterial solution was inoculated into a liquid medium for pure culture, and NH4 + -N, NO2 - -N, NO3 -The nitrogen fixation ability of the mycobacteria of the present invention is preliminarily determined from the change in -N. Next, the mycobacteria are streaked onto nitrogen-fixing plates and their growth is observed. If normal growth is observed, colonies are scraped off to detect the nitrogen fixation gene. If the nitrogen fixation gene is detected, it is determined that the mycobacterium has spontaneous nitrogen fixation activity.

[0097] 1.2.1 Liquid medium and culture method A: Liquid medium The medium contains glucose, CaCl2, MgSO4·7H2O, K2HPO4·3H2O, Na2MoO4·2H2O, HCl, and FeSO4·7H2O, and all reagents are prepared separately. Glucose: 11.25g + 45ml water CaCl2:0.625g+water 25ml MgSO4·7H2O: 1.25g + water 25ml K2HPO4·3H2O: 2.62g + water 25ml Na2MoO4·2H2O: 0.15g+water 25ml HCl:100mlHCl+water 900ml FeSO4·7H2O: 0.5g + 12.5ml water (mix, filter and sterilize before use) When preparing, 100 ml of deionized water is dispensed into a 250 ml Erlenmeyer flask and sterilized separately. After all the reagents are prepared separately, they are placed in a high-pressure steam cooker to sterilize and prepare for use. Liquid medium for spontaneous nitrogen fixation experiments was obtained by adding 2 ml of glucose, 0.6 ml of CaCl2, 0.4 ml of MgSO4·7H2O, 0.1 ml of FeSO4·7H2O, 0.1 ml of Na2MoO4·2H2O, 1 ml of K2HPO4·3H2O, and 0.16 ml of HCl to every 100 ml of sterile water under sterile conditions.

[0098] B: Culture system and culture conditions 1 ml of the PBS-JD / YT bacterial solution is inoculated into the above liquid medium and cultured in a shaker at 28°C and 180 rpm. + -N, NO2 - -N, NO3 -Changes in -N are measured qualitatively every two days to provide a preliminary assessment of nitrogen fixation capacity.

[0099] 1.2.2 Nitrogen fixation plate method The pure strain was streaked onto nitrogen-fixing medium and cultured in an incubator at 28°C. During the culture, the humidity of the culture conditions was maintained constant to prevent evaporation of water from the plate. After observing growth for a certain period, if normal growth was observed, all colonies were scraped with a sterile stainless steel spoon, suspended in phosphate buffer solution (PBS), centrifuged, and the supernatant removed. The centrifuged solids were collected, frozen in liquid nitrogen, and stored to detect their nitrogen fixation genes and nitrogen fixation ability.

[0100] 1.2.3 Detection of nitrogen fixation genes PCR amplification was performed using three pairs of nitrogen-fixing primers, with each primer pair repeated four times. All three primer pairs were capable of amplifying nitrogenase iron proteins from nitrogen-fixing bacteria. Letters other than A, T, C, and G in the primer sequences (e.g., Y, S, R, B, W, etc.) represent degenerate bases, allowing the primers to more comprehensively amplify the target gene. The first pair of primers had an amplification length of 360 bp and the primer sequences were as follows: PolyF:5'-TGCGAYCCSAARGCBGACTC-3'(SEQ ID NO:3) PolyR:5'-ATSGCCATCATYTCCRCGGA-3'(SEQ ID NO:4) The second pair of primers has an amplification length of 280 bp and the primer sequences are as follows: nifH F:5'-ACCCGCTGATCCTGCACGCCAAGG-3'(SEQ ID NO:5) nifH R:5'-ACGATGTAGATTTCCTGGGCCTTGTT-3'(SEQ ID NO:6) The third pair of primers has an amplification length of 450 bp and the primer sequences are as follows: nifH-F:5'-AAAGGYGGWATCGGYAARTCCACCAC-3'(SEQ ID NO:7) nifH-R:5'-TTGTTSGCSGCRTACATSGCCATCAT-3'(SEQ ID NO:8)

[0101] 1.3 Spontaneous nitrogen fixation in the presence of bound nitrogen The PBS-JD / YT bacterial solution is inoculated into a medium containing pyruvic acid as a carbon source and ammonium sulfate as a nitrogen source for pure culture. During the cultivation process, the growth of the bacterial cells and the NH4 + The change in -N concentration was measured periodically, and it was found that the bacteria grew normally and at the same time, NH4 + If the -N concentration increases rapidly, it is determined that nitrogen fixation is occurring. The spontaneous nitrogen fixation of the mycobacteria of the present invention in the presence of bound nitrogen only takes into account the ammonia secretion of the strain, not nitrogen fixation in the cellular synthesis process. Furthermore, the energy consumption of the spontaneous nitrogen fixation of the mycobacteria of the present invention can be calculated based on the amount of pyruvate consumed during this stage.

[0102] 1.3.1 Pyruvate as a carbon source and ammonium sulfate as a nitrogen source A: Modified Stevenson's medium: The nitrogen source is ammonium sulfate at a concentration of 2.0 g / L. B. Pyruvate-based medium: Add the prepared, sterilized pyruvate solution to the modified Stevenson medium to achieve a pyruvate concentration of 0.02 M. Adjust the pH value as needed with 1 M NaOH solution. 1 ml of the PBS-JD / YT bacterial solution is inoculated into 100 ml of a medium containing pyruvic acid as a carbon source and ammonium sulfate as a nitrogen source, and pure culture is carried out. Culture conditions: temperature 28°C, rotation speed 180 rpm, CO2 concentration 0.5%, RH 50%.

[0103] 1.3.2 Indicator analysis After 5 days of cultivation, the growth of the bacteria was measured every 2 days. + -N, NO2 - -N, NOx - -Measure the N content.

[0104] 1.4 Quantitative measurement of nitrogen fixation activity The nitrogen fixation activity of the experimental strains was measured by the acetylene reduction method. 100 mL of microbial cells were collected in a 100 mL glass bottle at mid-logarithmic growth phase (OD ). 600 Ten mL of bacterial suspension (pH = 0.4–0.8) was added and sealed. 10% of the air volume in the bottle was replaced with purified, high-purity acetylene using a syringe. After incubation in the dark at 28°C for 24 h, the gas in the bottle was collected and analyzed for ethylene concentration using a gas chromatograph (Shimadzu, GC-2014C) equipped with a flame ionization detector (FID) and a Porapak T 4 mm x 2 m packed column. The column temperature was set to 60°C, the injector temperature to 120°C, and the FID detector temperature to 220°C. The carrier gas was high-purity nitrogen gas. The protein content of the bacterial suspension was measured by the Bradford method (Bradford, 1976) to represent its biomass. Bovine serum albumin (BSA) was used as the protein standard. The ethylene production rate per unit mass of protein per unit time was calculated (nmol C2H4 / mg protein / h). The calculation formula is as follows:

[0105] (1) Calculation of headspace volume in the bottle (cm 3 ):V headspace =V total -V liquid .V total is 100cm 3 where V liquid is the volume occupied by 10 ml of bacterial solution.

[0106] (2) Calculation of sample ethylene concentration (ppm) using the external standard method based on the ethylene gas standard concentration:

number

[0107] (3) Calculation of the amount of ethylene produced during the incubation period (nmol C2H4):

number

[0108] (4) Calculation of ethylene production rate per unit mass of protein per unit time (nmol C2H4 / mg / h):

number

[0109] 1.5 Comparison of energy consumption by nitrogen fixation Representative nitrogen-fixing bacterial genera / species, such as Azotobacter chroococcum (e.g., Azotobacter chroococcum), Clostridium pasteurianum, Klebsiella pneumoniae, and Streptomyces thermoautotrophicus, were selected, and synthetic nitrogen fixation was also considered. Based on published data on the energy consumption of nitrogen fixation by related bacterial genera / species and synthetic nitrogen fixation, the nitrogen fixation ability of the mycobacteria of the present invention was analyzed and studied by comparing it with the experimental results of the nitrogen fixation ability of the mycobacteria JD and YT of the present invention, using mg of nitrogen fixed per mg of pyruvate consumed as a unified unit.

[0110] 2 Results 2.1 Spontaneous nitrogen fixation in the absence of bound nitrogen

[0111] 2.1.1 Growth in liquid medium When the JD and YT strains were inoculated into liquid media, both strains were able to grow, but their growth was slow. A ring of bacteria was found attached to the wall of the culture bottle, indicating that the two strains had strong adhesion to the wall, which affected their continued growth. As shown in Figure 3a, YT is growing on the left and JD on the right.

[0112] Table 4a shows the changes in pH and NH4 in liquid medium during pure culture of mycobacterial strains JD and YT. + -N, NO2 - -N, NO3 - -N accumulation. During the cultivation process, JD and YT produce acid, which lowers the pH of the liquid medium. If the pH of the medium is too low, the strains will stop growing. Therefore, during the cultivation process, the pH should be adjusted to 7.0-7.2 in a timely manner. Qualitative measurement shows that there is a high concentration of NH4 in the liquid medium. + -N accumulated, indicating that JD and YT have a certain nitrogen fixation ability.

[0113] [Table 4a]

[0114] 2.1.2 Growth on nitrogen-fixing plates As shown in Figure 3a and Table 4a, mycobacteria YT and JD grew slowly in liquid medium, and both strains exhibited NH4 + Although the bacteria accumulated -N, the amount of accumulation was very small. Because this bacterium has surface growth characteristics, it is not suitable for obtaining large numbers of cells in liquid culture. In this study, a nitrogen fixation plate experiment was also conducted.

[0115] As shown in Figure 3b (YT on the left, JD on the right), the two strains cultured in the above liquid medium were streaked onto nitrogen-free agar plates. After 11 days of culture, both strains grew normally on the plates, with single colonies. This indicates that the liquid medium and the strains inoculated therein are not contaminated. On the other hand, the turbidity observed in the growth experiment of the strains in liquid medium (Figure 3a) reflects normal growth of the strains and is not due to the death of the microorganisms.

[0116] Figure 3c shows YT (left) and JD (right) grown on nitrogen-fixing plates for 7 days. Both strains were growing on the plates, with milky white traces of bacteria visible to the naked eye. After 29 days of growth, both strains had grown further, with single colonies clearly visible, but the numbers were small and the growth was slow.

[0117] Figure 3d shows the cell morphology of JD bacteria after culturing on a nitrogen-fixing plate for more than 40 days (Nikon stereomicroscope, 100x magnification).

[0118] To promote the growth of the strains on nitrogen-fixing plates and obtain more growth to meet research and detection needs (single clones are not necessary), we used a coated rod to scrape the bacteria from the nitrogen-fixing plates shown in Figure 3c and then re-plated them onto nitrogen-fixing plates. However, growth was still poor. The resting cells were then centrifuged, the supernatant removed, and the colonies directly scraped and plated onto LB plates, ensuring as many bacteria as possible were collected at the time of plating. Figure 3e shows the culture after centrifuging the resting cells, removing the supernatant, and directly plating the bacteria onto nitrogen-fixing plates (YT on the left, JD on the right). After the above treatment, the two strains were observed to grow significantly on the nitrogen-fixing plates.

[0119] 2.1.3 Detection of nitrogen fixation genes PCR amplification was performed using three pairs of primers containing the nitrogenase iron protein. All three pairs of primers were able to amplify, with the amplification results for the second primer being particularly clear. The results are shown in Figure 3f. At the molecular level, both strains YT (liquid 1) and JD (liquid 2) have the ability to fix nitrogen.

[0120] 2.2 Spontaneous nitrogen fixation in the presence of bound nitrogen In the presence of bound nitrogen (2.0 g / L ammonium sulfate) and pyruvate as a carbon source, both mycobacteria JD and YT of the present invention grew normally. As shown in Figure 3g, as the two strains grew, the ammoniacal nitrogen concentration in the medium first rapidly decreased. After 7 days of growth, it dropped to a low level, then rapidly increased. After 9 days of growth, the ammoniacal nitrogen concentration in the medium decreased again. These results indicate that significant nitrogen fixation occurred between 7 and 9 days of growth.

[0121] As shown in the table below, in the nitrogen fixation process of mycobacteria of the present invention, in the culture system inoculated with JD, pyruvate decreased by 6.05 mg and ammonia nitrogen increased by 15.49 mg. That is, the JD strain fixed 2.56 mg of nitrogen (ammonia secretion) for every 1 mg of pyruvate consumed. In the culture system inoculated with YT, pyruvate decreased by 7.06 mg and ammonia nitrogen increased by 15.098 mg. That is, the YT strain fixed 2.13 mg of nitrogen (ammonia secretion) for every 1 mg of pyruvate consumed. The average amount of nitrogen fixed (ammonia secretion) for every 1 mg of pyruvate consumed by the two strains was 2.35 mg.

[0122] [Table 4b]

[0123] 2.3 Nitrogen fixation activity Analysis of the experimental results of nitrogen fixation activity of the two strains, YT and JD, revealed that the average ethylene production rates of the YT and JD strains were 0.827±0.552 nmol / mg protein / h and 0.502±0.337 nmol / mg protein / h, respectively.

[0124] 2.4 Comparison of the abilities of the mycobacteria of the present invention with representative nitrogen-fixing bacteria and synthetic nitrogen-fixing bacteria

[0125] 2.4.1 Energy consumption by nitrogen fixation by representative bacterial genera / species

[0126] (1) Azotobocter: The Azotobocter genus, represented by Azotobocter chroococcum, can fix 10–20 mg of nitrogen for every 1 g of glucose consumed (Zhu Zhaoliang et al., 1992). In the tricarboxylic acid cycle, one molecule of pyruvate produces a total of 15 molecules of ATP through dehydrogenation. One molecule of glucose produces two molecules of pyruvate, and one molecule of glucose produces a total of 36–38 ATP molecules through three steps: glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation (Shen Tong et al., 2000). Therefore, for ATP produced solely through the tricarboxylic acid cycle, 1 mole of glucose corresponds to 2 moles of pyruvate, which corresponds to 30 moles of ATP. Thus, for Azotobacter , 10–20 mg of nitrogen is fixed for every 1 g of glucose consumed, which corresponds to 0.978 g of pyruvate fixing 10–20 mg of nitrogen, i.e., 0.0102–0.0204 mg of nitrogen is fixed for every mg of pyruvate consumed.

[0127] (2) Clostridium pasteurianum: Clostridium pasteurianum fixes 1.5-7.0 mg of nitrogen for every 1 g of glucose consumed (Zhu Zhaoliang et al., 1992). Based on the above analysis, in terms of ATP production through the tricarboxylic acid cycle, 1 g of glucose is equivalent to 0.978 g of pyruvate, which is equivalent to 0.978 g of pyruvate fixing 1.5-7.0 mg of nitrogen, i.e., for every 1 mg of pyruvate consumed, 0.001534-0.007157 mg of nitrogen is fixed.

[0128] (3) Klebsiella pneumoniae: The basic enzymatic reaction of the nitrogen fixation enzyme represented by Klebsiella pneumoniae is as follows: N2 + 8H + +8e -+ 16MgATP → 2NH3 + H2 + 16MgADP + 16Pi (Han Bin et al., 2009). Therefore, 1 mol N2 corresponds to 16 mol of ATP. Because one molecule of pyruvate can generate a total of 15 molecules of ATP through the tricarboxylic acid cycle (Shen Tong et al., 2000), 1.07 mol of pyruvate is required to generate 16 mol of ATP. Therefore, Klebsiella pneumoniae requires at least 1.07 mol of pyruvate to fix 1 mol of N2, resulting in 0.298 g of nitrogen fixation for every 1 g of pyruvate consumed.

[0129] (4) Streptomyces thermoautotrophicus: The basic enzymatic reaction of the nitrogen fixation enzyme of Streptomyces thermoautotrophicus is: N2 + 8H + +8e - + 4-12MgATP → 2NH3 + H2 + 4-12MgADP + 4-12Pi (Markus Ribbeetal, 1997). Because one molecule of pyruvate can generate a total of 15 molecules of ATP via the tricarboxylic acid cycle (Shin Tong et al., 2000), in the case of Streptomyces thermoautotrophicus, at least 4-12 moles of ATP, generated by 0.27-0.8 moles of pyruvate, are required to fix 1 mole of N2. This translates to a nitrogen fixation capacity of 0.40-1.18 grams per gram of pyruvate consumed by Streptomyces thermoautotrophicus. In other words, 0.4-1.18 mg of nitrogen is fixed for every 1 mg of pyruvate consumed.

[0130] (5) Mycobacteria of the present invention: The section "Spontaneous nitrogen fixation in the presence of bound nitrogen" was calculated by studying the energy consumption of nitrogen fixation in JD and YT strains. Specifically, the JD strain fixed 2.56 mg of nitrogen (ammonia secretion) for every 1 mg of pyruvate consumed. The YT strain fixed 2.13 mg of nitrogen for every 1 mg of pyruvate consumed. The average amount of nitrogen fixed (ammonia secretion) for every 1 mg of pyruvate consumed by the two strains was 2.35 mg.

[0131] 2.4.2 Energy consumption by synthetic nitrogen fixation According to data, the current synthetic ammonia industry requires 48.2 x 10 energy to synthesize 1 ton of ammonia (NH3, molecular weight 17.03). 6 kJ, and the energy consumption to synthesize 1 ton of urea (CO(NH2)2, molecular weight 60.06) is 33.2 × 10 6 According to the amount of fixed nitrogen, synthetic ammonia consumes 48.2 kJ of energy to fix 0.822 g of nitrogen, which is equivalent to the energy released by hydrolyzing 1.58 molecules of ATP (ATP hydrolysis to produce ADP releases 30.54 kJ / mol of heat). This is equivalent to the amount of ATP produced through the tricarboxylic acid cycle from 9.28 g of pyruvate (based on the fact that 1 molecule of pyruvate produces 15 molecules of ATP). Therefore, when converting the energy consumption in synthetic ammonia, 0.08858 mg of nitrogen is fixed for every 1 mg of pyruvate consumed.

[0132] In synthetic urea, when converted into the amount of urea nitrogen, 33.2 kJ is required to fix 0.466 g of nitrogen, which is equivalent to the energy required for the hydrolysis of 1.087 molecules of ATP, and is equivalent to the amount of ATP produced by 6.38 g of pyruvic acid through the tricarboxylic acid cycle. Therefore, when converted into the energy consumption of synthetic urea, 0.073 mg of nitrogen is fixed for every 1 mg of pyruvic acid consumed.

[0133] 2.4.3 Comparison of nitrogen fixation capabilities of various bacterial species / genera and synthetics Analysis of the energy consumption of several representative nitrogen-fixing bacterial species / genera and synthetic nitrogen fixation revealed that, when comparing the amount of nitrogen fixed per unit weight of pyruvate consumed, the order of nitrogen fixation ability was as follows: mycobacteria of the present invention > Streptomyces thermoautotrophicus > Klebsiella pneumoniae > synthetic nitrogen fixation (synthetic ammonia and synthetic urea) > Azotoboctor genus represented by Azotoboctor crococcum > Clostridium pasteurianum. Meanwhile, the nitrogen fixation ability of the mycobacterium of the present invention was 2 to 6 times that of Streptomyces thermoautotrophicus, 8 times that of Klebsiella pneumoniae, 115 to 230 times that of Azotoboctor genus, and 328 to 1532 times that of Clostridium pasteurianum. In the case of synthetic nitrogen fixation (synthetic ammonia and synthetic urea), the nitrogen fixation capacity of the mycobacteria of the present invention is about 29 times that of the synthetic nitrogen fixation capacity, as shown in the table below.

[0134] [Table 4c]

[0135] 3 Conclusion (1) When cultured in liquid media, the two strains grew normally and accumulated ammoniacal nitrogen in the medium. When cultured on nitrogen-fixing plates, the two strains clearly grew. PCR amplification of the two strains using three pairs of primers containing the nitrogenase iron protein confirmed the presence of genes for nitrogen fixation in both strains. The mycobacteria of the present invention are capable of nitrogen fixation in the absence of bound nitrogen.

[0136] (2) In the presence of bound nitrogen (ammonium sulfate), the mycobacteria of the present invention have a high nitrogen fixation activity, with an average amount of nitrogen fixed (ammonia secretion) of 2.35 mg per 1 mg of pyruvic acid consumed.

[0137] (3) The average ethylene production rates of the two strains were 0.827 nmol / mg protein / h and 0.502 nmol / mg protein / h, respectively. The mycobacteria of the present invention have obvious nitrogen fixation activity.

[0138] (4) The mycobacteria of the present invention have a high nitrogen fixation capacity, which is 2 to 6 times that of Streptomyces thermoautotrophicus, 8 times that of Klebsiella pneumoniae, 115 to 230 times that of Azotobacter sp., and 328 to 1,532 times that of Clostridium pasteurianum. With respect to synthetic nitrogen fixation (synthetic ammonia and synthetic urea), the nitrogen fixation capacity of the mycobacteria of the present invention is approximately 29 times that of the synthetic nitrogen fixation capacity.

[0139] Example 2 Growth and nitrification activity of the mycobacterium of the present invention in pure culture using pyruvate as a carbon source

[0140] 1. Materials and Methods 1.1 Test strain: PBS-JD / YT bacterial solution.

[0141] 1.2 Culture media and indicator measurements

[0142] (1) Culture medium Modified Stevenson's medium with pyruvate as carbon The concentrations of pyruvate in the medium are designed to be 0.06 M and 0.1 M, respectively. Fine-tune the desired pH value with 1 M NaOH solution.

[0143] (2) Methods and indicators 1 ml of PBS-JD / YT bacterial solution was inoculated into the prepared sterilized liquid medium and cultured in a shaker at 28°C and 180 rpm. The growth rate of the bacterial cells, pH, and NH4 + -N, NO2 - -N, NO x - -Measure the N content regularly.

[0144] 2 Results As shown in Figure 4a (top graph: 0.06 M pyruvate, bottom graph: 0.1 M pyruvate) and the table below, when pure cultured with 0.06 M pyruvate as the carbon source, the OD of the two strains, JD and YT, was 0.06 M pyruvate after 24 d of culture. 600 The average values are 1.84 and 1.62, respectively. + The -N concentration decreased from the initial 424 mg / L to 93.08 mg / L and 66.87 mg / L, respectively, and NO2 - -N accumulated, with average concentrations reaching 139.90 mg / L and 96.31 mg / L, respectively. The experiment clearly showed that when purely cultured with 0.06 M pyruvate as the carbon source, both strains YT and JD grew well and had high nitrification activity. When purely cultured with 0.1 M pyruvate as the carbon source, the OD of both strains JD and YT was 0.06 M pyruvate after 33 days of culture. 600 The average values were all 2.04, and the NH4 + -N was almost absent, with average concentrations of only 4.52 mg / L and 3.88 mg / L, and NO2 - The concentrations of -N accumulated in the strains were 177.92 mg / L and 161.14 mg / L, respectively. The experiments clearly showed that when pure cultured with 0.1 M pyruvate as the carbon source, both strains YT and JD grew well and had high nitrification activity.

[0145] [Table 5]

[0146] 3 Conclusion: When purely cultured with different concentrations of pyruvate as the carbon source, all the test strains grew normally, had high ammonia oxidation activity, and exhibited significant nitrification activity.

[0147] Example 3 CO2 fixation by the mycobacteria of the present invention

[0148] 1. Materials and Methods

[0149] 1.1 Test strain: PBS-JD / YT bacterial solution

[0150] 1.2 Media and culture systems: A. Modified Stevenson's medium B. Pyruvic acid is used as a carbon source. First, prepare a 2M pyruvic acid solution (weigh 88.0701 g of pyruvic acid, add 400 ml of distilled water, stir, add 40 g of solid NaOH, cool, and then adjust the volume to 500 mL and adjust the pH to 7.17). Sterilize the solution separately and prepare it for use. Prior to use, 5 ml of a 2 M pyruvic acid solution is first added to 94 ml of modified Stevenson's medium.

[0151] 1 ml of the PBS-JD / YT bacterial solution was inoculated into the above medium to bring the total volume of the culture system to 100 ml. The concentration of pyruvic acid in the medium was set to approximately 0.1 M.

[0152] 1.3 Culture conditions: Culture in a shaker at 28°C, 180 rpm, and 5% CO2. Continuous culture for 21 days.

[0153] 1.4 Indicator measurement: The growth rate and pH of the bacterial cells are measured periodically. The supernatant is collected and NH4 + -N, NO2 - -N, NO x - The content of -N is measured. The content of pyruvate in the medium is measured periodically. The content of pyruvate is detected using high performance liquid chromatography (HPLC). After the cultivation is complete, the mixture is centrifuged at 4°C for 5 minutes at 10,000 rpm in a high-speed centrifuge. The resulting cells are then freeze-dried in a vacuum, and the dry weight and carbon and nitrogen content of the cells are measured. The amount of CO2 fixed is estimated by analyzing the changes in the carbon and nitrogen content in the culture system (culture medium supernatant and cells).

[0154] 2 Results 2.1 Changes in pyruvate concentration in the culture system As shown in Figure 4b (top panel: JD, bottom panel: YT), when JD and YT were cultured purely under high CO2 conditions with pyruvate as the carbon source, the pyruvate concentration in both strains decreased, then increased, and then decreased again. This indicates that the organisms independently complete the forward and reverse TCA cycle metabolic processes. In the first phase, the strains perform the forward TCA cycle, consuming pyruvate and accumulating arginine metabolic intermediates to generate energy for ammonia oxidation. In the second phase, the strains initiate ammonia oxidation, perform arginine metabolism, and use the energy generated by metabolism to proceed through the reverse TCA cycle, accumulating pyruvate and preparing for synthetic metabolism. In the third phase, the strains perform synthetic metabolism, consuming pyruvate, completing gluconeogenesis, and growing. Both strains fix CO2 through the reverse TCA cycle pathway.

[0155] 2.2 Analysis of carbon and nitrogen consumption and carbon fixation in metabolic processes

[0156] (1) Carbon-nitrogen ratio of bacterial cells Experimental results showed that the carbon-nitrogen ratio of the mycobacterial cells of the present invention was 5.65:1, which is not significantly different from the carbon-nitrogen ratio of general heterotrophic bacterial cells (empirical value is 5:1).

[0157] (2) Carbon and nitrogen consumption by the synthetic metabolism of the fungus The carbon and nitrogen consumption during synthetic metabolism in bacteria can be divided into two categories: material consumption for synthetic metabolism and energy consumption for synthetic metabolism. The materials and energy required for synthetic metabolism in typical heterotrophic bacteria come from organic carbon and nitrogen sources in the environment (usually derived primarily from the decomposition of other living organisms). Experience has shown that the total carbon-nitrogen mass ratio is usually about 25:1, with materials for synthetic metabolism accounting for 20% of the total carbon source and energy for synthetic metabolism accounting for 80% of the total carbon source.

[0158] (3) The effect of the mycobacteria of the present invention in reducing CO2 emissions The mycobacteria of the present invention differ from typical heterotrophic bacteria in the following respects: While typical heterotrophic bacteria obtain energy (and release CO2) through glycolysis of organic carbon sources and the TCA cycle, the mycobacteria of the present invention obtain energy through glycolysis of organic carbon sources and the TCA cycle, and also obtain the energy needed for assimilation through nitrogen metabolic pathways such as ammonia oxidation and nitrification. They can also assimilate CO2 through the reverse TCA cycle. As can be seen from the above, the mycobacteria of the present invention can reduce the organic carbon consumption required for the TCA cycle by 20 parts when synthesizing one part nitrogen and five parts carbon. Furthermore, by using the energy generated by nitrogen metabolism to drive the TCA cycle in the reverse direction, they can fix 5 parts CO2 and obtain organic carbon sources, which are substances needed for synthetic metabolism. Thus, compared to typical heterotrophic bacteria, the mycobacteria of the present invention reduce CO2 emission by 25 parts for every 1 part nitrogen consumed to synthesize substances through assimilation.

[0159] (4) Estimation of CO2 fixation equivalents by changing the substrate in the experiment NH4 + After continuous cultivation of JD bacteria for 21 days in a substrate solution containing 424 mg of -N and 0.1 mol of pyruvic acid, NH4 + -N 4.52mg, NO x - 287.8 mg of N and <0.1 mg of organic carbon remain. Therefore, the mass of elemental nitrogen for assimilative growth is 131.68 mg. Converted at a molar ratio of 25:1, the mass of elemental carbon consumed by assimilating 131.68 mg of elemental nitrogen is 2822 mg, which translates to a mass reduction in CO2 emissions of 10.35 g. Similarly, after 21 days of continuous cultivation of YT bacteria, NH4 + -N 3.88mg, NO x -260.6 mg of N and <0.1 mg of organic carbon remain. Therefore, the mass of elemental nitrogen for assimilative growth is 159.52 mg. Based on a 25:1 molar ratio, the mass of elemental carbon consumed by assimilating 159.52 mg of elemental nitrogen is reduced by 3418 mg, which translates to a mass reduction in CO2 emissions of 12.53 g.

[0160] 3 Conclusion (1) The mycobacteria of the present invention have a reverse TCA cycle pathway.

[0161] (2) The mycobacteria of the present invention fix CO2 through the reverse TCA cycle and have the function of reducing CO2 emissions.

[0162] Example 4 Growth and nitrification activity of the mycobacterium of the present invention in pure culture using hydrazine sulfate as a nitrogen source

[0163] 1. Materials and Methods

[0164] 1.1 Test strain: Resting cells of JD and YT obtained in Production Example 1. That is, the cultured bacteria were centrifuged to remove the supernatant, washed repeatedly with physiological saline, and placed in a refrigerator at 4°C. In the Griess reagent spot test, NO2 - When -N no longer develops color, the pure bacteria can be confirmed by streaking. After inoculation, the supernatant is removed by centrifugation, and the mixture is suspended in medium and then cultured in a shake flask.

[0165] 1.2 Culture system (1) Culture medium A. Modified Stephenson's medium with hydrazine sulfate as the nitrogen source The remaining ingredients and treatments were the same except that 2.0 g of (NH4)2SO4 in the modified Stevenson medium was replaced with an equal amount of nitrogen, N2H4·H2SO4, i.e., 1.97 g of N2H4·H2SO4. B. Add 6 ml of a separately sterilized 1.0 M pyruvate solution to 100 ml of A to adjust the pyruvate concentration in the medium to approximately 0.06 M. Adjust the pH with 1 M NaOH.

[0166] (2)Culture system 1 ml of resting cell suspension is inoculated into the above medium and cultured in a shaker at 28°C and 180 rpm. (3) Indicator measurement: OD (the amount of bacterial growth in the culture medium) 600 , pH value and NH4 + -N, NO2 - -N, NO x - -Measure the N content.

[0167] 2 Results The test strain was inoculated into a liquid medium containing hydrazine sulfate as a nitrogen source, and the culture medium was clear (Fig. 5, left). After 7 days of growth, the culture medium containing hydrazine sulfate was inoculated onto a solid plate again and allowed to grow for 14 days on the solid plate. The test strain was found to grow normally on the plate containing hydrazine sulfate as a nitrogen source, but the NO2 - When measuring the -N qualitatively, the plates did not develop color (Fig. 5). After 38 days of growth in liquid medium, the culture medium was visually observed to be turbid, indicating that the strains were growing (Fig. 5, right panel). At this time, the OD values of the two strains, YT and JD, were 1.09 and 1.12, respectively (Table 6b).

[0168] During the cultivation, NH4 + -N, NO2 - -N, NO3 - The change in the -N content was analyzed periodically, and the qualitative analysis results (Table 6a) and quantitative measurement results (Table 6b) were combined to determine the NH4 + -N formation and NO2 - -N, NO3 - -N, NO x -No accumulation of -N was observed, and the pH value increased significantly, initially at 6.4, but after 25 days of growth it was found to be between 7.7 and 8.0. From the above, it was found that this bacterium could grow on hydrazine sulfate as a substrate, but did not produce nitrite or nitrate.

[0169] [Table 6a]

[0170] [Table 6b]

[0171] 3. Conclusion: The mycobacteria of the present invention can grow using hydrazine sulfate as a nitrogen source, but it does not convert it to ammonium nitrogen, and does not produce nitrite or nitrate, so it has no nitrification activity. A metabolic pathway in which the strain utilizes hydrazine sulfate dehydrogenation to produce hydroxylamine is possible.

[0172] Example 5 Growth and nitrification activity of the mycobacterium of the present invention in pure culture with hydroxylamine as the nitrogen source

[0173] 1. Materials and Methods

[0174] 1.1 Culture medium (1) Iron ion-containing medium (g / L): NaH2PO4 0.25, K2HPO4 0.75, MgSO4·7H2O 0.03, MnSO4·H2O 0.01, FeSO4·7H2O 0.01. Adjust the pH of the medium to 7.0-7.2 using sulfuric acid and sodium hydroxide before sterilization. (2) Iron-free medium (g / L): NaH2PO4 0.25, K2HPO4 0.75, MgSO4·7H2O 0.03, MnSO4·H2O 0.01. Adjust the pH of the medium to 7.0-7.2 using sulfuric acid and sodium hydroxide before sterilization. The nitrogen sources were 0.2 mM and 1.8 mM hydroxylamine hydrochloride, and the carbon sources were 0 and 0.06 M pyruvate.

[0175] 1.2 Cultures JD and YT resting cells Specifically, the cultured JD and YT were centrifuged to remove the supernatant, washed repeatedly with saline, and placed in a refrigerator at 4°C. - When -N no longer develops color, the pure bacteria can be confirmed by streaking. After suspending the bacteria in the medium at the time of inoculation, place the bacteria in a shake flask and culture.

[0176] 1.3 Culture conditions The cells were cultured in a 180 rpm shaker at 28°C for 21 days.

[0177] 1.4 Culture system and indicator measurements As a control, non-inoculated bacteria were cultured in iron-containing and iron-free media with two nitrogen source concentrations, 0.2 mM hydroxylamine and 1.8 mM hydroxylamine, and two types of media, no carbon source added and 0.06 M pyruvate, as shown in the table below.

[0178] [Table 7a]

[0179] After the cultivation, the growth rate of the bacterial cells in the culture solution (OD 600 ) and pH were measured, and the supernatant was frozen and centrifuged (4°C, 10,000 rpm). + -N, NO2 - -N, NO3 - The -N content of each strain was measured, and after centrifugation, the cells were quickly frozen in liquid nitrogen and stored at -80°C for transcriptome sequencing. Each batch was repeated three times.

[0180] 1.5 Transcriptome sequencing of cultures As shown in the table below, the sequencing was performed by classifying the cells as follows: (1) W-0: starved resting cells; (2) W-1: 0.2 mM hydroxylamine hydrochloride + 0 M pyruvate; (3) W-2: 0.2 mM hydroxylamine hydrochloride + 0.06 M pyruvate.

[0181] [Table 7b]

[0182] 2 Results

[0183] 2.1 Growth and nitrification activity of mycobacteria of the present invention in pure culture with hydroxylamine as nitrogen source

[0184] As can be seen from Table 7c, under the conditions of pure culture with hydroxylamine hydrochloride as a nitrogen source (0.2 mM and 1.8 mM) and pyruvate as a carbon source (0.06 M concentration), the two strains JD and YT could grow in both iron-containing and iron-free media, and NH4 + -N, NO2 - -N, NO3 - Analysis of changes in -N concentration showed that neither of the two strains exhibited nitrification activity.

[0185] [Table 7c]

[0186] Table 7d shows the growth and nitrification activity of the two strains JD and YT when cultured purely in a medium containing hydroxylamine hydrochloride as a nitrogen source and without pyruvate as a carbon source. At concentrations of 0.2 mM and 1.8 mM of the two hydroxylamines, JD and YT can grow in both iron-containing and iron-free media and exhibit nitrification activity.

[0187] [Table 7d]

[0188] 2.3 Transcriptome sequencing of cultures Analysis of the transcriptome sequencing results of YT cultures revealed that at least five types of dehydrogenases exist in the mycobacteria of the present invention, and obvious differences were observed in the expression of hydroxylamine dehydrogenase in the strains under various treatments, such as the addition or absence of a pyruvate carbon source (Fig. 6).

[0189] Among the five dehydrogenases, two increases in enzyme activity may be related to the pathways from hydroxylamine to nitrogen gas and from hydroxylamine to ammonia. While "hydrogen dehydrogenase" was identified as the pathway to nitrogen gas, the other was identified as the pathway to ammonia. The expression of the remaining four dehydrogenases may be related to the subsequent oxidation products of hydroxylamine (nitrous oxide, nitrous oxide to nitric oxide, nitric oxide to nitrite, hydroxylamine to hyponitrite, and nitrite to nitrate).

[0190] 3 Conclusion (1) The mycobacteria of the present invention can grow using hydroxylamine as a nitrogen source regardless of the presence or absence of organic carbon sources and iron ions in the medium, but there is a clear difference in the expression of nitrification activity.

[0191] (2) Iron 2+ Addition and Fe 2+ The results for the case without Fe are consistent with those for the case without Fe. 2+ This indicates that the arginine pathway is not involved in the reaction.

[0192] (3) The presence or absence of a carbon source has a significant effect on the reaction results. When a carbon source is added, there is the highest possibility of nitrogen gas loss and other products may also be produced, but no nitrite is produced. When a carbon source is not added, nitrite is produced, indicating nitrification activity.

[0193] (4) The same results were obtained with both concentrations of hydroxylamine and two different bacterial samples, which further supports the above results.

[0194] (5) Transcriptome sequencing analysis of the cultures showed that the mycobacteria of the present invention have at least five types of dehydrogenases, and there are clear differences in the expression of hydroxylamine dehydrogenase in the strains under various treatments, such as with or without the addition of a pyruvate carbon source.

[0195] Example 6 Hyponitrite (sodium trans hyponitrite) incubation experiment

[0196] 1. Materials and Methods

[0197] 1.1 Cultures JD and YT resting cells.

[0198] 1.2 Culture medium The modified Stevenson's medium was kept at a natural pH, except that ammonium sulfate was replaced with sodium hyponitrite with an equal amount of nitrogen. The carbon source was pyruvate, and the concentration of pyruvate in the medium was set at 0.06M.

[0199] 1.3 Culture conditions The cells are cultured in a 180 rpm shaker at 28°C and 5% CO2 for approximately 24 days.

[0200] 1.4 Observation Indicators Observe the growth of the bacteria and check the NH4 + -N, NO2 - -N, NO3 - -N is measured qualitatively.

[0201] 2 Results As shown in Figure 7a, in a pure culture system using trans-sodium hyponitrite as a nitrogen source, a slight film was observed on the surface at the beginning of the culture, and a small amount of filamentous fungi was clearly visible upon visual inspection. This indicates that the fungi were growing but not reaching their maximum growth potential with further cultivation. This is thought to be because trans-sodium hyponitrite is a strongly basic, weakly acidic salt (stable at a pH of approximately 12). When the pH was adjusted to approximately 7, a large amount of bubbles formed in the solution, indicating the decomposition of trans-sodium hyponitrite. Figure 7b shows the results of observing the cell morphology using a stereomicroscope.

[0202] In an experiment in which resting cells were cultured using sodium trans-hyponitrite as a nitrogen source, colorimetric titration of relevant indicators in the liquid medium was performed. As a result, YT and JD showed a slow nitrification reaction and a weakly positive diphenylamine reaction (Fig. 7c, and in the right panel of Fig. 7c, from left to right, NH4 + -N, NO2 - -N, NO3 - -N).

[0203] 3 Conclusion

[0204] The mycobacteria of the present invention can grow using hyponitrite (sodium trans-hyponitrite) and exhibit nitrification activity.

[0205] Example 7 Effect of iron ions on growth and nitrification activity of mycobacteria of the present invention

[0206] 1. Materials and Methods

[0207] 1.1 Culture medium: PBS-JD and PBS-YT bacterial solution.

[0208] 1.2 Culture medium

[0209] A. Modified Stevenson's medium B. Iron-free modified Stevenson's medium: FeSO4·7H2O in the modified Stevenson's medium was removed, but the remaining ingredients and treatments were the same. C. Iron-free and manganese-free modified Stevenson's medium: All FeSO4·7H2O and MnSO4·H2O in the modified Stevenson's medium were removed, but the remaining ingredients and treatment were the same. Pyruvate is used as the carbon source, and the concentration of pyruvate in the medium is 0.06M.

[0210] 1.3 Effect of iron ions on the growth and nitrification performance of the bacterial strain 1 ml of the PBS-JD / YT bacterial solution was inoculated into each of the three media A+pyruvic acid, B+pyruvic acid, and C+pyruvic acid, and cultured in a shaker at 180 rpm and a constant temperature of 28°C. After 24 days of continuous culture, the OD of the bacterial solution was measured. 600 The culture medium was centrifuged at 4°C for 15 min (5000 g) and the NO2 - -N, NH4 + -N, and NO x - -N(NO2 - -N+NO3 - The ammonia oxidation and nitrification abilities of the test strains are analyzed by measuring the content of nitrification factors (N) and calculating the ammonia oxidation rate and total nitrification rate.

[0211] For all three treatment media, the experiment is designed so that there are three replicates per detection, with an uninoculated medium as the control (CK). Calculation of nitrification rate: Total nitrification rate = [NO x - -N]×100 / [NH4 + -N(CK)] Nitrification rate=[NO2 - -N]×100 / [NH4 + -N(CK)] Shortcut nitrification rate = [NO2 - -N]×100 / [NO x - -N] Ammonia oxidation rate / NH4 + -N conversion rate = ([NH4 + -N(CK)]-[NH4 + -N(residual)])×100 / [NH4+ -N(CK)]

[0212] 1.4 Treatment of laboratory equipment: All glassware used in the above experiments should be soaked in a 1:1 nitric acid solution for 24 hours, thoroughly rinsed with tap water, and then thoroughly rinsed with deionized water to eliminate the influence of heavy metals on the laboratory equipment on the experimental results.

[0213] 2 Results Table 8 shows the growth and nitrification activity of the test strains in iron-manganese-containing basal medium, iron-free medium, and iron-free manganese-free medium. After 21 days of culture, YT and JD were found to be able to grow in all three treatment media, and there was obvious turbidity in the inoculated culture medium compared to the uninoculated culture medium (Table 8 and Figure 8). In Figure 8, from left to right, the OD values are: 1: JD strain + iron-free medium; 2: YT strain + iron-free manganese-free medium; 3: YT strain + iron-free medium; and 4: JD strain + iron-free manganese-free medium. Compared to the iron-free medium and iron-free manganese-free medium, the OD values in the iron-manganese-containing basal medium were significantly higher. 600 The values are higher, with mean values of 1.84 and 1.62 for strains JD and YT, respectively.

[0214] The two strains, JD and YT, showed high nitrification activity in the basal culture, and in the cultures grown for 24 days, NO2 - The average concentrations of -N reached 139.9 mg / L and 96.31 mg / L, respectively, and NO x - The mean contents of -N were 219.04 mg / L and 152.39 mg / L, respectively. On the other hand, the NO2 - -N, NO x - -N was not produced, indicating that the test strain had no nitrification activity in the absence of iron.

[0215] [Table 8]

[0216] 3 Conclusion: (1) In iron-free medium, the test strain can grow but does not show any nitrification activity.

[0217] (2) In iron-free, manganese-free medium, the test strain can grow but does not show any nitrification activity.

[0218] (3) Iron ions are an important factor influencing the nitrification activity of this bacterium, but manganese ions have no effect on its nitrification activity.

[0219] Example 8 Growth and nitrification activity of the mycobacterium of the present invention in pure culture with arginine as the nitrogen source

[0220] 1. Materials and Methods

[0221] 1.1 Culture: PBS-JD / YT bacterial solution

[0222] 1.2 Culture medium

[0223] A. Modified Stevenson's medium with arginine as the nitrogen source Preparation of nitrogen-free modified Stevenson's medium The ammonium sulfate in the modified Stevenson's medium was removed, but the remaining ingredients were the same, and the pH was 7.0 to 7.2, resulting in a nitrogen-free modified Stevenson's medium. Preparation of constant concentration L-arginine mother solution Weigh out 5.28 g of L-arginine (relative molecular mass 174.20, solubility in water at 20°C 148.7 g / L) and completely dissolve it in 50 ml of deionized water. The pH is adjusted to 7.0-7.2, and the volume is adjusted to 80 ml. Because L-arginine, an amino acid, is easily decomposed under high-pressure steam sterilization conditions, a filtration membrane sterilization method is used for sterilization. During cultivation, 2 ml of the prepared L-arginine solution was added to 100 ml of nitrogen-free modified Stevenson medium, and the nitrogen content in the culture system was the same as that when ammonium sulfate was used as the nitrogen source (nitrogen content: 424 mg / L). In this way, a modified Stevenson medium with the same nitrogen content as that when arginine was used as the nitrogen source was obtained.

[0224] B: Prepare 1 M pyruvic acid solutions using pyruvic acid as the carbon source, sterilize them separately, and finely adjust the pH value to 7.0-7.2 with 1 M NaOH solution before sterilization. Before use, if necessary, externally add pyruvate as a carbon source, by adding 6 ml of 1 M pyruvate solution to approximately 100 ml of the above-mentioned modified Stevenson medium containing arginine as a nitrogen source, to adjust the pyruvate concentration in the culture system to approximately 0.06 M.

[0225] 1.3 Culture system and indicator measurements The following two processes are set up.

[0226] (1) Treatment using arginine as the sole carbon and nitrogen source (arginine medium), i.e., A of the above media.

[0227] (2) Treatment using arginine as a carbon and nitrogen source and pyruvate as an external carbon source (arginine + pyruvate medium), i.e., medium A + B described above. 1 ml of the PBS-JD / YT bacterial solution was inoculated into each of the two media mentioned above, and for each treatment, an uninoculated control was used. The culture was then placed in a shaker at 180 rpm and a constant temperature of 28°C. After culturing for a certain period of time, the OD 600 The NO2 value and pH were measured. - -N, NH4 + -N, and NO x - -Measure the N content.

[0228] 1.4 Detection of NOS genes YT and JD bacterial liquids purely cultured with L-arginine and pyruvate are amplified by PCR to detect their nitric oxide synthase (NOS) genes. For PCR, TransStart Fastpfu DNA Polymerase is used in a 20 μl reaction volume. 5×FastPfu Buffer..........4μl 2.5mM dNTPs..........2μl Forward Primer(5μM)..........0.8μl Reverse Primer(5μM)..........0.8μl FastPfu Polymerase..........0.4μl Template DNA....10ng ddH2O ........................Remaining volume up to 20 μl

[0229] PCR device: ABI GeneAmp® 9700 model PCR reaction parameters: a. 1×(5min at 95℃) b. cycles×(30s at 95℃;30s at Tm℃;45s at 72℃) c. 10min at 72℃, 10℃ until halted by user Primer sequences: nosF:5'-CGM(C / T) TGT TCN(A / C) TCG ACA GCC AG-3'(SEQ ID NO:9) nosR:5'-CAT GTG CAG D(A / C / G / T)GC H(A / G)TG GCA GAA-3'(SEQ ID NO:10)

[0230] 2 Results 2.1 Growth and nitrification activity

[0231] The results of the experiment in which L-arginine was used as the sole carbon and nitrogen source showed that neither of the JD nor YT strains grew, and the pH of the culture solution did not change. - -N, NH4 + -N, and NO x - Qualitative detection of the -N content was negative and showed no changes. External observation revealed no changes in the color or morphology of the culture medium (shown in the two left columns of Figure 9a).

[0232] As shown in the table below and the two right columns of Figure 9a, the two strains JD and YT grow rapidly when inoculated into L-arginine + pyruvate medium, respectively, and the pH obviously improves during the growth process. For the two strains JD and YT, the NH4 + The concentrations of -N were 59.27mg / L and 56.88mg / L, respectively, and the NH4 + The concentration of -N is obviously improved.

[0233] When pyruvate was used as a carbon source, the nitrification activity of JD and YT was high, and NO2 - -N and NO x - -N accumulated in large amounts, and after 18 days of incubation, NO2 in the culture solution - The concentrations of -N were 122.0 mg / L and 136.55 mg / L, respectively, and NO x - The concentrations of -N are 198.67 mg / L and 221.0 mg / L, respectively.

[0234] [Table 9]

[0235] 2.2 PCR amplification of nitric oxide synthase The genes of the YT and JD cultures were detected and analyzed. The PCR amplification results showed that both JD and JT clearly showed the desired bands, revealing the presence of nitric oxide synthase (NOS) in both JD and YT, as shown in Figure 9b (M is the standard, 1 is JD, 2 is YT, and CK is the control).

[0236] 3 Conclusion (1) The Icobacteria of the present invention did not grow in a medium containing L-arginine as the sole carbon and nitrogen source, and no nitrification activity was observed, indicating the absence of an arginine hydrolysis pathway.

[0237] (2) When cultured using L-arginine as a nitrogen source and pyruvate as a carbon source, the mycobacteria of the present invention grow vigorously, nitrite nitrogen and nitrate nitrogen accumulate in large amounts, the nitrification activity of the test strain is high, and the L-arginine oxidation pathway is definitely present.

[0238] (3) The mycobacteria of the present invention utilize arginine, have high nitrification activity, and also have nitric oxide synthase, indicating that the arginine biosynthesis pathway is one of the pathways of nitrogen metabolism in the strain.

[0239] Example 9 Aerobic nitrite oxidation and assimilatory nitrate reduction in mycobacteria of the present invention

[0240] 1. Materials and Methods

[0241] 1.1 Culture medium Based on the modified Stevenson medium, except that ammonium sulfate was replaced with sodium nitrite (content 0.25 g / L) with an equal nitrogen content as the nitrogen source, the remaining ingredients and treatment were the same, i.e., modified Stevenson medium with sodium nitrite as the nitrogen source was obtained. First, prepare a 0.4 M sodium pyruvate solution using sodium pyruvate as a carbon source and sterilize it separately. Before use, add 10 ml of the 0.4 M sodium pyruvate solution to 100 ml of modified Stevenson's medium containing sodium nitrite as a nitrogen source to adjust the pyruvic acid concentration in the medium to approximately 0.04 M.

[0242] 1.2 Culture: PBS-JD / YT bacterial solution

[0243] 1.3 Culture system and conditions One ml of PBS-JD / YT was inoculated into modified Stevenson's medium containing sodium nitrite as a nitrogen source, and 1 ml of sterile water was added without inoculation as a control. Each measurement was repeated three times.

[0244] The culture was carried out in a shaker at a rotation speed of 180 rpm, a temperature of 28°C, a CO2 content of 5%, and an RH of 50%.

[0245] 1.4 Metrics From the 8th day of cultivation, samples were taken and analyzed every 2 days, with 3 parallel samples taken each time. The growth rate of the culture medium (OD) was measured. 600 ), pH value, and NH4 + -N, NO2 - -N, NO x - -Measure the content of N and TN. x - -N content and NO2 - The difference between the -N content and NO3 - It becomes -N. After the cultivation is completed, the dry weight of the cells and their nitrogen content (N%) and carbon content (C%) are measured.

[0246] 2 Results Figure 10a shows the growth of JD (left) and YT (right) under aerobic culture conditions using sodium nitrite as a nitrogen source, as well as the change in the pH of the medium. Both JD and YT can grow, and the pH of the medium changes as the strains grow. In the early stages of culture, the growth of both strains was extremely slow, and especially after about the 8th day of culture, the OD600 The change is extremely small.

[0247] At the initial stage of cultivation, the growth of the two strains, JD and YT, was extremely slow, but the NO2 - -N content decreased obviously, and NO3 - As can be seen from Table 10a, after 8 days of cultivation, the NO2 - The average content of -N in all samples decreased from the initial approximately 50 mg / L to 15 mg / L, and NO3 - -N is present and reaches approximately 50 mg / L, and nitrite can be oxidized to nitrate.

[0248] After culturing for 14 days, NO2 in JD medium - -N, NO3 - The concentrations of -N were 0.36 mg / L and 1.51 mg / L, respectively, and NO2 in the YT medium - -N, NO3 - The concentrations of -N were 0.30 mg / L and 1.54 mg / L, respectively, and the two forms of nitrogen almost disappeared. + -N was observed, but its concentration was not high. As for TN, the measurement results showed that the TN content of the two culture systems did not change significantly in the early stage of the culture (i.e., the stage when nitrite was oxidized to nitrate), and the rapid growth of the bacteria and NO2 - -N, NO3 - The TN concentration also decreased rapidly with the rapid decrease in the -N concentration (Fig. 10b). After 14 days of cultivation, the TN concentrations in the medium were 4.29 mg / L and 4.58 mg / L, respectively. From the above, it can be seen that nitrate reduction occurs during the above stages.

[0249] [Table 10a]

[0250] During the nitrate reduction process, the JD and YT fungal cells grew. After 14 days of cultivation, the culture was centrifuged, and the resulting fungal cells were freeze-dried under vacuum. They were then weighed and their nitrogen and carbon contents were measured. The nitrogen contents of the JD and YT fungal cells were approximately 6.5% and 7.4%, respectively (Table 10b). From the dry weight and nitrogen content, the amount of nitrogen converted into the fungal cells could be calculated. As a result, after the cultivation was completed, the amount of organic nitrogen in the fungal cells matched the amount of nitrogen source added to the medium, indicating that the nitrate reduction pathway was an assimilation pathway.

[0251] [Table 10b]

[0252] 3 Conclusion In the mycobacteria of the present invention, when cultured in a shaker using sodium nitrite as a nitrogen source, nitrite is oxidized to nitrate. During the culture process, the bacterial cells grow, and inorganic nitrogen is almost entirely synthesized in the medium, resulting in assimilation and nitrate reduction.

[0253] Example 10 Anaerobic denitrification (dissimilatory nitrate reduction) of the mycobacteria of the present invention

[0254] 1. Materials and Methods

[0255] 1.1 Culture: PBS-JD bacterial solution.

[0256] 1.2 Culture medium: Based on the modified Stevenson medium, the nitrogen source was changed from ammonium sulfate to sodium nitrite (0.25 g / L) with an equal nitrogen content, while the other ingredients and treatments were the same. In other words, a modified Stevenson medium with sodium nitrite as the nitrogen source was obtained. First, prepare a 0.4 M sodium pyruvate solution using sodium pyruvate as a carbon source and sterilize it separately. Before use, add 10 ml of the 0.4 M sodium pyruvate solution to 100 ml of modified Stevenson's medium containing sodium nitrite as a nitrogen source to adjust the pyruvic acid concentration in the medium to approximately 0.04 M.

[0257] 1.3 Culture system One ml of PBS-JD was inoculated into modified Stevenson's medium containing sodium nitrite as a nitrogen source, and 1 ml of sterile water was added as a control without inoculation. The experiment was designed so that each measurement was repeated three times.

[0258] 1.4 Anaerobic Treatment and Culture Conditions: The culture system used in this experiment should be free of O2 and N2 in the bottle. The specific procedure is as follows: The culture bottle containing the culture system is placed in an aspirator and vacuum-suctioned for 10 minutes, then helium gas is introduced for 15 seconds. The above procedure is repeated six times. During the aspirating process, a 0.25 μm filter membrane must be added to prevent contamination by foreign bacteria and ensure a pure culture. Once the air pressure is balanced, the suction hole is smeared with sealant and the cells are placed in a shaker at 180 rpm and 28°C for incubation.

[0259] 1.5 Metrics From the 8th day of cultivation, samples were taken and analyzed every 2 days, with three parallel samples taken each time. The growth rate of the culture medium (OD) was measured. 600 ), pH value, and NH4 + -N, NO2 - -N, NO3 - -N(NO x - -N content and NO2 - -Measure the TN content (difference from the N content), and measure the N2, CO2, and N2O contents.

[0260] 2 Results In culture systems, NO2 - The initial content of -N is about 50 mg / L. After 6 days of cultivation, JD --N concentration decreased to 0.5 mg / L, and NH4 + -N, NO x - The N content was all less than 0.5 mg / L, and the TN content was about 2.0 mg / L. At this time, the pH of the medium was 7.18, and the OD 600 The value of JD was 0.005, and the bacterial cells showed almost no growth (Figure 11). Analysis of the gas content in the culture system revealed that three gases, CO2, N2, and N2O, accumulated in the culture bottle (see the table below). JD indicates that anaerobic denitrification and dissimilatory nitrate reduction are occurring.

[0261] [Table 11]

[0262] 3 Conclusion In an anaerobic environment where nitrite is used as a nitrogen source, the NO2 - -N, NO3 - -N has almost disappeared, N2, N2O, and CO2 gases are produced, the mycobacteria of the present invention are undergoing anaerobic denitrification, the bacterial cells are not growing, and there is dissimilatory nitrate reduction.

[0263] Example 11 Direct denitrification by ammonia oxidation in the mycobacteria of the present invention

[0264] 1. Materials and Methods

[0265] 1.1 Culture: PBS-JD bacterial solution

[0266] 1.2 Culture medium: Formulate modified Stevenson's medium, adjust the pH to 6.4, make the volume of medium 50ml in one bottle, sterilize at high temperature and high pressure for 20 minutes, and prepare for use. Using sodium pyruvate as the carbon source, first prepare a 0.2 M sodium pyruvate solution (concentration calculated as pyruvic acid), adjust the pH to 6.4, sterilize separately, and prepare for use. During cultivation, add 2.5 ml of the 0.2 M sodium pyruvate solution to 50 ml of modified Stevenson's medium to make the sodium pyruvate concentration 0.01 M.

[0267] 1.3 Culture system One ml of the PBS-JD bacterial solution was inoculated into the modified Stevenson medium (pH 6.4) containing sodium pyruvate as a carbon source. As a control, 1 ml of sterile water was added without inoculation of the bacteria. The experiment was designed so that each measurement was repeated three times. A sterile rubber stopper is placed on the culture bottle and pressed down to ensure a sealed environment.

[0268] 1.4 Cultivation conditions in a closed environment The above culture system was placed in an aspirator and subjected to vacuum for 10 minutes, after which a mixture of 20% oxygen and 80% helium was introduced for 15 seconds, and this procedure was repeated six times. During the aspirating process, a 0.25 μm filter membrane was added to ensure a pure culture and to prevent contamination by various bacteria. Once the air pressure is balanced, sealant is applied to the air intake vent and the cells are placed in a shaker at 28°C and 180 r / min for incubation.

[0269] 1.5 Indicator measurement Samples were taken and analyzed every two days, with 3 t of parallel samples taken each time. The growth rate of the culture medium (OD) was measured. 600 ), measure the pH value, collect the culture supernatant, and add NH4 + -N, NO2 - -N, NO x - -Measure the contents of N and TN, measure the contents of four gases, N2, CO2, O2, and N2O, in the culture system, and measure the concentration of pyruvic acid in the culture medium.

[0270] 2 Results Figure 12a shows the growth and pH changes of the liquid medium during the JD cultivation process. During the 7-day cultivation, JD grew rapidly and the pH tended to improve. After 11 days of cultivation, the pH was 6.73.

[0271] As shown in Fig. 12b, in the culture process, NO2 - When the change in -N was qualitatively observed, no color was observed in the Griess reagent spot test. This was because there was NO2 in the culture medium. - -N is not accumulated. Also, NO2 - Quantitative measurement of -N revealed that NO x - The content of -N did not increase compared to the control, and the NH4 + The concentrations of -N and TN decreased, indicating that under the culture conditions, JD oxidized ammonia but did not nitrify.

[0272] Additionally, we measured the changes in the concentrations of four gases: CO2, O2, N2, and N2O. Table 12 shows the statistical changes in the concentrations of CO2, O2, N2, and N2O in the JD culture process compared to the control. Figures 12c and 12d show the changes in gas concentrations over JD culture time. Analysis revealed that, with the passage of culture time, the O2 concentration in the JD culture system decreased, while the CO2 concentration gradually increased. N2 and N2O were clearly observed in the JD culture system, and the cumulative amount of N2O gas tended to increase with the number of culture days. Under these culture conditions, pyruvate was oxidized to CO2, and ammonia was oxidized to N2 and N2O in the JD metabolic process.

[0273] [Table 12]

[0274] 3 Conclusion

[0275] The nitrogen metabolism of the mycobacteria of the present invention includes a pathway for direct denitrification through the oxidation of ammonia.

[0276] Example 12

[0277] Inhibition of nitrification activity of mycobacteria of the present invention by nitrification inhibitors

[0278] 1. Materials and Methods

[0279] 1.1 Culture: PBS-YT bacterial solution.

[0280] 1.2 Culture medium Modified Stevenson's medium ((NH4)2SO4 content 2.0 g / L) containing (NH4)2SO4 as a nitrogen source, and L-arginine hydrochloride (C6H 14 Modified Stevenson's medium (C6H) containing N4O2·HCl as a nitrogen source 14 The nitrogen content of these two nitrogen sources is equal. When adding sodium pyruvate as a carbon source to a medium, first prepare a 1.0 M sodium pyruvate solution (concentration is calculated as pyruvic acid), adjust the pH to 7.0-7.2, sterilize it separately, and prepare it for use. Before use, add 1 ml of the 1.0 M sodium pyruvate solution to modified Stevenson's medium with (NH4)2SO4 as the nitrogen source or L-arginine hydrochloride (CH6H4). 14 Add approximately 100 ml of modified Stevenson's medium containing N4O2·HCl as the nitrogen source to bring the concentration of pyruvate in the culture system to 0.01 M.

[0281] 1.3 Nitrification inhibitors Select four nitrification inhibitors: 4-amino-1,2,4-triazole (ATC), dicyandiamide (DCD, C2H4N4), aminoguanidine hydrochloride (CH6N4 HCl), and guanylthiourea (C2H6N4S). Prepare each of the four nitrification inhibitors at a concentration of 500 mM, filter, and sterilize. Add 1 ml of each of the four nitrification inhibitors at a concentration of 500 mM to each of the two nitrogen source media listed above, resulting in a 5 mM concentration of the corresponding nitrification inhibitor for each system.

[0282] 1.4 Establishment of the culture system Two media containing different nitrogen sources were set up with five treatments: no inhibitor, ATC, DCD, aminoguanidine hydrochloride, and guanylthiourea, as shown in the table below. The YT system without inhibitor and a system without YT but with inhibitor only were used as controls. One ml of PBS-YT bacterial solution was inoculated; if no bacteria were inoculated, 1 ml of sterile water was used instead. The total volume of each culture system was the same, approximately 100 ml.

[0283] [Table 13a] In addition, we will design a culture medium treatment without adding any nitrogen source, with each of the four inhibitors as the nitrogen source and pyruvate as the carbon source, and observe the growth and nitrogen metabolism conditions.

[0284] 1.5 Culture conditions: Shaker 180 rpm, culture at 28°C.

[0285] 1.6 Indicator Measurement and Calculation: Weekly growth rate of the culture medium (OD) 600 , measure the pH value, NH4 + -N, NO2 - -N, NO x - The -N content will be measured, and the inhibitory effect on the activity of nitric oxide synthase (nitrification) in various culture systems will be calculated and compared. Nitric oxide synthase (nitrification) inhibition rate (%) = (NO2 - -NYT concentration -NO2 - -N inhibitor added culture concentration) x 100 / NO2 - -NYT concentration

[0286] 2 Research results

[0287] 2.1 Effects of aminoguanidine hydrochloride and guanylthiourea on YT growth and nitrification activity When YT was cultured purely using ammonium sulfate and L-arginine hydrochloride as nitrogen sources, it exhibited nitrification activity in both cases, and the NO2- When cultured for 35 days with ammonium sulfate as the nitrogen source, the amount of NO2 accumulated in the culture system without adding any nitrification inhibitors increased gradually. - The concentration of -N was 6.88 mg / L, and the NO2 in the system was increased after 7, 14, 21, 28, and 35 days of incubation after adding aminoguanidine hydrochloride. - The concentrations of -N in the system were all lower than those in the system without the inhibitor. After the addition of guanylthiourea, the NO2 in the system was cultured for 7, 14, 21, 28, and 35 days. - The concentrations of -N were all lower than those in the system without inhibitors. From the above, it was found that aminoguanidine hydrochloride has an inhibitory effect on the nitrification activity of YT, and guanylthiourea also has a nitrification inhibitory effect, but that this nitrification inhibitory effect gradually weakens as the incubation time passes. Details are shown in the table below.

[0288] When L-arginine hydrochloride was used as a nitrogen source and cultured for 7, 14, 21 and 35 days, the culture system without any nitrification inhibitors added showed that NO2 - The concentrations of -N were 0, 101.59, 113.72, and 113.65 mg / L, respectively. When aminoguanidine hydrochloride was added and the culture was continued for 14, 21, 28, and 35 days, the NO2 - The concentrations of -N in the system were all lower than those in the system without the addition of inhibitors. When guanylthiourea was added and the incubation was continued for 14, 21, 28, and 35 days, the NO2 - The concentrations of -N were all lower than those in the control system without inhibitors. From the above, it can be seen that both aminoguanidine hydrochloride and guanylthiourea have inhibitory effects on the nitrification activity of YT. The nitrification inhibitory effect of aminoguanidine hydrochloride is clear within 35 days, while the nitrification inhibitory effect of guanylthiourea weakens as the incubation time progresses. Details are shown in the table below.

[0289] [Table 13b]

[0290] As can be seen from Figures 13a and 13b, when YT was cultured purely with two nitrogen sources, ammonium sulfate (Figure 13a) and L-arginine hydrochloride (Figure 13b), growth of YT was faster when cultured purely with L-arginine hydrochloride than when cultured purely with ammonium sulfate. The addition of aminoguanidine hydrochloride inhibited the growth of YT cultured purely with both nitrogen sources, and the inhibitory effect remained significant throughout the culture period. The addition of guanylthiourea significantly inhibited the growth of YT cultured purely with both nitrogen sources at the early stage of culture, but after 14 days of culture, the inhibition of growth when ammonium sulfate was used as the nitrogen source weakened.

[0291] 2.2 Effects of ATC and DCD on YT growth and nitrification activity

[0292] First, as shown in the table below, when YT was purely cultured with L-arginine hydrochloride as the nitrogen source, the growth and nitrification activity were stronger than when it was purely cultured with ammonium sulfate as the nitrogen source. - Analysis of changes in the concentration of -N revealed that ATC maintained its inhibitory effect on the nitrification activity of YT purely cultured with the two nitrogen sources, but DCD did not inhibit the nitrification activity. On the other hand, neither of the two nitrification inhibitors, ATC nor DCD, inhibited the growth of YT.

[0293] [Table 13c]

[0294] 2.4 Comparison of the effects of various inhibitors on the inhibition of YT nitric oxide synthase (nitrification) activity As shown in the table below, aminoguanidine hydrochloride inhibited the nitrification activity of YT with L-arginine hydrochloride as the nitrogen source by over 99% and with ammonium sulfate as the nitrogen source by over 97%. Guanylthiourea inhibited YT nitric oxide synthase (nitrification) activity with both nitrogen sources by over 95% by 14 days of incubation, and then the inhibition rate decreased. After 35 days of incubation with ammonium sulfate as the nitrogen source, the inhibition rate of nitric oxide synthase (nitrification) activity decreased to 11% and after 35 days of incubation with L-arginine hydrochloride as the nitrogen source, the inhibition rate of nitric oxide synthase (nitrification) activity decreased to 72%.

[0295] [Table 13d]

[0296] ATC clearly inhibited YT nitric oxide synthase (nitrification) activity. When L-arginine hydrochloride was used as the nitrogen source, nitric oxide synthase (nitrification) activity was almost completely inhibited. On the other hand, when ammonium sulfate was used as the nitrogen source, the inhibitory effect was relatively weak and tended to decrease with the passage of incubation time. As shown in Table 13e, DCD had almost no effect on YT nitric oxide synthase (nitrification) activity.

[0297] [Table 13e]

[0298] 2.5 Growth and metabolism of YT in pure culture with four nitrification inhibitors as nitrogen sources YT could grow in pure culture with the four nitrification inhibitors as the nitrogen source and pyruvate as the carbon source, and the TN content in the culture medium was lower than the initial content in the medium before inoculation, indicating that the nitrification inhibitors were involved in nitrogen metabolism (Fig. 13c).

[0299] 3 Conclusion (1) All four nitrification inhibitors used can grow YT using arginine as the sole nitrogen source instead of the nitrogen source substrate.

[0300] (2) Aminoguanidine hydrochloride inhibits both the growth and nitrification activity of YT. Guanylthiourea inhibits the nitrification activity of YT, and the effect is clear in the early stages of cultivation, but the inhibitory effect on nitrification activity weakens as the cultivation time progresses. ATC clearly inhibits the nitrification activity of YT, but does not inhibit its growth. DCD has no clear inhibitory effect on either the growth or nitrification activity of YT.

[0301] (3) When YT was fed with L-arginine hydrochloride as a nitrogen source, both the growth and nitrification activity were higher than when it was fed with ammonium sulfate as a nitrogen source. (4) Nitrification inhibitors essentially inhibit the activity of nitric oxide synthase.

[0302] Example 13 Antagonism of other mycobacteria by the mycobacteria of the present invention

[0303] 1. Materials and Methods

[0304] 1.1 Test strains: YT obtained in Example 1; Mycobacterium austroafricanum (Accession No. CMCC(B)95044), Mycobacterium vaccae (ATCC No. 15483TM), Mycolicibacterium vanbaalenii (DSM 7251) standard strains. All of the above strains are prepared as PBS solutions.

[0305] 1.2 LB plate: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, agar powder 20g / L, pH 7.2.

[0306] 1.3 Experimental Method The following two will be adopted:

[0307] Type 1: LB flat plate drawing method The specific procedure is as follows: two or three test standard strains are evenly streaked onto the same LB plate, and YT is streaked between each pair. The plates are then placed in an incubator and grown at 28°C for one week, and the growth of the test strains on each plate is observed.

[0308] Type 2: KB method, i.e., filter paper method The specific steps are as follows: (1) Select uniform filter paper and punch several disks of the same diameter (6 mm). After sterilization and baking, a certain amount was soaked in PBS-YT bacterial solution before use. (2) Take one loopful of each of the three standard strains and place it in three Erlenmeyer flasks containing 100 ml of PBS. Then, evenly spread 200 microliters of each on separate LB plates to create test plates. (3) Place the filter paper soaked in PBS-YT bacterial solution on the test plates, placing it three times on each test plate and inverting it after 5 minutes. (4) Use the test plate with the saline-soaked filter paper attached as a control. (5) Place each treated plate in an incubator and culture at 28°C for one week. Remove the plates, measure the size of the inhibition zone, and photograph the inhibition zone using a Nikon AZ100 stereomicroscope.

[0309] 2 Results When M. vaccae and M. bambaarenii were inoculated onto an LB plate and YT was streaked between the two standard strains, M. vaccae and M. bambaarenii clearly spread and grew on the side not inoculated with YT. As shown in Figure 14a, when three standard strains were streaked onto an LB plate and YT was inoculated between M. austroafricanum and M. bambaarenii, and between M. bambaarenii and M. vaccae, M. austroafricanum and M. vaccae on both sides of the LB plate spread and grew on the side not inoculated with YT, but M. bambaarenii in the center did not spread and grew.

[0310] As shown in Figure 14b (without removing the YT-soaked filter paper, three LB plates were inoculated with Mycobacterium bambaarenii, Mycobacterium vaccae, and Mycobacterium austroafricanum, from left to right) and Figure 14c (with the YT-soaked filter paper removed), electron microscopy after one week of incubation using the KB method revealed that inhibition zones exceeding 6 mm in diameter formed on the LB plates where the YT-soaked filter paper had been attached, and the three standard strains, Mycobacterium austroafricanum, Mycobacterium bambaarenii, and Mycobacterium vaccae, did not grow at all. Adding more drops of Griess's reagent to the filter paper revealed a pink color, indicating nitrification. The three standard strains grew uniformly on the control plate, where filter paper not soaked in YT was attached. This indicates that YT has antagonistic effects against other mycobacteria.

[0311] 3 Conclusion The mycobacteria of the present invention have antagonistic activity against mycobacteria including Mycobacterium bambaareni, Mycobacterium vaccae, and Mycobacterium austroafricanum. Although the preferred embodiments of the present invention have been described in detail above, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, the technical solutions of the present invention may be modified in a number of simple ways, and may be combined in any other suitable manner including each technical feature. These simple modifications and combinations shall also be regarded as the disclosure content of the present invention, and all belong to the protection scope of the present invention.

[0312] References Markus Ribbe, Dilip Gadkari, Ortwin Meyer. N2Fixation by Streptomyces thermoautotrophicus Involves a Molybdenum-Dinitrogenase and a Manganese-Superoxide Oxidoreductase That Couple N2Reduction to the Oxidation of Superoxide Produced from O2by a Molybdenum-CO Dehydrogenase. The Journal of Biological Chemistry, 1997, 272(42):26627-26633. Marcel MM Kuypers, Hannah K. Marchant and Boran Karta. The microbial nitrogen-cycling network. Nature, 2018. doi:10.1038 / nrmicro.2018.9, Published online 5 Feb 2018. Pertti JM Heterotrophic nitrification--An eternal mystery in the nitrogen cycle. Soil Biology and Biochemistry, 2022, 168. https: / / doi.org / 10.1016 / j.soilbio.2022.108611. Aina Soler-Jofra, Julio Perez, Mark CM van Loosdrecht. Hydroxylamine and the nitrogen cycle: A review. Water Research, 2021, 190, 116723. https: / / doi.org / 10.1016 / j.watres.2020.116723. Holger Daims, Elena V. Lebedeva, Petra Pjevac, Ping Han, Craig Herbold, et al. Complete nitrification by Nitrospira bacteria. Nature, 2015, 528. doi: 10.1038 / nature 16461. Zhang Miao-miao, Shen Ju-pei, He Ji-zheng, Zhang Li-mei. Mechanism of microbial inhibition by nitrification inhibitors and its application. Journal of Agricultural and Environmental Sciences, 2014,33(11):2077-2083. Zhang Hao, Zhang Jinxing, Liu Peixun. Research and development of inducible nitric oxide synthase inhibitors. China Journal of New Drugs, 2012, 22(6):665-669. Luo Hui-ting, Kong Baohua, Li Peijun, and Li Muzi. Bacterial nitric oxide synthase and its use in food science research. Food Science, 2014, 35(17):266-271. Zhang Yang, Li Yaying, Zheng Ningguo, Yao Huaiying. Inhibition principle of biological nitrification inhibitors and its research and development. Jiangsu Agricultural Science, 2019,47(1):21-26. Zhaoliang Zhu, Qixiao Wen. Nitrogen in China. Nanjing: Jiangsu Science and Technology Publishing House, 1992. Shen Tong, Wang Jiayan. Biochemistry. Beijing: Higher Education Publishing House, 2000. Han Bin, Kong Jijun, Zou Xiaoming, Gong Hede. Current status and prospects of research on biological nitrogen fixation. Shanxi Agricultural Science, 2009, 37(10): 86-89, 85.

Claims

1. A mycobacterium sp. having the deposit number CGMCC No. 21272 or CGMCC No. 21273.

2. A bactericidal agent comprising the mycobacterium of claim 1.

3. A denitrification method comprising contacting the mycobacterium according to claim 1 or the bacterial agent according to claim 2 with a target system for denitrification.

4. The nitrogen in the denitrification target system is at least one of molecular nitrogen, ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen, And / or the method according to claim 3, wherein the target system for denitrification is wastewater, waste gas, or waste residue.

5. 10. A method for treating nitrogen contamination, comprising contacting a mycobacterium according to claim 1 or a bacterial agent according to claim 2 with a nitrogen-containing contaminant.

6. CO 2 10. A method for capturing mycobacteria according to claim 1 or the bacterial agent according to claim 2, comprising: 2 with a system comprising:

7. A method for selecting a pesticide, comprising inoculating and culturing the mycobacteria described in claim 1 in an ammonia nitrogen medium containing the pesticide to be selected, and determining whether the pesticide to be selected has the function of inhibiting the loss of ammonia nitrogen based on the change in nitrogen during the culture process and / or the growth status of the mycobacteria.

8. Use of the mycobacterium of claim 1 or the fungus of claim 2 in nitrogen pollution treatment, carbon capture, pesticide screening, treatment and / or prevention of skin diseases, treatment and / or prevention of diseases associated with low nitrite levels, treatment and / or prevention of body odor, treatment by providing nitric oxide to a subject, or inhibition of microbial growth.

9. the pesticide is a nitrification inhibitor and / or a nitric oxide synthase inhibitor; and / or said inhibition is in vitro inhibition; and / or said use is for the treatment and / or prevention of skin diseases, for the treatment and / or prevention of diseases associated with low nitrite levels, for the treatment and / or prevention of body odor, for the treatment of a subject by providing nitric oxide, or for the manufacture of a medicament or formulation for the inhibition of microbial growth, And / or the microorganism is a Mycobacterium microorganism, more preferably Mycobacterium vaccae and / or Mycobacterium bambaarenii, use according to claim 8.

10. 1. A method for isolating mycobacteria having nitrifying activity, comprising the steps of: (1) Preliminary screening of mycobacteria having nitrification activity; (2) further enriching the mycobacteria having high nitrification activity using a medium supplemented with an antibiotic.

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