Mycobacterium based on multiple carbon and nitrogen metabolic pathways, separation method and application

Abstract

The invention provides a Mycobacterium sp., a separation method of the Mycobacterium sp. And application of the Mycobacterium sp. The preservation number of the mycobacterium is CGMCC (China General Microbiological Culture Collection Center) No. 21272 or CGMCC No. 21273. The mycobacterium has obvious nitrogen fixation, carbon sequestration and ammonia oxidation capacities, can directly oxidize ammonia nitrogen into nitrogen, and has an antagonistic effect on other microorganisms.

Description

Mycobacteria based on multiple carbon and nitrogen metabolic pathways, isolation methods and uses

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Chinese patent application No. 202210989249.4 filed on August 17, 2022, the contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of wastewater treatment and is used in the fermentation industry, in particular to mycobacteria based on multiple carbon and nitrogen metabolic pathways, as well as separation methods and uses. Background Art

[0004] In nature, nitrification 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 nitric acid. A 2015 study proposed that nitrification is carried out by a single bacterium. Due to the characteristics of nitrifying bacteria, they are widely used in the treatment of nitrogen-containing wastewater and the screening of nitrification inhibitors. However, the existing technology believes that nitrifying bacteria are "autotrophic" bacteria, and the presence of organic matter is harmful to the growth of nitrifying bacteria. Based on this understanding, the application of nitrifying bacteria is greatly restricted. For example, based on the understanding that nitrifying bacteria are autotrophic bacteria, the microbial basis and process principles of existing sewage biochemical treatment technologies for removing C and N pollutants are based on a combination of carbon removal through the "ammoniation" reaction of heterotrophic bacteria, the "nitrification" reaction of ammonia oxidation and nitrite oxidation by autotrophic bacteria, and nitrogen removal through the "denitrification" reaction of anaerobic (facultative) heterotrophic bacteria. However, the above combination has problems such as slow bacterial growth rate and low total nitrogen removal rate.

[0005] Mycobacteria are elongated, slightly curved organisms that sometimes exhibit branching or filamentous structures. Their primary characteristic is that their cell walls contain a high amount of lipids, primarily mycolic acids. They lack flagella, spores, and do not produce endotoxins or exotoxins. They are aerobic saprophytes that grow on surfaces and strongly adhere to solid surfaces. Most research on mycobacteria has focused on pathogenic bacteria such as Mycobacterium tuberculosis and Mycobacterium leprae. Currently, there are no reports of mycobacteria exhibiting nitrifying activity.

[0006] In addition, 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 different soils through steps such as nutrient agar plate separation and Gliese reagent color development. Although this method can screen out many heterotrophic strains with nitrifying activity and is universal, it lacks specificity and makes it difficult to specifically identify strains with high nitrifying activity.

[0007] Summary of the Invention

[0008] The present invention aims to overcome the problems of the prior art by providing a mycobacterium strain based on multiple carbon and nitrogen metabolic pathways, an isolation method, and uses thereof. The strain or bacterial agent of the present invention has good application prospects in sewage treatment, screening of nitrification inhibitors, and other aspects.

[0009] In order to achieve the above object, the present invention provides a Mycobacterium sp. in a first aspect. The Mycobacterium sp. has a deposit number of CGMCC No. 21272 or CGMCC No. 21273.

[0010] A second aspect of the present invention provides a bacterial agent comprising the mycobacterium as described above.

[0011] Other aspects of the present invention relate to the use of the mycobacteria or bacterial agents.

[0012] The present invention also provides a method for separating microorganisms with nitrification activity.

[0013] The mycobacterium of the present invention is different from existing mycobacteria and has at least the following characteristics: 1) significant nitrogen fixation ability, 2) strong ammonia oxidation and significant nitrification activity, capable of heterotrophic removal of C (carbon capture) and N organic pollutants without the need for cooperation with other microorganisms, 3) the ability to dehydrogenate hydrazine sulfate to produce hydroxylamine, 4) the presence of at least five dehydrogenases; 5) the presence of a nitric oxide synthase gene capable of oxidizing arginine, with Fe being an important element affecting its nitrification activity, and 6) a significant antagonistic effect on other microorganisms.

[0014] The separation method of the present invention can stably and specifically separate strains with high nitrification activity from samples (such as soil) with good repeatability.

[0015] Biological Deposits

[0016] The Mycobacterium sp. provided by the present invention was deposited on December 1, 2020 at the General Microbiology Center of the China Culture Collection Administration (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101). The abbreviation of the depository unit is CGMCC, and the strain deposit number is CGMCC No. 21272, abbreviated as YT.

[0017] The Mycobacterium sp. provided by the present invention was deposited on December 1, 2020 at the General Microbiology Center of the China Culture Collection Administration (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101). The abbreviation of the depository unit is CGMCC, and the strain deposit number is CGMCC No. 21273, abbreviated as JD. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figures 1a-1b are circle diagrams of the whole genome of mycobacteria of the present invention;

[0019] Figures 1c-1d are schematic diagrams of nitrogen metabolism pathways;

[0020] Figure 2a shows the use of 10 -7 Bacterial growth on LB-malachite green plates under dilution gradient;

[0021] Figure 2b is a transmission electron microscope photo of the target bacteria;

[0022] Figures 2c-2e are color images of Griess reagent;

[0023] Figure 2f is a distribution diagram of target bacteria on the separation plate;

[0024] FIG3 a is a photograph of mycobacteria of the present invention cultured in a liquid culture medium;

[0025] Figures 3b-3c are photographs of mycobacteria of the present invention grown on solid culture medium;

[0026] FIG3 d is a morphological image of mycobacteria of the present invention under a Nikon stereomicroscope;

[0027] FIG3e is a photograph of mycobacteria of the present invention cultured on a solid medium;

[0028] Figure 3f shows the PCR amplification results of the nitrogenase ferritin gene;

[0029] Figure 3g shows the OD of the culture during the cultivation of mycobacteria using ammonium sulfate as the nitrogen source and pyruvate as the carbon source. 600 With NH4 + -N concentration change diagram;

[0030] Figure 4a shows the OD during the culture process. 600 Graph showing changes with pH;

[0031] FIG4 b is a graph showing changes in pyruvate concentration during the culture process;

[0032] FIG5 shows a photograph of the mycobacteria of the present invention cultured in a culture medium containing hydrazine sulfate as a nitrogen source;

[0033] FIG6 shows the transcriptome sequencing results of YT culture obtained using hydroxylamine as the nitrogen source;

[0034] FIG7 a is a photograph of the initial stage of pure culture of JD using sodium hyponitrite as a nitrogen source and ammonium sulfate as a nitrogen source;

[0035] FIG7 b is a morphological diagram of mycobacteria of the present invention under a Nikon stereomicroscope;

[0036] FIG7 c is a diagram showing the qualitative determination results of nitrification activity of the culture using sodium hyponitrite as the nitrogen source;

[0037] FIG8 is a photograph of mycobacteria of the present invention cultured in an iron-free liquid culture medium;

[0038] Figure 9a is a comparison of YT pure culture for 13 days and the control;

[0039] FIG9 b is a diagram of NOS gene amplification of mycobacteria of the present invention;

[0040] Figure 10a shows the OD of the culture during the cultivation of mycobacteria using sodium nitrite as the nitrogen source. 600 Graph showing changes with pH;

[0041] FIG10 b is a graph showing changes in total nitrogen concentration in the culture during the cultivation of mycobacteria of the present invention using sodium nitrite as a nitrogen source;

[0042] Figure 11 shows the NO2 content after JD was grown for 6 days under anaerobic culture with sodium nitrite as nitrogen source. - -N change diagram and culture photos;

[0043] Figure 12a shows the OD of JD during the culture process 600 and pH change graph;

[0044] Figure 12b shows the JD culture NO2 after 11 days of culture. - -N qualitative determination result diagram;

[0045] Figure 12c is a graph showing the changes in CO2 and O2 concentrations in the JD culture system over culture time;

[0046] Figure 12d shows the changes in N2 and N2O concentrations in the JD culture system over culture time;

[0047] Figures 13a-13b show the effects of different nitrification inhibitors on YT growth;

[0048] FIG13c is a graph comparing TN concentrations in YT cultures grown with different nitrification inhibitors as nitrogen sources;

[0049] Figure 14a shows the antagonistic effect of YT on other strains;

[0050] FIG14b is an electron micrograph of the inhibition zones formed by the antagonistic effect of YT on three standard strains;

[0051] FIG14c shows the inhibition zones formed by the antagonistic effect of YT on three standard strains and their morphological images under a stereomicroscope. DETAILED DESCRIPTION

[0052] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0053] In the present invention, "mM" refers to "mmol / L", "M" refers to "mol / L"; "ammonia nitrogen" and "ammonium nitrogen" can be used interchangeably.

[0054] I. Strains and Inoculants

[0055] A first aspect of the present invention provides a Mycobacterium sp., characterized in that the deposit number of the Mycobacterium sp. is CGMCC No. 21272 or CGMCC No. 21273.

[0056] The inventors of the present invention have verified through further experiments that the strain CGMCC No. 21272 (abbreviated as YT) or CGMCC No. 21273 (abbreviated as JD) of the present invention is different from any existing mycobacterium. Therefore, the strain of the present invention is a new mycobacterium species, which is named Mycobacterium guanghaopengii sp.nov.

[0057] In the present invention, the cell morphology and physicochemical characteristics of the Mycobacterium CGMCC No. 21272 are shown in Table 1a.

[0058] Table 1a

[0059] In the present invention, the 16S rRNA sequence of the Mycobacterium CGMCC No. 21272 is shown as SEQ ID NO: 1.

[0060] In the present invention, the phylogenetic analysis results based on the 16S rRNA gene sequence showed that CGMCC No. 21272 was a Mycobacterium strain, and its whole genome circle map is shown in FIG1a .

[0061] In the present invention, the antibiotic sensitivity test results of the Mycobacterium CGMCC No. 21272 are shown in Table 1b.

[0062] Table 1b

[0063] In the present invention, the cell morphology and physicochemical characteristics of the Mycobacterium CGMCC No. 21273 are shown in Table 2a.

[0064] Table 2a

[0065] In the present invention, the 16S rRNA sequence of the Mycobacterium CGMCC No. 21273 is shown as SEQ ID NO: 2.

[0066] In the present invention, the phylogenetic analysis results based on the 16S rRNA gene sequence showed that CGMCC No. 21273 was a Mycobacterium strain, and its whole genome circle map was shown in FIG1 b.

[0067] In the present invention, the antibiotic sensitivity test results of the Mycobacterium CGMCC No. 21273 are shown in Table 2b.

[0068] Table 2b

[0069] The nitrogen metabolic pathway of the mycobacterium of the present invention is shown in Figure 1c (the upper figure is a schematic diagram of the nitrogen metabolic pathway of the mycobacterium of the present invention, and the lower figure is a schematic diagram of the currently known nitrogen metabolic pathway) and Figure 1d. It can be seen that in addition to the conventional metabolic pathway, the mycobacterium of the present invention also includes additional direct ammonia oxidation pathways and organic nitrogen (such as arginine) oxidation pathways.

[0070] The second aspect of the present invention provides a bacterial agent, characterized in that the bacterial agent contains the mycobacterium as described above. The bacterial agent can be a solid bacterial agent or a liquid bacterial agent, and can be prepared using various auxiliary materials or carriers commonly used in the art to obtain a suitable form of bacterial agent. For example, in order to prepare a solid bacterial agent, the auxiliary materials can include but are not limited to at least one of non-water-soluble carbonates (such as calcium carbonate), rice bran, sawdust, cake powder, straw powder, etc., and the number of viable mycobacteria in the solid bacterial agent can be 10 5 -10 18 CFU / g.

[0071] II. Environmental Protection Applications

[0072] The strain of the present invention has nitrogen fixation, nitrification and denitrification capabilities. Based on this, the third aspect of the present invention provides a denitrification method, which comprises: contacting the mycobacterium as described above or the bacterial agent as described above with the system to be denitrified. The system to be denitrified refers to various common systems that need to remove nitrogen. In the system to be denitrified, nitrogen can exist in the form of common organic nitrogen and / or inorganic nitrogen, preferably in the form of molecular nitrogen (N2), ammonia nitrogen (NH3 and / or NH4 + ), nitrate nitrogen (NO3 - ) or nitrite nitrogen (NO2 - ) in the form of, therefore, the system to be denitrified can refer to various systems containing ammonia nitrogen (such as ammonia nitrogen (NH3 and / or ammonium salt (NH4 + )) waste), various systems containing nitrate nitrogen (such as NO3 - waste), various systems containing nitrite (such as NO2 - The system to be denitrified can be a system containing nitrogenous pollutants, and can be any system that needs to be denitrified, such as wastewater, waste gas or waste residue. The contact enables the mycobacterium to grow with the N in the system to be denitrified as a nitrogen source, thereby changing the form in which N exists, which is convenient for the removal of N. A suitable carbon source (such as at least one of glucose, pyruvic acid and pyruvate) can also be added to the system according to the characteristics of the mycobacterium. Wherein, when N in the system to be denitrified is molecular nitrogen, the contact converts the molecular nitrogen into ammoniacal nitrogen, nitrate nitrogen and / or nitrite nitrogen (playing a nitrogen-fixing role). In addition to the carbon source and nitrogen source, inorganic salts can also be added to the contact system, for example, calcium chloride 0.1-1g / L, magnesium sulfate 0.05-0.5g / L, dipotassium hydrogen phosphate 0.5-5g / L, sodium molybdate 0.005-0.05g / L, and ferrous sulfate 0.01-0.1g / L. When the nitrogen in the system to be denitrified is ammoniacal nitrogen, the contact converts the ammoniacal nitrogen into nitrate nitrogen and / or nitrite nitrogen (performing a nitrification effect). When the nitrogen in the system to be denitrified is nitrate nitrogen and / or nitrite nitrogen, the contact is carried out under anaerobic conditions and converts the nitrate nitrogen and / or nitrite nitrogen into N2 and / or NO (performing a denitrification effect).

[0073] The present invention also provides the use of the aforementioned mycobacteria or the aforementioned 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, characterized in that the method comprises: contacting the aforementioned mycobacteria or the aforementioned bacterial agent with the nitrogen-containing pollutant. The nitrogen-containing pollutant can be various common pollutants containing ammonia nitrogen. The contact enables the mycobacteria to grow using the nitrogen in the nitrogen-containing pollutant as a nitrogen source, thereby changing the form in which the N exists and facilitating the removal of the N. The contact converts the ammonia nitrogen into nitrate nitrogen, nitrite nitrogen, and N2. In the method for treating nitrogen pollution, in order to remove ammonia nitrogen, the contact system can first be placed under conditions that are conducive to the mycobacteria performing nitrification (converting ammonia nitrogen into nitrate nitrogen and / or nitrite nitrogen), and then placed under conditions that are conducive to the mycobacteria performing denitrification (converting nitrate nitrogen and / or nitrite nitrogen into N2), thereby converting the environmentally harmful ammonia nitrogen into colorless and odorless nitrogen gas.

[0074] The mycobacteria of the present invention are capable of single-path denitrification (directly oxidizing ammonia nitrogen (such as hydrazine and / or hydroxylamine) into nitrogen-containing gas (direct ammonia oxidation). Specifically, under aerobic conditions in the presence of organic matter, the mycobacteria may oxidize ammonia nitrogen via the pathway of 'ammonia nitrogen → hydrazine → hydroxylamine → nitrogen and nitrous oxide'. This process can be completed before nitrification occurs. The mycobacteria can complete the entire denitrification process under aerobic conditions). This overcomes the problem that traditional biological denitrification technology requires aerobic nitrification and anaerobic denitrification to be carried out in two stages, greatly simplifies the denitrification process flow, and saves processing space and cost. Therefore, the method of using mycobacteria as described above to perform ammonia oxidation is applicable to various types of ammonia nitrogen-containing wastewater (especially hydroxylamine-containing wastewater), including single-path denitrification and nitrification. Single-path denitrification is to directly oxidize ammonia nitrogen into nitrogen-containing gas by controlling pH. In some embodiments of the present invention, the ammonia nitrogen-containing wastewater is propellant wastewater and / or hydroxylamine wastewater. In order to enable the mycobacterium to better achieve single-pathway denitrification, pyruvate is preferably used as the carbon source.

[0075] The mycobacteria described above can be used to remove various hydrazines and / or their derivatives (such as hydrazine, hydrazine sulfate, unsymmetrical dimethylhydrazine, etc.). The mycobacteria described above can also be used to remove hydroxylamine and / or its derivatives (such as oxime, etc.).

[0076] Preferably, the nitrogen-containing pollutant is at least one of hydroxylamine, hydroxylamine derivatives, hydrazine and hydrazine derivatives.

[0077] More preferably, the hydroxylamine derivative is an oxime.

[0078] More preferably, the hydrazine derivative is hydrazine sulfate and / or unsymmetrical dimethylhydrazine.

[0079] The mycobacteria of the present invention can carry out the reverse oxidation tricarboxylic acid cycle to convert CO2 into amino acids, sugars and lipids. Therefore, the present invention also provides the use of the mycobacteria as described above or the bacterial agent as described above in carbon capture. The fifth aspect of the present invention provides a method for capturing CO2, characterized in that the method comprises: contacting the mycobacteria as described above or the bacterial agent as described above with a system containing CO2. The contact enables the mycobacteria to grow using CO2 as a carbon source, thereby achieving carbon capture. A suitable nitrogen source (such as ammonium salt) can also be added to the system according to the characteristics of the mycobacteria. The system containing CO2 can be at least one of coal-fired power plant exhaust gas, natural gas production exhaust gas, synthetic fuel plant exhaust gas, fossil fuel-based hydrogen production plant exhaust gas, etc.

[0080] For the above-mentioned contacting, unless otherwise specified, the amount of carbon source used is such that the C content in the contact system is preferably 0.5-5 g / L; the amount of nitrogen source used is such that the N content in the contact system is preferably 0.2-2 g / L. Inorganic salts may also be added to the contact system, for example, 0.1-1 g / L of sodium dihydrogen phosphate, 0.5-5 g / L of dipotassium hydrogen phosphate, 0.01-0.1 g / L of magnesium sulfate, 0.005-0.05 g / L of manganese sulfate, and 0.005-0.05 g / L of ferrous sulfate. The above-mentioned contacting can be carried out at a temperature suitable for the growth of the mycobacteria (e.g., 15-35°C, preferably 25-32°C). A pH adjuster may be used to adjust the initial pH of the contact system to 6-9, preferably neutral (e.g., in the range of 7-7.2). The contact time can be determined as needed, for example, from 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 (culturing / growth) is carried out under aerobic conditions.

[0081] WO2009018686A1 (Method for removing C and N pollutants in sewage using heterotrophic ammonia-oxidizing bacteria) still belongs to a mixed bacteria treatment process and cannot maximize the ammonia oxidation rate. However, the mycobacteria of the present invention can be applied in the form of a pure culture, first maximizing the absolute number of bacteria and then adding them, thereby maximizing the ammonia oxidation rate.

[0082] III. Application in Agriculture

[0083] The inventors of the present invention have discovered that, in addition to the traditional ammonia nitrogen metabolic pathway, the ammonia nitrogen metabolism pathway of the strain of the present invention also includes the arginine oxidation pathway. Therefore, in addition to screening for nitrification inhibitors based on the traditional pathway, substances that can act at any node in the above pathway to inhibit the conversion of ammonia nitrogen to other forms and the resulting loss can be used as target agricultural chemicals. Therefore, the present invention also provides the use of the aforementioned mycobacteria or the aforementioned bacterial agent in screening agricultural chemicals. A sixth aspect of the present invention provides a method for screening agricultural chemicals based on the aforementioned mycobacteria, characterized in that the method comprises: inoculating the aforementioned mycobacteria into an ammonia nitrogen culture medium containing the agricultural chemical to be screened and culturing the agrochemical; and determining whether the agrochemical to be screened has the function of inhibiting ammonia nitrogen loss based on changes in nitrogen and / or the growth of the mycobacteria during the culture process. Monitoring changes in nitrogen during the culture process can include detecting the ammonia nitrogen content in the culture, detecting the nitrate nitrogen and / or nitrite nitrogen content in the culture, or detecting the production of nitrogen and / or nitrogen dioxide in the culture system. If the ammonia nitrogen content remains almost unchanged after cultivation, no nitrate nitrogen, nitrite nitrogen, N2 or N2O is detected during cultivation, or the growth of mycobacteria during cultivation is poor, the agricultural chemical to be screened can be considered a product that inhibits ammonia nitrogen loss. The concentration of the agricultural chemical to be screened in the ammonia nitrogen culture medium containing the agricultural chemical to be screened can be 1-20 mM, which can be a modified Stephenson medium supplemented with the agricultural chemical to be screened. The specific composition can be: 1-20 mM of the agricultural chemical to be screened, 0.5-5 g / L of carbon source (as C), 0.2-2 g / L of nitrogen source (as N), 0.1-1 g / L of sodium dihydrogen phosphate, 0.5-5 g / L of potassium dihydrogen phosphate, 0.01-0.1 g / L of magnesium sulfate, 0.005-0.05 g / L of manganese sulfate, and 0.005-0.05 g / L of ferrous sulfate. The carbon source is preferably at least one of glucose, pyruvic acid, and pyruvate. The nitrogen source can be ammoniacal nitrogen (NH3 and / or NH4 + ), preferably at least one of ammonia, ammonium salt and amino acid, more preferably ammonium sulfate and / or arginine. The culture temperature may be 15-35°C, preferably 25-32°C. The culture time may be 10 hours to 50 days (e.g., 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 thereof).

[0084] In the present invention, the agricultural chemicals mainly refer to potential products that can inhibit ammonia nitrogen loss, especially products that can reduce nitrogen fertilizer loss, improve nitrogen fertilizer utilization, and reduce the content of nitrite in crops. They can be called ammonia nitrogen loss inhibitors, including nitrification inhibitors and / or nitric oxide synthase inhibitors. Because the strains of the present invention have more ammonia nitrogen metabolic pathways as described above, using them to screen agricultural chemicals is conducive to screening inhibitors with richer effects. The nitrification inhibitors can inhibit the activity of enzymes in the nitrification process or the growth of nitrifying bacteria to inhibit nitrification. The ammonia nitrogen loss inhibitor may include non-heterocyclic inhibitors (such as aminoguanidines, acetamidines, cycloalkylamidines, thioureas, etc.) and heterocyclic inhibitors (such as pyridines, imidazoles, indazoles, triazoles, coumarins, piperazines, piperidines, pteridines, tetrahydropteridines, sulfur nitrogen heterocycles, thiazoles, etc.). Specific examples may include but are not limited to ammonium sulfate, ammonium nitrate, potassium nitrate, calcium sulfate, copper sulfate, acetylene, acetylene pyridine, phenylacetylene, 4-amino-1,2,4-triazole (ATC), aminoguanidine, At least one of 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-dichlorobenzene succinamide, allylthiourea, 4-amino-1,2,3-triazole hydrochloride, and 2-sulfathiazole (ST).

[0085] The inventors of the present invention have discovered that non-amino acid nitration inhibitors have a strong inhibitory effect on nitric oxide synthase. Therefore, the present invention also provides a method for inhibiting the activity of nitric oxide synthase (especially the nitric oxide synthase of the mycobacterium of the present invention) (in vivo, in vitro, non-therapeutic purpose), characterized in that the method comprises: contacting at least one of the non-amino acid nitration inhibitors with nitric oxide synthase. The non-amino acid nitration inhibitor can be selected from at least one of 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 nitration inhibitor can be directly added to the culture system of the mycobacteria, and the addition amount can be 2-10mM.

[0086] IV. Medical Applications

[0087] In a seventh aspect, the present invention provides the use of the mycobacterium as described above or the bacterial agent as described above in the treatment and / or prevention of skin disorders, the treatment and / or prevention of diseases associated with low nitrite levels, the treatment and / or prevention of body odor, the supply of nitric oxide to a subject, or the inhibition of microbial growth.

[0088] In some embodiments, the mycobacterium or bacterial agent can be used to treat at least one of HIV, dermatitis, infection in ulcers (e.g., venous ulcers, e.g., leg ulcers, e.g., venous leg ulcers), (e.g., infection in diabetic foot ulcers), atopic dermatitis, acne (e.g., acne vulgaris), eczema, contact dermatitis, allergic reactions, psoriasis, urticaria, rosacea, skin infections, vascular diseases, vaginal yeast infections, sexually transmitted diseases, heart disease, atherosclerosis, alopecia, leg ulcers secondary to diabetes or bed rest, angina (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 disease, epidermolysis bullosa, or cancer.

[0089] In some embodiments, the mycobacterium or bacterial agent can be used to treat at least one of: infection in diabetic foot ulcers, atopic dermatitis, acne (eg, acne vulgaris), eczema, psoriasis, urticaria, rosacea, and skin infections.

[0090] In some embodiments of the present invention, the use is for preparing a drug or preparation for the treatment and / or prevention of skin disorders, the treatment and / or prevention of diseases associated with low nitrite levels, the treatment and / or prevention of body odor, the supply of nitric oxide to a subject, or the inhibition of microbial growth.

[0091] In some embodiments of the invention, the inhibition is in vitro inhibition.

[0092] In some embodiments of the present invention, the microorganism is a Mycobacterium microorganism. Since the strain of the present invention can produce nitric oxide, the strain of the present invention can inhibit various microorganisms (including fungi, bacteria and viruses) that are sensitive to nitric oxide, especially 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 van Barensis, Mycobacterium vaccae and Mycobacterium southafrica.

[0093] In the present invention, the drug or preparation may further contain common pharmaceutical excipients to be formulated into a specific dosage form. The dosage form of the drug may be a tincture, ointment, cream, paste, aerosol, spray, powder, ear preparation, lotion, douche, liniment, paint, film coating, gel, patch, etc.

[0094] V. Separation Methods

[0095] In an eighth aspect, the present invention provides a method for isolating mycobacteria with nitrifying activity, characterized in that the method comprises: (1) enriching mycobacteria with nitrifying activity; and (2) screening mycobacteria with high nitrifying activity using a culture medium supplemented with antibiotics.

[0096] In some embodiments of the present invention, the culture medium used for enrichment can be a culture medium commonly used in the art for isolating Mycobacterium tuberculosis. In other embodiments of the present invention, the culture medium used for enrichment is preferably a modified Stephenson medium, more preferably containing: an optional organic carbon source (such as glucose) 0.5-5g / L (in terms of C), a carbonate (such as calcium carbonate) 2-10g / L (in terms of carbonate ions), ammonium sulfate 0.2-2g / L (in terms of N), sodium dihydrogen phosphate 0.1-1g / L, potassium dihydrogen phosphate 0.5-5g / L, magnesium sulfate 0.01-0.1g / L, manganese sulfate 0.005-0.05g / L, and ferrous sulfate 0.005-0.05g / L. In step (1), the culture temperature for enrichment can be 15-35°C, preferably 25-32°C. The culture time for enrichment can be based on NH4 + -N, NO2 - -N and NO3 - -N qualitative test results to determine, it can be 4-30 days.

[0097] In the present invention, if the target strain has been isolated by enrichment with the modified Stephenson medium, the strain can be picked for screening in step (2). If the target strain has not been isolated by enrichment with the modified Stephenson medium, the sample can be further treated with acid (to remove impurities) before performing step (2). Therefore, in a preferred embodiment of the present invention, step (1) includes: inoculating the sample into the modified Stephenson medium for culturing, mixing the culture with acid for acid treatment, and neutralizing the acid in the system after treatment. Relative to 1L of culture, the amount of acid used for acid treatment can be 0.2-2 mol (in terms of hydrogen ions), preferably 0.3-0.5 mol (in terms of hydrogen ions). The acid used for acid treatment can be various common inorganic acids, preferably HCl. The temperature for acid treatment is preferably 3-8°C. The time for acid treatment is preferably 1-5 min. The base used for neutralization can be a common inorganic base, such as an alkali metal hydroxide (such as sodium hydroxide).

[0098] In some embodiments of the present invention, in step (2), the content of antibiotics in the culture medium added with antibiotics is preferably 0.1-0.5 mg / L. The antibiotics can be various antibiotics commonly used in the art, preferably malachite green, penicillin, etc. Preferably, the culture medium added with antibiotics is LB culture medium added with antibiotics, more preferably containing: peptone 5-15 g / L, yeast extract 5-15 g / L, sodium chloride 5-15 g / L, antibiotic 0.1-0.5 mg / L. The culture temperature of the enrichment can be 15-35°C, preferably 25-32°C. The culture time of the enrichment can be 4-30 days. After enrichment in step (2), the dominant bacteria are the target bacteria, which have higher nitrification activity. The inventors of the present invention have found that the time of acid treatment and the concentration of antibiotics within the above-mentioned preferred range can better separate the target strain, otherwise it may lead to separation failure. The method of the present invention is particularly beneficial for the separation of mycobacteria of the present invention.

[0099] The present invention will be described in detail below through examples. In the following preparation examples and examples, some culture media and test methods used are as follows:

[0100] 1. Preparation of basal culture medium

[0101] 1.1 Preparation of modified Stephenson medium (ST medium)

[0102] Weigh 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, and sterilize with high-pressure steam before use.

[0103] 1.2 Ordinary nutrient agar plate (PM plate)

[0104] 3g beef extract, 5g peptone, 20g agar, 1000ml distilled water, pH 7.0-7.2. Before sterilization, adjust the pH of each culture medium to the required pH range with 1M NaOH or 25% (V / V) HCl solution.

[0105] 1.3 Culture medium with sodium pyruvate as carbon source

[0106] Prepare a sodium pyruvate solution of desired concentration based on the experimental concentration requirements. Sterilize the solution separately. Before use, add a predetermined volume (determined by the desired concentration) to modified Stephenson's medium to ensure the desired sodium pyruvate concentration in the medium meets the experimental requirements. Due to partial decomposition of sodium pyruvate during the preparation process, the actual concentration may be lower than the desired concentration. Fine-tune the desired pH using 1M NaOH solution.

[0107] 2 Nitrite nitrogen (NO2 - -N) Qualitative determination

[0108] 2.1 Preparation of Griess reagent

[0109] (1) Sulfanilic acid reagent (Solution A): Dissolve 0.5 g of sulfanilic acid in 150 ml of 20% dilute acetic acid solution, store in a brown bottle, and refrigerate until use.

[0110] (2) α-naphthylamine reagent (Solution B): Add 0.5 g of α-naphthylamine to 20 ml of distilled water and 150 ml of 20% dilute acetic acid solution, store in a brown bottle, and refrigerate until use.

[0111] 2.2 NO2 - -N qualitative determination: Take a few drops of culture medium on a white porcelain colorimetric plate, add two drops each of Griess reagent A and B. If nitrite is present, it will turn red.

[0112] Color and its corresponding concentration range: "+ - " is trace pink, with a concentration lower than 0.5 mg / L; "+" is light pink, with a concentration of about 0.5 mg / L; "++" is pink, with a concentration of about 1 mg / L; "+++" is dark magenta, with a concentration of about 5 mg / L; "++++" is light brown, with a concentration of about 10 mg / L; ">++++" is brown, with a concentration higher than 20 mg / L.

[0113] 3 Ammonium nitrogen (NH4 + Qualitative determination of -N)

[0114] 3.1 Preparation of Nessler's reagent

[0115] Weigh 17g of potassium mercuric iodide and dissolve it in 10ml of water. Dissolve 24.4g of KOH in a 100ml volumetric flask containing 70ml of water. Cool to room temperature. Slowly add the potassium mercuric iodide to the volumetric flask while shaking. Add water to the mark and shake well. Let it sit for two days before use. Store in a brown bottle in a dark place.

[0116] 3.2 NH4 +-N qualitative determination: Take a few drops of culture medium on a white porcelain colorimetric plate and add two drops of Nessler's reagent. If ammonium nitrogen is present, it will turn yellow.

[0117] Color and its corresponding concentration range: "+" indicates slightly yellow, with a concentration lower than 0.5 mg / L; "+++" indicates a concentration of around 5 mg / L; "++++" indicates a concentration of around 10 mg / L; ">++++" indicates dark yellow, with a concentration higher than 20 mg / L.

[0118] 4 Nitrate nitrogen (NO3 - Qualitative determination of -N)

[0119] 4.1 Preparation of diphenylamine reagent

[0120] 0.5g diphenylamine, 100ml concentrated sulfuric acid, 20ml distilled water. Dissolve the diphenylamine in distilled water, then slowly add the concentrated sulfuric acid. Store in a brown bottle until ready for use. For qualitative determination, place a few drops of the culture medium on a white porcelain colorimetric plate and add two drops of diphenylamine reagent. If nitrate is present, a blue color will appear.

[0121] 4.2 NO3 - -N qualitative test: Place several drops of culture medium on a white porcelain colorimetric plate and add two drops of diphenylamine reagent. If nitrate is present, a blue color will appear. "-" indicates a negative diphenylamine drop, and "+" indicates a positive diphenylamine drop.

[0122] 5 Quantitative determination of various forms of nitrogen

[0123] When quantitatively measuring different indicators, the culture medium was centrifuged at 4°C for 15 min (5000 g) and the supernatant was taken for measurement.

[0124] NH4 + -N was quantitatively determined by indigo blue colorimetry, NO2 - -N was quantitatively determined by Griess reagent colorimetry. TN was determined by potassium persulfate oxidation-flow analyzer. x - -N(NO2 - -N+NO3 - -N) quantitative determination was carried out by hydrazine sulfate reduction and flow analysis-spectrometry. x - -N(NO2 - -N+NO3 - -N) and NO2 - -N content, NO3 - -N content is NO x - -N and NO2 -The basic principle is to compress pump tubes of different diameters using a peristaltic pump, inject the reaction reagent and the sample to be tested into a closed, continuous flow carrier in proportion. A color reaction occurs in the chemical reaction unit, and its signal value is measured in the detector. The concentration of the sample to be tested is determined according to the standard curve method.

[0125] 6. Gas determination in culture system

[0126] The contents of N2, CO2, O2, N2O, etc. in the culture system gas were determined by gas chromatograph (Agilent).

[0127] 7 Determination of bacterial growth: turbidimetric method, expressed as OD value at a wavelength of 600nm.

[0128] 8pH value determination: Combine precision pH test paper with pH meter.

[0129] Pyruvate concentration determination: The culture supernatant (centrifuged at 5000 rpm for 15 min at 4°C) was analyzed using an Agilent 1260 liquid chromatograph (UV detector) using the following conditions: wavelength 214 nm; mobile phase: phosphate buffer:methanol (98:2); flow rate 1.0 ml / min.

[0130] 10 Culture processing and analysis: When analyzing the cultured bacteria, first obtain them by centrifugation at 4°C and 10,000 rpm for 5 minutes in a high-speed centrifuge and store them at 4°C until use.

[0131] When determining the dry weight and carbon and nitrogen content of the culture, the bacteria must be freeze-dried and vacuum-dried after centrifugation and then weighed.

[0132] 11 Experimental replicates: Each experiment used a culture medium without bacteria as a control (CK), and the experiment was arranged with 3 replicates for each test.

[0133] Preparation Example 1 Isolation of Mycobacteria

[0134] 1. Isolation and identification methods of target bacteria

[0135] 1.1 Culture medium

[0136] Required culture media: modified Stephenson's medium, LB-malachite green solid medium, PM plates.

[0137] Preparation of LB-malachite green solid medium

[0138] (1) Preparation of malachite green mother liquor: Weigh 0.125 g of malachite green solid and dissolve it in 500 ml of deionized water. After complete dissolution, adjust the pH to 7.0 and sterilize with high pressure steam before use.

[0139] (2) Preparation of LB solid medium: Weigh 10 g of peptone, 10 g of yeast extract, and 10 g of sodium chloride, dissolve in 1000 ml of deionized water, adjust the pH to 7-7.2, and sterilize by high-pressure steam before use. Add 100 μl of 0.25 g / L malachite green to each 100 ml of LB solid medium, so that the concentration of malachite green in the LB solid medium is 0.25 mg / L.

[0140] 1.2 Separation method

[0141] (1) Enrichment culture: Inoculate 2g of sample (original sample or enrichment culture sample) into 100ml of modified Stephenson medium containing calcium carbonate, with the content of calcium carbonate being 0.5g / 100ml medium; or inoculate the sample into modified Stephenson medium containing calcium carbonate and organic carbon source (concentration is 0.01M); culture at 28℃ and 180rpm shaking table to increase the abundance of target bacteria in the culture medium. Generally, enrichment culture is performed until NH4 + -N concentration Nessler reagent drop test is negative, NO2 - -N Griess reagent is strongly positive; or in NH4 + -N and NO2 - -N drops are all negative, NO3 - -Separate when N-diphenylamine spot is positive.

[0142] (2) Removal of impurities: Add 3 ml of cold concentrated hydrochloric acid (4°C, 3% of the volume of the culture medium) to the above culture system and shake well to remove other bacteria; then quickly neutralize with cold sodium hydroxide solution (4°C) within 3 minutes to adjust the system pH to about 7.0.

[0143] (3) Isolation of target bacteria through selective (positive) plates: The bacterial solution expressing nitrification activity after impurity treatment is diluted tenfold and spread on LB-malachite green solid plates to isolate the target bacteria in a targeted manner.

[0144] 1.3 Purification

[0145] After 7 days of incubation at 28°C, single colonies were picked onto PM plates and streaked for purification. Purity was verified by microscopic examination.

[0146] 1.4 Activity Identification

[0147] The isolated and purified target bacteria were inoculated on PM plates and cultured at 28°C for 10 days. Griess reagent was directly dropped onto the plates to confirm nitrification activity, and a plate without bacteria was used as a blank control. Within 1 minute, the Griess reagent turned red, indicating that nitrite was generated. Repeated verification showed that the color reaction was still positive, and it was preliminarily confirmed to be a nitrifying active bacteria. PM culture medium was analyzed by high-pressure liquid chromatography, which showed that the contents of nitrite and nitrate were both trace amounts, of which nitrite was not detected and NO3 - The -N content is less than 0.2 mg / L and will not interfere with the results.

[0148] 1.5 Verification of nitrification activity in liquid pure culture

[0149] Scrape 1 ring of pure bacterial moss growing on PM plate into 100mL PBS solution, ultrasonicate for 2min, fully disperse and make PBS-target bacterial suspension. Take 1ml of bacterial suspension and inoculate it into 50ml modified Stephenson medium (container is 250ml conical flask) with different organic matter as carbon source, with three replicates per group. And use uninoculated culture medium as blank control. Cultivate at 28℃, 180rpm shaker for more than 14 days, take the culture supernatant to measure NO2 - -N content. NO2 - The -N content was higher than 5 mg / L compared with the control, which was judged to have obvious high nitrification activity.

[0150] 2 Isolation, purification and identification of target bacteria in the soil of the drawdown zone of the Three Gorges Reservoir on the Yangtze River in Kaizhou District, Chongqing

[0151] The test soil samples were taken from the drawdown zone of the Three Gorges Reservoir in Kaizhou District, Chongqing (latitude and longitude 31°07'56-57", 108°28'34-35").

[0152] 5 ml of 0.1 M glucose (concentration in the culture medium 0.01 M) and 1 g of a 20-mesh sieved soil sample were added to 45 ml of modified Stephenson's medium (supplemented with calcium carbonate), and the mixture was cultured in a shaking incubator at 28° C. and 180 rpm for 14 days.

[0153] Take about 200ul of sample every 2 or 3 days, place it on a white porcelain colorimetric plate, and use Griess reagent to drip (qualitative determination) to observe NO2 - -N changes, after about 7 days of cultivation, it showed obvious nitrification activity, with a content of about 1.0ppm, and after 13 days of cultivation, NO2 - -N exceeds 20ppm. The results are shown in the table below:

[0154] Table 3a

[0155] After 14 days of culture, concentrated HCl (3% of the volume of the culture medium) was added to the culture medium, shaken for 3 minutes, and then spread on LB-malachite green plates according to a 10-fold gradient dilution, with 3 replicates for each dilution gradient. -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.

[0156] Single colonies were picked onto PM plates and streaked for purification. After incubation at 28°C, microscopic examination confirmed that the bacteria had a uniform external morphology and were pure (Figure 2b).

[0157] The isolated and purified bacteria were inoculated on PM plates and cultured at 28°C for 10 days. Griess reagent was directly applied to the plates, and the color immediately changed to brown (Figure 2c). - -N content exceeds 10mg / L, and nitrification activity is strong.

[0158] Scrape the pure bacterial lawn growing on the PM plate to prepare a PBS-target bacterial suspension. Take 1 ml of the bacterial suspension and inoculate it into 100 ml of modified Stephenson medium (container: 250 ml conical flask) with 0.06 M pyruvate as the carbon source. Each group is repeated three times. The uninoculated medium is used as a blank control. Incubate at 28°C, 180 rpm in a shaker for 24 days. The supernatant of the culture is then collected to measure NO2 - -N content exceeds 100 mg / L. Figure 2d shows the color development of Griess reagent after 10-fold and 100-fold dilution of the supernatant of the target bacteria culture medium, indicating that the target bacteria has high nitrification activity.

[0159] 3. Isolation of target bacteria from fluvo-aquic soil in North China

[0160] Take 1g of North China fluvo-aquic soil that has passed through a 100-mesh sieve and inoculate it into 50ml of modified Stephenson medium with calcium carbonate for cultivation. Use Griess reagent to detect NO2 regularly. - -N changes, indicating nitrification activity. After 14 days of culture, add concentrated HCl to the culture system at 3% of the culture volume and shake thoroughly. Within 3 minutes, quickly neutralize with sodium hydroxide solution to a pH of approximately 7.0. Add 1 ml of the neutralized culture to 9 ml of sterile water and spread onto LB-malachite green plates in a 10-fold dilution series, with three replicates for each dilution series. Target colonies should appear on the plates. The following table shows the distribution of target bacteria.

[0161] Table 3b

[0162] Single colonies were picked onto PM plates and streaked for purification. Microscopic examination confirmed that the cells were cultured at 28°C for 14 days and that the cells turned brown when spotted with Griess reagent (Figure 2e), indicating strong nitration activity.

[0163] Scrape the pure bacterial lawn growing on the PM plate to prepare a PBS-target bacterial suspension. Take 1 ml of the suspension and inoculate it into 100 ml of modified Stephenson medium (container: 250 ml conical flask) with 0.06 M pyruvate as the carbon source. Each group is repeated three times. Use the uninoculated medium as a blank control. Incubate at 28°C, 180 rpm in a shaker for 28 days. Take the culture supernatant to measure NO2 - -N content exceeds 100 mg / L. The target bacteria have high nitrification activity.

[0164] 4. Isolate target bacteria from various other soils

[0165] Using the target bacteria isolation scheme formulated by the present invention, the target bacteria can also be isolated from the soil of farmland in Zhaogang Township, Fengqiu County, Xinxiang City, Henan Province (latitude and longitude 35°02', 114°5'), Wushan soil (latitude and longitude 31°32'58.98", 120°41'55.37") and loess soil (latitude and longitude 31°48.87", 120°38'32.22") in Changshu, Jiangsu Province, and red soil in Yingtan, Jiangxi Province.

[0166] Isolation revealed that while contaminants typically emerge within 24 hours, the target bacteria only emerge after 5-7 days. Figure 2f shows the emergence of the target bacteria from soil or soil suspension (modified Stephenson's medium). The target bacteria, circled, can be clearly distinguished from other contaminants.

[0167] Example 1

[0168] Autogenous nitrogen-fixing properties of the mycobacteria of the present invention

[0169] 1 Materials and Methods

[0170] 1.1 Preparation of Experimental Strains and PBS Solution: Strains JD and YT were obtained according to Preparation Example 1 and deposited at the CGMCC. Strain JD was isolated from soil in the drawdown zone of the Three Gorges Reservoir on the Yangtze River in Kaizhou District, Chongqing (latitude and longitude 31°07'56-57", 108°28'34-35"); strain YT was isolated from soil in farmland in Zhaogang Township, Fengqiu County, Xinxiang City, Henan Province (latitude and longitude 35°02', 114°05').

[0171] Take 1 loop of pure bacteria JD or YT in 100 mL PBS solution, ultrasonicate for 2 min, and disperse evenly to about 1×10 7 CFU / mL, and prepare PBS-JD / YT bacterial solution for later use.

[0172] 1.2 Autogenous nitrogen fixation in the absence of combined nitrogen

[0173] 1 ml of PBS-YT / JD bacterial solution was inoculated into liquid culture medium for pure culture, and NH4 + -N, NO2 - -N, NO3 - The nitrogen fixation ability of the mycobacterium of the present invention is preliminarily determined by the change in -N. Then, the mycobacterium is streaked on a nitrogen-fixing plate and its growth is observed. If it grows normally, the colony is scraped and tested for nitrogen fixation genes. If nitrogen fixation genes are detected, it is determined that spontaneous nitrogen fixation occurs.

[0174] 1.2.1 Liquid culture medium and culture method

[0175] A: Liquid culture medium

[0176] The culture medium contained glucose, CaCl2, MgSO4·7H2O, K2HPO4·3H2O, Na2MoO4·2H2O, HCl, and FeSO4·7H2O, and all reagents were prepared separately.

[0177] Glucose: 11.25g + 45ml water

[0178] CaCl2: 0.625g + 25ml water

[0179] MgSO4·7H2O: 1.25g + 25ml water

[0180] K2HPO4·3H2O: 2.62g + 25ml water

[0181] Na2MoO4·2H2O:0.15g+25ml water

[0182] HCl: 100ml HCl + 900ml water

[0183] FeSO4·7H2O: 0.5g + 12.5ml water (prepare immediately and filter sterilize)

[0184] During preparation, 100 ml of deionized water was dispensed into a 250 ml Erlenmeyer flask and sterilized separately. After all reagents were prepared separately, they were sterilized in a high-pressure steam cooker and set aside.

[0185] Under sterile conditions, add 2ml glucose, 0.6ml CaCl2, 0.4ml MgSO4·7H2O, 0.1ml FeSO4·7H2O, 0.1ml Na2MoO4·2H2O, 1ml K2HPO4·3H2O, and 0.16ml HCl to every 100ml sterile water to obtain the liquid culture medium used for the autogenous nitrogen fixation experiment.

[0186] B: Culture system and culture conditions

[0187] Inoculate 1 ml of PBS-JD / YT bacterial solution into the above liquid culture medium and culture it in a shaking platform at 28°C and 180 rpm. Qualitatively determine the NH4 in the culture medium every two days. + -N, NO2 - -N, NO3 - -N changes to preliminarily determine its nitrogen fixation capacity.

[0188] 1.2.2 Nitrogen fixation plate method

[0189] The pure experimental strain was streaked onto nitrogen-fixing medium and cultured in a 28°C incubator. The humidity was maintained constant during the culture to prevent evaporation of water from the plate. The growth of the strain was observed for a period of time. If normal growth was observed, all colonies were scraped with a sterilized stainless steel spatula and suspended in phosphate-buffered saline (PBS). The supernatant was removed by centrifugation. The solid matter was collected and frozen in liquid nitrogen for testing for nitrogen-fixing genes and nitrogen-fixing ability.

[0190] 1.2.3 Detection of nitrogen-fixing genes

[0191] PCR amplification was performed using three pairs of nitrogen-fixing primers, with four replicates for each pair. All three pairs of primers were capable of amplifying the nitrogenase ferritin of nitrogen-fixing bacteria. Letters other than A, T, C, and G in the primer sequences, such as Y, S, R, B, and W, represent substituted bases, which enable the primers to more comprehensively amplify the target gene.

[0192] The first pair of primers amplifies 360 bp in length, and the primer sequences are:

[0193] PolyF: 5'-TGCGAYCCSAARGCBGACTC-3' (SEQ ID NO: 3)

[0194] PolyR: 5'-ATSGCCATCATYTCRCCGGA-3' (SEQ ID NO: 4)

[0195] The second pair of primers amplifies 280 bp in length, and the primer sequences are:

[0196] nifH F: 5'-ACCCGCCTGATCCTGCACGCCAAGG-3' (SEQ ID NO: 5)

[0197] nifHR: 5'-ACGATGTAGATTTCCTGGGCCTTGTT-3' (SEQ ID NO: 6)

[0198] The third pair of primers amplifies 450 bp in length, and the primer sequences are:

[0199] nifH-F: 5'-AAAGGYGGWATCGGYAARTCCACCAC-3' (SEQ ID NO: 7)

[0200] nifH-R: 5'-TTGTTSGCSGCRTACATSGCCATCAT-3' (SEQ ID NO: 8)

[0201] 1.3 Autogenous nitrogen fixation in the presence of combined nitrogen

[0202] The PBS-JD / YT bacterial solution was inoculated into a culture medium with pyruvate as carbon source and ammonium sulfate as nitrogen source for pure culture. During the culture process, the bacterial growth and the NH4 + -N concentration changes, such as normal bacterial growth, while NH4 + A sudden increase in -N concentration indicates the presence of nitrogen fixation. The spontaneous nitrogen fixation of the Mycobacterium spp. in the presence of combined nitrogen only considers ammonia secretion by the strain and does not account for nitrogen fixation during cellular synthesis. The energy consumption of spontaneous nitrogen fixation by the Mycobacterium spp. can also be calculated based on the corresponding pyruvate consumption during this stage.

[0203] 1.3.1 Culture medium with pyruvate as carbon source and ammonium sulfate as nitrogen source

[0204] A: Modified Stephenson's medium: The nitrogen source is ammonium sulfate, with a concentration of 2.0 g / L.

[0205] B. Medium with pyruvate as the carbon source: Add the prepared, separately sterilized pyruvate solution to the modified Stephenson medium to achieve a pyruvate concentration of 0.02 M. Fine-tune the pH to the desired value with 1 M NaOH solution.

[0206] 1 ml of PBS-JD / YT bacterial solution was inoculated into 100 ml of culture medium with pyruvate as carbon source and ammonium sulfate as nitrogen source for pure culture.

[0207] Culture conditions: temperature 28°C, rotation speed 180 rpm, CO2 concentration 0.5%, RH 50%.

[0208] 1.3.2 Indicator Analysis

[0209] After 5 days of culture, the growth of the bacteria was measured every 2 days. The culture supernatant was used to measure the NH4 + -N, NO2 - -N and NO x - -N content.

[0210] 1.4 Quantitative determination of nitrogen fixation activity

[0211] The nitrogen-fixing activity of the experimental strains was determined using the acetylene reduction method. 10 mL of bacterial culture at the mid-logarithmic growth stage (OD600 = 0.4-0.8) was added to a 100 mL glass vial. The vial was sealed and 10% of the air volume in the vial was replaced with purified high-purity acetylene using a syringe. The vial was incubated in the dark at 28°C for 24 h. The gas in the vial was collected and analyzed for ethylene concentration using a gas chromatograph (Shimadzu, GC-2014C) equipped with a flame ionization detector (FID). The column temperature was set at 60°C, the injector temperature at 120°C, and the FID detector temperature at 220°C. High-purity nitrogen was used as the carrier gas. The protein content of the bacterial culture was determined using the Bradford method (Bradford, 1976) to characterize the biomass, using bovine serum albumin (BSA) as a protein standard. The ethylene production rate per unit mass of protein per unit time (nmol C2H4 / mg protein / h) was calculated.

[0212] The calculation formula is as follows:

[0213] (1) Calculate the headspace volume in the bottle (cm 3 ): V headspace =V total -V liquid . V total 100cm 3 , V liquid The volume occupied by 10 ml of bacterial solution.

[0214] (2) Calculate the sample ethylene concentration (ppm) using the external standard method based on the ethylene standard gas concentration: P C2H4 is the peak height of the sample, C standard C2H4 / P standard C2H4 It is the ethylene concentration corresponding to the unit ethylene peak height.

[0215] (3) Calculate the amount of ethylene produced during the incubation time (nmol C2H4):

[0216] (4) Calculate the ethylene production rate per unit mass of protein per unit time (nmol C2H4 / mg / h):

[0217] 1.5 Comparison of energy consumption of nitrogen fixation

[0218] Typical nitrogen-fixing bacterial genera / species, such as Azotobocter (Azotobocter chroococcum, etc.), Clostridium pasteurianum, Klebsiella pneumoniae, and Streptomyces thermoautotrophicus, were selected. Artificial nitrogen fixation was also considered. Based on published nitrogen fixation energy consumption data for related bacterial genera / species and artificial nitrogen fixation, the number of milligrams of nitrogen fixed per milligram of pyruvate consumed was used as a unified unit and compared with the nitrogen fixation ability experimental results of the mycobacteria JD and YT of the present invention to analyze and study the nitrogen fixation ability of the mycobacteria of the present invention.

[0219] 2 Results

[0220] 2.1 Autogenous nitrogen fixation in the absence of combined nitrogen

[0221] 2.1.1 Growth in liquid culture medium

[0222] The JD and YT strains were inoculated into liquid culture media respectively. Both strains were able to grow, but the growth was relatively slow. A circle of bacteria was observed to adhere to the wall of the culture bottle, indicating that the two strains had strong wall adhesion, thus affecting the continued growth of the bacteria, as shown in Figure 3a, with YT on the left and JD on the right.

[0223] Table 4a lists the changes in pH of the liquid culture medium and the NH4 + -N, NO2 - -N, NO3 - -N accumulation. During the culture process, JD and YT will produce acid, causing the pH of the liquid culture medium to decrease. When the pH of the culture medium is too low, the strain will stop growing. Therefore, the pH should be adjusted to 7.0-7.2 during the culture process. Through qualitative analysis, there will be NH4 + The accumulation of -N indicated that JD and YT had certain nitrogen fixation ability.

[0224] Table 4a Changes in pH and ammonia nitrogen during pure culture of Mycobacterium JD and YT nitrogen-fixing liquid media

[0225] 2.1.2 Growth of nitrogen-fixing plates

[0226] As shown in Figure 3a and Table 4a, Mycobacterium YT and JD grew slowly in liquid culture medium. + -N accumulation was observed, but the amount was very low. Because the bacterium grows on surfaces, it is not suitable for obtaining large numbers of cells through liquid culture. This study further conducted nitrogen fixation plate experiments.

[0227] Figure 3b (YT on the left and JD on the right) shows that the two strains cultured in the above liquid culture medium were streaked onto a nitrogen-free agar plate. After 11 days of culture, the two strains grew normally on the plate with a single colony morphology. This indicates that the liquid culture medium and the strains inoculated therein were not contaminated. On the other hand, it indicates that the turbidity that appeared in the growth experiment of the strains in the liquid culture medium (Figure 3a) was a manifestation of normal growth of the strains rather than the death of the microorganisms.

[0228] Figure 3c shows YT (left) and JD (right) strains grown on nitrogen-fixed plates for 7 days. Both strains are clearly growing on the plates, with milky white bacterial colonies visible to the naked eye. By day 29, both strains had further growth, with distinct single colonies visible, but the bacterial population was small and growth remained slow.

[0229] FIG3 d shows the cell morphology of JD bacteria after being cultured on a nitrogen-fixed plate for more than 40 days (Nikon stereo microscope, 100 times magnification).

[0230] To promote growth of the strains on nitrogen-fixing plates and obtain a larger amount of growth to meet research and testing needs (no single clones are required), this study used a spreading rod to scrape the bacteria from the nitrogen-fixing plate shown in Figure 3c and then spread them on the plate again. However, growth was still unsatisfactory. Resting cells were then centrifuged to remove the supernatant, and the colonies were directly scraped and spread on LB plates, selecting as many bacteria as possible. Figure 3e shows the results of cultures (YT on the left and JD on the right) obtained by centrifuging resting cells to remove the supernatant and then directly spreading them on nitrogen-fixing plates. It can be seen that after the above treatment, both strains grew significantly on the nitrogen-fixing plate.

[0231] 2.1.3 Detection of nitrogen-fixing genes

[0232] By using three pairs of primers containing nitrogenase iron protein for PCR amplification, all three pairs of primers were able to amplify, especially the amplification result using the second primer was more obvious, as shown in Figure 3f, thus demonstrating at the molecular level that both strains YT (bacterial solution 1) and JD (bacterial solution 2) have the ability to fix nitrogen.

[0233] 2.2 Autogenous nitrogen fixation in the presence of combined nitrogen

[0234] In the presence of combined nitrogen (2.0 g / L ammonium sulfate) and using pyruvate as a carbon source, both Mycobacterium strains JD and YT of the present invention grew normally. The ammonia nitrogen concentration in the culture medium of both strains initially decreased rapidly during growth, then decreased to a low level after 7 days of growth, then rapidly increased. After 9 days of growth, the ammonia nitrogen concentration in the culture medium decreased again (see Figure 3g). This indicates that significant nitrogen fixation occurs between 7 and 9 days of growth.

[0235] As shown in the table below, during nitrogen fixation by the mycobacteria of the present invention, the culture system inoculated with JD decreased 6.05 mg of pyruvate and increased 15.49 mg of ammonia nitrogen. This means that the JD strain fixed 2.56 mg of nitrogen (and secreted 2.56 mg of ammonia) for every mg of pyruvate consumed. The culture system inoculated with YT decreased 7.06 mg of pyruvate and increased 15.098 mg of ammonia nitrogen. This means that the YT strain fixed 2.13 mg of nitrogen (and secreted 2.35 mg of ammonia) for every mg of pyruvate consumed.

[0236] Table 4b NH4 in the culture medium between the 7th and 9th days of growth of mycobacteria of the present invention + -N and pyruvate concentration changes

[0237] 2.3 Nitrogen fixation activity

[0238] Through the analysis of the nitrogen fixation activity experimental results of the two strains YT and JD, the average ethylene production rates of YT and JD were 0.827±0.552nmol / mg protein / h and 0.502±0.337nmol / mg protein / h, respectively.

[0239] 2.4 Comparison of the ability of the mycobacteria of the present invention to typical nitrogen-fixing bacteria and artificial nitrogen-fixing

[0240] 2.4.1 Energy consumption of nitrogen fixation by typical bacterial genera / species

[0241] (1) Azotobocter: Azotobocter, represented by Azotobocter chroococcum, fixes 10-20 mg of nitrogen per 1 g of glucose consumed (Zhu Zhaoliang et al., 1992). In the tricarboxylic acid cycle, 1 molecule of pyruvate produces 15 molecules of ATP starting from dehydrogenation. Each molecule of glucose can produce 2 molecules of pyruvate, so each molecule of glucose produces a total of 36-38 ATP molecules through the three stages of glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation (Shen Tong et al., 2000). Therefore, for the ATP generated by the tricarboxylic acid cycle alone: ​​1 mol of glucose corresponds to 2 mol of pyruvate, which corresponds to 30 mol of ATP. Therefore, for Azotobacter, 10-20 mg of nitrogen is fixed for every 1 g of glucose consumed, which is equivalent to 10-20 mg of nitrogen fixed for every 0.978 g of pyruvate, that is, 0.0102-0.0204 mg of N is fixed for every 1 mg of pyruvate consumed.

[0242] (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, from the perspective of ATP production through the tricarboxylic acid cycle, 1 g of glucose corresponds to 0.978 g of pyruvate, which is equivalent to 0.978 g of pyruvate fixing 1.5-7.0 mg of nitrogen, that is, every 1 mg of pyruvate consumed fixes 0.001534-0.007157 mg of nitrogen.

[0243] (3) Klebsiella pneumoniae: The basic enzymatic reaction of nitrogenase represented by Klebsiella pneumoniae is: N2+8H + +8e - +16MgATP → 2NH3 + H2 +16MgADP +16Pi (Han Bin et al., 2009), then: 1 mol N2 corresponds to 16 mol ATP. Since 1 molecule of pyruvate generates 15 molecules of ATP through the tricarboxylic acid cycle (Shen Tong et al., 2000), 1.07 mol of pyruvate is required to produce 16 mol of ATP. In other words, Klebsiella pneumoniae requires at least 1.07 mol of pyruvate to produce 16 mol of ATP to fix 1 mol of N2. By conversion, Klebsiella pneumoniae can fix 0.298 g of N for every 1 g of pyruvate consumed, that is, 0.298 mg of N for every 1 mg of pyruvate consumed.

[0244] (4) Streptomyces thermoautotrophicus: The basic enzymatic reaction of nitrogenase in Streptomyces thermoautotrophicus is: N2+8H + +8e - +4-12MgATP → 2NH3 + H2 + 4-12MgADP + 4-12Pi (Markus Ribbe et al., 1997). Since 1 molecule of pyruvate generates 15 molecules of ATP through the tricarboxylic acid cycle (Shen et al., 2000), thermoautotrophic Streptomyces requires at least 4-12 mol of ATP, produced by 0.27-0.8 mol of pyruvate, to fix 1 mol of N2. By conversion, every gram of pyruvate consumed by thermoautotrophic Streptomyces fixes 0.40-1.18 g of nitrogen, meaning that every mg of pyruvate consumed fixes 0.4-1.18 mg of nitrogen.

[0245] (5) Mycobacterium of the present invention: The study on the "autogenous nitrogen fixation in the presence of combined nitrogen" section calculated the nitrogen fixation energy consumption of JD and YT, that is, the JD strain fixed nitrogen (secreted ammonia) 2.56 mg for every 1 mg of pyruvate consumed, and the YT strain fixed nitrogen (secreted ammonia) 2.13 mg for every 1 mg of pyruvate consumed. The average amount of nitrogen fixed (secreted ammonia) per mg of pyruvate consumed by the two strains was 2.35 mg.

[0246] 2.4.2 Energy consumption of artificial nitrogen fixation

[0247] According to the data, in the current artificial ammonia industry, the energy consumption for synthesizing 1 ton of ammonia (NH3, molecular weight 17.03) is 48.2×10 6 kJ, the energy consumption for synthesizing 1 ton of urea (CO(NH2)2, molecular weight 60.06) is 33.2×10 6 kJ. Calculated based on the amount of fixed nitrogen, artificial ammonia synthesis consumes 48.2 kJ of energy to fix 0.822 g of N, equivalent to the energy released by the hydrolysis of 1.58 molecules of ATP (ATP hydrolysis to produce ADP releases heat of 30.54 kJ / mol). This is equivalent to the amount of ATP generated through the tricarboxylic acid cycle by 9.28 g of pyruvate (based on the assumption that 1 molecule of pyruvate produces 15 molecules of ATP). Therefore, the energy consumption in artificial ammonia synthesis can be converted to 0.08858 mg of N fixed per mg of pyruvate consumed.

[0248] In synthetic urea, based on the amount of urea nitrogen, 33.2 kJ is required to fix 0.466 g of N, which is equivalent to the energy produced by the hydrolysis of 1.087 molecules of ATP. This is equivalent to the amount of ATP produced by 6.38 g of pyruvate through the tricarboxylic acid cycle. Therefore, the energy consumption in artificial urea synthesis can be converted into 0.073 mg of N fixed for every 1 mg of pyruvate consumed.

[0249] 2.4.3 Comparison of nitrogen fixation abilities of different bacterial species / genera and artificial nitrogen fixation

[0250] By analyzing the energy consumption of several typical nitrogen-fixing bacteria species / genuses and artificial nitrogen fixation, and comparing the nitrogen fixation amount per unit weight of pyruvic acid consumed, the order of nitrogen fixation ability is: Mycobacterium of the present invention > Thermoautotrophic Streptomyces > Klebsiella pneumoniae > artificial nitrogen fixation (synthetic ammonia and synthetic urea) > Azotobacter genus represented by Azotobacter rotundifolia > Clostridium pasteurianum. The nitrogen fixation ability of the mycobacterium of the present invention is 2-6 times that of the thermoautotrophic Streptomyces, 8 times that of Klebsiella pneumoniae, 115-230 times that of the Azotobacter genus, and 328-1532 times that of Clostridium pasteurianum. For artificial nitrogen fixation (synthetic ammonia and synthetic urea), the nitrogen fixation ability of the mycobacterium of the present invention is approximately 29 times that of its own nitrogen fixation ability, as shown in the table below.

[0251] Table 4c Comparison of nitrogen fixation ability of the mycobacteria of the present invention with typical nitrogen-fixing bacteria genera / species and artificial nitrogen fixation

[0252] 3 Conclusion

[0253] (1) When cultured in liquid culture medium, both strains grew normally and ammonia nitrogen accumulated in the culture medium. When cultured in nitrogen-fixing plate culture medium, both strains grew significantly. PCR amplification of both strains using three pairs of primers encoding nitrogenase-ferritin confirmed the presence of genes encoding nitrogen fixation. The mycobacteria of the present invention have nitrogen fixation in the absence of combined nitrogen.

[0254] (2) In the presence of combined nitrogen (ammonium sulfate), the mycobacterium of the present invention has a strong nitrogen-fixing effect, with an average nitrogen-fixing (ammonia-secreting) amount of 2.35 mg per 1 mg of pyruvate consumed.

[0255] (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 mycobacterium of the present invention has significant nitrogen fixation activity.

[0256] (4) The mycobacteria of the present invention have a strong nitrogen-fixing ability, which is 2-6 times that of thermoautotrophic Streptomyces, 8 times that of Klebsiella pneumoniae, 115-230 times that of Azotobacter, and 328-1532 times that of Clostridium pasteurianum. Compared to artificial nitrogen fixation (synthetic ammonia and urea), the nitrogen-fixing ability of the mycobacteria of the present invention is approximately 29 times that of synthetic nitrogen.

[0257] Example 2

[0258] Growth and nitrification activity of the mycobacterium of the present invention based on pure culture with pyruvate as carbon source

[0259] 1 Materials and Methods

[0260] 1.1 Test bacteria: PBS-JD / YT bacterial solution.

[0261] 1.2 Culture medium and index determination

[0262] (1) Culture medium

[0263] Modified Stephenson's medium with pyruvate as the carbon source, with the designed pyruvate concentrations of 0.06 M and 0.1 M in the medium. 1 M NaOH solution was used to fine-tune the required pH value.

[0264] (2) Methods and indicators

[0265] Inoculate 1 ml of PBS-JD / YT bacterial solution into the prepared and sterilized liquid culture medium, culture at 28°C and 180 rpm on a shaking table, and regularly measure bacterial growth, pH, and NH4 + -N, NO2 - -N, NO x - -N content.

[0266] 2 Results

[0267] As shown in Figure 4a (0.06M pyruvate in the upper figure and 0.1M pyruvate in the lower figure) and the table below, when pure culture was carried out with 0.06M pyruvate as the carbon source, the OD values ​​of the JD and YT strains were significantly different after 24 days of culture. 600 The average values ​​were 1.84 and 1.62 respectively. At this time, the NH4 + -N concentration decreased from the initial 424mg / L to 93.08mg / L and 66.87mg / L respectively; NO2 - -N accumulation, the average concentration reached 139.90mg / L and 96.31mg / L respectively. The experiment showed that when pure cultured with 0.06M pyruvate as carbon source, both strains YT and JD could grow and produce strong nitrification. When pure cultured with 0.1M pyruvate as carbon source, after 33 days of culture, the OD of strains JD and YT was 600 The average value is 2.04. At this time, the NH4 + -N content almost disappeared, with average concentrations of only 4.52mg / L and 3.88mg / L, and NO2 - -N accumulated to 177.92mg / L and 161.14mg / L. The experiment showed that when the YT and JD strains were cultured in pure culture with 0.1M pyruvate as carbon source, they could grow and produce strong nitrification.

[0268] Table 5 Growth and nitrification activity of strains YT and JD when pure cultured with different concentrations of pyruvate as carbon source

[0269] 3 Conclusion: When pure cultured with different concentrations of pyruvate as the carbon source, the test strains were able to grow normally, and had strong ammonia oxidation and significant nitrification activity.

[0270] Example 3

[0271] Fixation of CO2 by Mycobacterium of the Present Invention

[0272] 1 Materials and Methods

[0273] 1.1 Test bacteria: PBS-JD / YT bacterial solution

[0274] 1.2 Culture medium and culture system:

[0275] A. Modified Stephenson's medium

[0276] B. Using pyruvate as the carbon source: First, prepare a 2 M pyruvate solution (weigh 88.0701 g of pyruvate, add 400 mL of distilled water, stir, add 40 g of solid NaOH, cool, and dilute to 500 mL. Adjust the pH to 7.17). Sterilize the solution separately for later use.

[0277] Before use, first take 5 ml of 2 M pyruvic acid solution and add it to 94 ml of modified Stephenson's medium.

[0278] Inoculate 1 ml of PBS-JD / YT bacterial solution into the above culture medium, with a total culture volume of 100 ml. The designed concentration of pyruvate in the culture medium is approximately 0.1 M.

[0279] 1.3 Culture conditions: Temperature 28°C, rotation speed 180 rpm, CO2 concentration 5%, shaker culture. Continuous culture for 21 days.

[0280] 1.4 Index determination: Regularly measure bacterial growth and pH; take the supernatant to determine NH4 + -N, NO2 - -N, NO x - -N content; the pyruvate content in the culture medium was regularly determined, and the pyruvate content was detected by high performance liquid chromatography (HPLC).

[0281] After the incubation period, the cells were centrifuged at 4°C and 10,000 rpm for 5 minutes. The cells were then freeze-dried and vacuum-dried, and their dry weight, carbon content, and nitrogen content were determined. The amount of CO2 fixed was estimated by analyzing the changes in carbon and nitrogen content in the culture system (culture supernatant and cells).

[0282] 2 Results

[0283] 2.1 Changes in pyruvate concentration in the culture system

[0284] As shown in Figure 4b (top: JD, bottom: YT), under high CO2 conditions, pure cultures of JD and YT using pyruvate as the carbon source showed a decrease, then an increase, and finally a decrease in pyruvate concentrations in the culture media of both strains, reflecting the independent completion of both the forward and reverse TCA cycles. In the first phase, the strain undergoes the forward TCA cycle, consuming pyruvate to generate energy for accumulating arginine metabolic intermediates in preparation for ammonia oxidation. In the second phase, the strain initiates ammonia oxidation, undergoes arginine metabolism, and uses the energy generated by metabolism to drive the reverse TCA cycle, accumulating pyruvate in preparation for anabolism. In the third phase, the strain undergoes anabolism, consuming pyruvate to complete gluconeogenesis and achieve growth. Both strains fix CO2 via the reverse TCA cycle.

[0285] 2.2 Analysis of carbon and nitrogen consumption and carbon fixation during metabolism

[0286] (1) Carbon-nitrogen ratio of bacteria

[0287] The experimental results show that the carbon-nitrogen ratio of the mycobacterium cells of the present invention is 5.65:1, which is not much different from the carbon-nitrogen ratio of general heterotrophic bacteria cells (the empirical value is 5:1).

[0288] (2) Carbon and nitrogen consumption during bacterial anabolism

[0289] Carbon and nitrogen consumption during bacterial anabolism can be categorized into two types: the consumption of substances used for anabolism and the energy consumed for anabolism. Generally, heterotrophic bacteria derive their anabolism energy and substances from organic carbon and nitrogen sources in the environment (usually derived primarily from the decomposition of other life forms). The overall carbon-nitrogen mass ratio is typically around 25:1, with substances used for anabolism accounting for 20% of the total carbon source, and the energy used to generate anabolism accounting for 80%.

[0290] (3) CO2 emission reduction effect of mycobacteria of the present invention

[0291] The difference between the mycobacteria of the present invention and general heterotrophic bacteria is that general heterotrophic bacteria obtain energy from organic carbon sources through glycolysis and the TCA cycle (while releasing CO2), while the mycobacteria of the present invention can obtain energy from organic carbon sources through glycolysis and the TCA cycle, and can also obtain the energy required for assimilation through nitrogen metabolism pathways such as ammonia oxidation and nitrification, promote the reverse TCA cycle, and assimilate CO2. It can be seen from this that when the mycobacteria of the present invention want to complete the anabolism of one part nitrogen and five parts carbon, it can reduce the consumption of twenty parts of organic carbon required to complete the TCA cycle. At the same time, it also uses the energy generated by nitrogen metabolism to reversely drive the TCA cycle to fix five parts of CO2 to obtain the substance required for anabolism - the organic carbon source. In this way, compared with general heterotrophic bacteria, the mycobacteria of the present invention consume one part of nitrogen for the synthesis of substances for assimilation, which is equivalent to reducing the emission of 25 parts of CO2.

[0292] (4) Calculate the equivalent amount of fixed CO2 based on the changes in substrate during the experiment

[0293] At 424 mg NH4 + -N and 0.1mol pyruvate in the matrix solution, the residual NH4 + -N4.52mg, NO X -N 287.8mg and organic carbon <0.1mg. Therefore, the mass of nitrogen used for assimilation growth is 131.68mg. According to the molar ratio of 25:1, the mass of carbon element consumption reduced by assimilating 131.68mg of nitrogen is 2822mg, which is equivalent to the mass of CO2 reduced by 10.35g. In the same way, after 21 days of continuous cultivation of YT bacteria, the residual NH4 + -N 3.88mg, NO X-N 260.6mg and organic carbon <0.1mg. Therefore, the mass of nitrogen used for growth is 159.52mg. Based on a 25:1 molar ratio, assimilating 159.52mg of nitrogen reduces carbon consumption by 3418mg, which translates to a reduction of 12.53g of CO2 emissions.

[0294] 3 Conclusion

[0295] (1) The mycobacterium of the present invention has a reverse TCA cycle pathway.

[0296] (2) The mycobacterium of the present invention fixes CO2 through a reverse TCA cycle and has the function of reducing CO2 emissions.

[0297] Example 4

[0298] Growth and nitrification activity of the mycobacterium of the present invention in pure culture using hydrazine sulfate as a nitrogen source

[0299] 1 Materials and Methods

[0300] 1.1 Test bacteria: Prepare the resting cells of JD and YT obtained in Example 1. Centrifuge the cultured bacteria, remove the supernatant, wash repeatedly with physiological saline, and place in a 4°C refrigerator until the Griess reagent is used to drip NO2. - -N until no color is developed and the cells are confirmed to be pure by streaking. When inoculating, the supernatant is removed by centrifugation and suspended in culture medium before being added to a shake flask for culture.

[0301] 1.2 Training system

[0302] (1) Culture medium

[0303] A. Modified Stephenson's medium with hydrazine sulfate as the nitrogen source

[0304] The 2.0 g (NH4)2SO4 in the modified Stephenson's medium was replaced with an equal amount of N2H4·H2SO4, i.e., 1.97 g N2H4·H2SO4, while the other components and treatments remained unchanged.

[0305] B. Using pyruvate as the carbon source: Add 6 ml of a separately sterilized 1.0 M pyruvate solution to 100 ml of medium A to achieve a designed pyruvate concentration of approximately 0.06 M. Adjust the pH with 1 M NaOH.

[0306] (2) Training system

[0307] 1 ml of resting cell fluid was inoculated into the above culture medium and cultured on a shaking platform at 28°C and 180 rpm.

[0308] (3) Index determination: Determine the OD of bacterial growth in the culture medium 600 , pH value and NH4+ -N, NO2 - -N, NO x - -N content.

[0309] 2 Results

[0310] The test strain was inoculated into a liquid culture medium with hydrazine sulfate as the nitrogen source. At this time, the culture medium was clear (Figure 5, left). After 7 days of growth, the bacterial solution grown in hydrazine sulfate was inoculated onto a solid plate. After 14 days of growth on the solid plate, it was clearly found that the test strain grew normally on the plate with hydrazine sulfate as the nitrogen source, but the qualitative determination of NO2 - -N, the plate did not show color (Figure 5, middle panel); after 38 days of growth in liquid culture medium, the culture medium became obviously turbid when observed with the naked eye, and the strain grew (Figure 5, right panel). At this time, the OD values ​​of the YT and JD strains were 1.09 and 1.12, respectively (Table 6b).

[0311] During the culture period, the NH4 in the culture medium was analyzed regularly. + -N, NO2 - -N, NO3 - -N content changes, combined with the qualitative analysis results (Table 6a) and quantitative determination results (Table 6b), it can be seen that there is no NH4 + -N formation and NO2 - -N, NO3 - -N, NO X - -N accumulation; at the same time, the pH value increased significantly, that is, it was 6.4 at the beginning and reached 7.7-8.0 after 25 days of growth. This shows that the bacteria can grow when using hydrazine sulfate as a substrate, but does not produce nitrite and nitrate.

[0312] Table 6a NH4 in the culture medium of pure culture of YT and JD using hydrazine sulfate as nitrogen source + -N, NO2 - -N, NO3 - -N content changes

[0313] Table 6b Growth and nitrification activity of strains YT and JD using hydrazine sulfate as nitrogen source

[0314] 3 Conclusion: The Mycobacterium of the present invention can use hydrazine sulfate as a nitrogen source for growth, but it does not convert into ammonium nitrogen, nor does it produce nitrite and nitrate, and has no nitrification activity at all; the metabolic pathway of the strain using hydrazine sulfate dehydrogenation to produce hydroxylamine is possible.

[0315] Example 5

[0316] Growth and nitrification activity of pure cultured mycobacteria of the present invention using hydroxylamine as a nitrogen source

[0317] 1 Materials and Methods

[0318] 1.1 Culture medium

[0319] (1) Iron ion culture 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 culture medium to 7.0-7.2 with sulfuric acid and sodium hydroxide before sterilization.

[0320] (2) Iron-free culture medium (g / L): NaH2PO4 0.25, K2HPO4 0.75, MgSO4·7H2O 0.03, MnSO4·H2O 0.01. Adjust the pH of the culture medium to 7.0-7.2 with sulfuric acid and sodium hydroxide before sterilization.

[0321] There were two nitrogen sources, 0.2 mM and 1.8 mM hydroxylamine hydrochloride; and the carbon sources were 0 and 0.06 M pyruvate.

[0322] 1.2 Culture

[0323] Resting cells JD and YT, that is, centrifuge the cultured JD and YT to remove the supernatant, wash repeatedly with physiological saline and place in a 4℃ refrigerator until the Gliese reagent is used to drip NO2 - -N until no color is developed and the bacteria are confirmed to be pure by streaking. When inoculating, suspend the bacteria in culture medium and then add them to the shake flask for culture.

[0324] 1.3 Culture conditions

[0325] The cells were cultured at 28°C in a shaker at 180 rpm for 21 days.

[0326] 1.4 Culture system and indicator determination

[0327] Using the uninoculated bacteria as a control, at two nitrogen source concentrations of 0.2 mM hydroxylamine and 1.8 mM hydroxylamine, two types of culture were set according to the table below, no carbon source and 0.06 M pyruvate, and cultured using iron-containing medium and iron-free medium, respectively.

[0328] Table 7a Culture system settings

[0329] After the culture was completed, the bacterial growth rate (OD 600 ) and pH, and the supernatant was measured for NH4 + -N, NO2 - -N, NO3 -After centrifugation, the cells were quickly frozen in liquid nitrogen and stored at -80°C for transcriptome sequencing. Each run was repeated three times.

[0330] 1.5 Culture transcriptome sequencing

[0331] According to the table below, the sequencing was classified as follows: (1) W-0: resting cells treated with starvation; (2) W-1: 0.2 mM hydroxylamine hydrochloride + 0 M pyruvate; (3) W-2: 0.2 mM hydroxylamine hydrochloride + 0.06 M pyruvate.

[0332] Table 7b Culture transcriptome sequencing

[0333] 2 Results

[0334] 2.1 Growth and Nitrification Activity of Mycobacteria of the Present Invention in Pure Culture Using Hydroxylamine as a Nitrogen Source

[0335] As shown in Table 7c, under pure culture conditions with hydroxylamine hydrochloride as nitrogen source (0.2mM and 1.8mM) and pyruvate as carbon source (concentration of 0.06M), strains JD and YT can grow in both iron-containing and iron-free medium. + -N, NO2 - -N, NO3 - -N concentration changes analysis showed that both strains showed no nitrification activity.

[0336] Table 7c Comparison of strain growth and nitrification activity under pure culture of hydroxylamine + 0.06M pyruvate

[0337] Table 7d shows the growth and nitrification activity of strains JD and YT when pure cultured with hydroxylamine hydrochloride as the nitrogen source and without the addition of pyruvate as the carbon source. At hydroxylamine concentrations of 0.2 mM and 1.8 mM, JD and YT were able to grow and exhibit nitrification activity in both iron-containing and iron-free culture media.

[0338] Table 7d Comparison of growth and nitrification activity of pure cultured strains of hydroxylamine + 0 pyruvate

[0339] 2.3 Culture transcriptome sequencing

[0340] Analysis of the transcriptome sequencing results of the YT culture clearly showed that the Mycobacterium of the present invention has at least five dehydrogenases, and the expression of hydroxylamine dehydrogenase in the strains was significantly different under different treatments such as the addition of pyruvate as a carbon source ( FIG. 6 ).

[0341] For the five dehydrogenases, the enzyme that increased twice may be related to the pathways from hydroxylamine to nitrogen gas and from hydroxylamine to ammonia. If "hydrogen dehydrogenase" is determined to be the pathway to nitrogen gas, then the other one is determined to be the pathway to ammonia; the expression of the other four dehydrogenases may be related to the subsequent oxidation products of hydroxylamine (to nitrous oxide, nitrous oxide to nitric oxide, nitric oxide to nitrous acid, hydroxylamine to dinitric acid, and nitrous acid to nitric acid).

[0342] 3 Conclusion

[0343] (1) With hydroxylamine as the nitrogen source, the mycobacteria of the present invention can grow regardless of the presence or absence of an organic carbon source or iron ions in the culture medium, but there are significant differences in the expression of nitrification activity.

[0344] (2) Adding Fe 2+ and without Fe 2+ The results are consistent, indicating that Fe 2+ No effect, the reaction did not follow the arginine pathway.

[0345] (3) Whether or not a carbon source is added has a huge impact on the reaction results. After adding a carbon source, nitrogen gas is most likely to be lost, and other products may also be generated, but nitrous acid will not be produced. Without adding a carbon source, nitrous acid can be produced, showing nitrification activity.

[0346] (4) The same results were obtained for two concentrations of hydroxylamine and two different bacterial samples, which strengthened the above results.

[0347] (5) Transcriptome sequencing analysis of the culture showed that the mycobacteria of the present invention have at least five dehydrogenases, and the expression of hydroxylamine dehydrogenase in the strains was significantly different under different treatments such as with and without the addition of pyruvate as a carbon source.

[0348] Example 6

[0349] Experiment on the cultivation of secondary nitric acid (anti-sodium hyponitrite)

[0350] 1 Materials and Methods

[0351] 1.1 Culture

[0352] Resting cells JD and YT.

[0353] 1.2 Culture medium

[0354] The ammonium sulfate in the modified Stephenson's medium was removed and replaced with an equal amount of sodium hyponitrite in the nitrogenous fraction. All other components remained unchanged and the pH was kept natural. The carbon source was pyruvate, and the designed pyruvate concentration in the medium was 0.06 M.

[0355] 1.3 Culture conditions

[0356] The cells were cultured in a shaker at 180 rpm, 28°C, and 5% CO2 for about 24 days.

[0357] 1.4 Observation indicators

[0358] Observe the growth of bacteria and qualitatively determine the NH4 in the culture medium + -N, NO2 - -N, NO3 - -N.

[0359] 2 Results

[0360] As shown in Figure 7a, a film was initially visible on the surface of the culture system using sodium hyponitrite as the nitrogen source, with a small amount of filamentous bacteria clearly visible to the naked eye, indicating bacterial growth. However, continued cultivation did not maximize growth, likely due to the strong base and weak acid salt of sodium hyponitrite (stable around pH 12). Adjusting the pH to around 7 generated a large number of bubbles in the solution, decomposing the sodium hyponitrite. Stereoscopic observation of cell morphology is shown in Figure 7b.

[0361] By colorimetric titration of relevant indicators in the liquid culture medium of the resting cell culture experiment using sodium hyponitrite as the nitrogen source, YT and JD had slow nitration reactions, and the diphenylamine reaction was weakly positive (Figure 7c, in the right figure of Figure 7c, from left to right, NH4 + -N, NO2 - -N, NO3 - -N).

[0362] 3 Conclusion

[0363] The mycobacterium of the present invention can grow using dinitrate (trans-sodium hyponitrite) and exhibit nitrification activity.

[0364] Example 7

[0365] Effects of iron ions on the growth and nitrification activity of mycobacteria of the present invention

[0366] 1 Materials and Methods

[0367] 1.1 Culture medium: PBS-JD and PBS-YT bacterial solution.

[0368] 1.2 Culture medium

[0369] A. Modified Stephenson's medium

[0370] B. Iron-free modified Stephenson's medium: Remove FeSO4·7H2O from the modified Stephenson's medium, while keeping other components and treatments unchanged.

[0371] C. Iron-free and manganese-free modified Stephenson's medium: Remove FeSO4·7H2O and MnSO4·H2O from the modified Stephenson's medium, while keeping other components and treatments unchanged.

[0372] Pyruvate was used as the carbon source, and the designed concentration of pyruvate in the culture medium was 0.06M.

[0373] 1.3 Effect of iron ions on strain growth and nitrification performance

[0374] 1 ml of PBS-JD / YT bacterial solution was inoculated into the above three culture media A+pyruvate, B+pyruvate, and C+pyruvate, respectively, and cultured in a shaking incubator at 180 rpm and a constant temperature of 28°C. Culture was continued for 24 days, and the OD value of the bacterial solution was measured regularly. 600 At the same time, the culture medium was centrifuged at 4°C for 15 min (5000 g) and the NO2 in the supernatant was determined. - -N、NH4 + -N and NO x - -N(NO2 - -N+NO3 - -N) content, and the ammonia oxidation and nitrification abilities of the test strains were analyzed by calculating the ammonia oxidation rate and total nitrification rate.

[0375] The non-inoculated culture medium was used as the control (CK) in all three treatment culture media, and the experiment was repeated three times for each test.

[0376] Nitrification rate calculation:

[0377] Total nitrification rate = [NO x - -N]×100 / [NH4 + -N(CK)]

[0378] Nitrite rate = [NO2 - -N]×100 / [NH4 + -N(CK)]

[0379] Short-range nitrification rate = [NO2 - -N]×100 / [NO x - -N]

[0380] Ammonia oxidation rate / NH4 + -N conversion rate = ([NH4 + -N(CK)]-[NH4 + -N(residual)])×100 / [NH4 + -N(CK)]

[0381] 1.4 Treatment of laboratory instruments: All glassware used in the above experiments were soaked in a 1:1 nitric acid solution for 24 h, washed thoroughly with natural water, and then washed thoroughly with deionized water to eliminate the influence of heavy metal adsorption on the experimental results.

[0382] 2 Results

[0383] Table 8 lists the growth and nitrification activity of the test strains in the basic medium containing iron and manganese and in the iron-free medium and the iron-free and manganese-free medium. After 21 days of culture, it can be clearly observed that YT and JD can grow in the three treatment mediums. Compared with the uninoculated culture medium, the culture medium of the inoculated strains becomes obviously turbid (Table 8 and Figure 8). Figure 8 is from left to right 1: JD strain + iron-free medium; 2: YT strain + iron-free and manganese-free medium; 3: YT strain + iron-free medium, 4: JD strain + iron-free and manganese-free medium. Compared with the iron-free medium and the iron-free and manganese-free medium, the OD 600 The values ​​were higher, with the average values ​​of JD strain and YT strain being 1.84 and 1.62 respectively.

[0384] JD and YT strains showed high nitrification activity in the basic culture. After 24 days of growth, the NO2 - The average concentrations of -N reached 139.9 mg / L, 96.31 mg / L, and NO x - The average contents of -N were 219.04 mg / L and 152.39 mg / L respectively; while there was no NO2 in the uninoculated control, iron-free medium, iron-free and manganese-free medium. - -N, NO x - The production of -N indicated that the test strain had no nitrification activity in the absence of iron.

[0385] Table 8 Comparison of growth and nitrification activity of the test strains in iron-containing and iron-free media

[0386] 3 Conclusions:

[0387] (1) The test strain was able to grow in iron-free medium but had no nitrification activity at all.

[0388] (2) The test strain was able to grow in an iron-free and manganese-free medium, but had no nitrification activity at all.

[0389] (3) Iron ions are an important factor affecting the nitrification activity of this bacterium, while manganese ions have no effect on its nitrification activity.

[0390] Example 8

[0391] Growth and nitrification activity of pure cultured mycobacteria of the present invention using arginine as a nitrogen source

[0392] 1 Materials and Methods

[0393] 1.1 Culture medium: PBS-JD / YT bacterial solution

[0394] 1.2 Culture medium

[0395] A. Modified Stephenson's medium with arginine as the nitrogen source

[0396] First, prepare a nitrogen-free modified Stephenson medium: remove ammonium sulfate from the modified Stephenson medium, keep other components unchanged, and adjust the pH to 7.0-7.2 to obtain the nitrogen-free modified Stephenson medium.

[0397] Next, prepare a stock solution of L-arginine at a specific concentration: weigh 5.28g of L-arginine (relative molecular weight 174.20, solubility in water at 20°C: 148.7g / L) and dissolve it completely in 50ml of deionized water. Adjust the pH to 7.0-7.2, and finally adjust the volume to 80ml. Because L-arginine, as an amino acid, is easily decomposed under autoclaving conditions, sterilize it using a membrane filter.

[0398] During the culture, add 2 ml of the prepared L-arginine solution to every 100 ml of the nitrogen-free modified Stephenson medium. At this time, the nitrogen content in the culture system is the same as the nitrogen content when the original nitrogen source is ammonium sulfate (nitrogen content 424 mg / L), that is, the modified Stephenson medium with arginine as the nitrogen source with equal nitrogen content.

[0399] B: Carbon source is pyruvate. Prepare 1 M pyruvate solution separately, sterilize separately, and adjust the pH to 7.0-7.2 with 1 M NaOH solution before sterilization.

[0400] Before use, if an external pyruvate carbon source is needed, add 6 ml of 1 M pyruvate solution to the above-mentioned approximately 100 ml of the modified Stephenson medium with arginine as the nitrogen source. The designed concentration of pyruvate in the culture system is approximately 0.06 M.

[0401] 1.3 Culture system and indicator determination

[0402] Set up 2 processing:

[0403] (1) Treatment with arginine as the sole carbon and nitrogen source (arginine medium): that is, only A in the above medium is used.

[0404] (2) Treatment with arginine as the carbon and nitrogen sources and pyruvate as the external carbon source (arginine + pyruvate medium): that is, the above-mentioned medium A + B.

[0405] 1 ml of PBS-JD / YT bacterial solution was inoculated into the above two culture media respectively, and each treatment was treated with a non-inoculated culture medium as a control. The culture was placed in a shaking incubator at a speed of 180 rpm and a constant temperature of 28°C.

[0406] After a certain period of culture, the OD of the bacterial solution was measured. 600value, pH, and simultaneously determine NO2 in the supernatant - -N、NH4 + -N and NO x - -N content.

[0407] 1.4 NOS gene testing

[0408] PCR amplification was performed on YT and JD bacterial cultures cultured under L-arginine + pyruvate to detect their nitric oxide synthase (NOS) genes.

[0409] PCR was performed using TransStart Fastpfu DNA Polymerase in a 20 μl reaction system:

[0410] 5×FastPfu Buffer............4μl

[0411] 2.5mM dNTPs........................2μl

[0412] Forward Primer(5μM)............0.8μl

[0413] Reverse Primer(5μM).........0.8μl

[0414] FastPfu Polymerase............0.4μl

[0415] Template DNA........................10ng

[0416] Add ddH2O to 20μl

[0417] PCR instrument: ABI Type 9700

[0418] PCR reaction parameters:

[0419] a.1×(5min at 95℃)

[0420] b.cycles×(30s at 95℃; 30s at Tm℃; 45s at 72℃)

[0421] c.10min at 72℃,10℃ until halted by user

[0422] The primer sequences are as follows:

[0423] nosF:5'-CGM(C / T)TGT TCN(A / C)TCG ACA GCC AG-3'(SEQ ID NO: 9)

[0424] nosR:5'-CAT GTG CAG D(A / C / G / T)GC H(A / G)TG GCA GAA-3'(SEQ ID NO: 10)

[0425] 2 Results

[0426] 2.1 Growth and nitrification activity

[0427] The results of the culture experiment using L-arginine as the only carbon and nitrogen source showed that both strains JD and YT did not grow, the pH of the culture medium did not change, and the qualitative detection of NO2 in the culture medium - -N、NH4 + -N and NO x - -N content was negative, showing no change. From external observation, the color and morphology of the culture medium also showed no change (as shown in the two left columns of Figure 9a).

[0428] As shown in the table below and the two right columns of Figure 9a, strains JD and YT were inoculated in L-arginine + pyruvate medium and grew rapidly. The pH increased significantly during the growth process. After 18 days of growth in medium with pyruvate as the carbon source, the culture medium NH4 + -N concentrations were 59.27 mg / L and 56.88 mg / L, respectively. Compared with the control, NH4 + -N concentration increased significantly.

[0429] Using pyruvate as carbon source, JD and YT have strong nitrification effect, and NO2 in the culture medium - -N and NO x - -N accumulates in large quantities: After 18 days of culture, NO2 - -N concentrations were 122.0 mg / L and 136.55 mg / L, respectively, and NO x - -N concentrations were 198.67 mg / L and 221.0 mg / L respectively.

[0430] Table 9 Growth and nitrification activity of strains JD and YT inoculated in arginine + pyruvate medium (cultured for 18 days) Note: The data in the table are averages, and the number of repetitions n=4

[0431] 2.2 PCR amplification of nitric oxide synthase

[0432] Through genetic detection and analysis of the culture system YT and JD bacterial liquid, according to the PCR amplification results, strong target bands were found in both JD and JT, as shown in Figure 9b (M is the standard, 1 is JD, 2 is YT, and CK is the control), indicating that nitric oxide synthase NOS is present in both JD and YT.

[0433] 3 Conclusion

[0434] (1) The mycobacteria of the present invention do not grow in a culture medium containing L-arginine as the sole carbon and nitrogen source, and no nitrification activity occurs, indicating that the arginine hydrolysis pathway does not exist.

[0435] (2) When cultured with L-arginine as the nitrogen source and pyruvate as the carbon source, the mycobacterium of the present invention grew vigorously, accumulated a large amount of nitrite nitrogen and nitrate nitrogen, and had a strong nitrification effect of the test strain, indicating that the L-arginine oxidation pathway actually existed.

[0436] (3) The mycobacterium of the present invention utilizes arginine and has a strong nitrification effect, and there is nitric oxide synthase, which proves that the arginine biosynthesis pathway is one of the pathways of nitrogen metabolism of the strain.

[0437] Example 9

[0438] Aerobic nitrite oxidation and assimilative nitrate reduction of mycobacteria of the present invention

[0439] 1 Materials and Methods

[0440] 1.1 Culture medium

[0441] On the basis of the modified Stephenson medium, the nitrogen source ammonium sulfate is replaced with an equal amount of sodium nitrite (content is 0.25g / L), and the other components and treatments remain unchanged, that is, the modified Stephenson medium with sodium nitrite as the nitrogen source.

[0442] The carbon source is sodium pyruvate. First, prepare a 0.4M sodium pyruvate solution and sterilize it separately. Before use, take 10ml of the 0.4M sodium pyruvate solution and add it to 100ml of modified Stephenson medium with sodium nitrite as the nitrogen source, so that the designed pyruvate concentration of the medium is about 0.04M.

[0443] 1.2 Culture medium: PBS-JD / YT bacterial solution

[0444] 1.3 Culture system and conditions

[0445] Inoculate 1 ml of PBS-JD / YT into modified Stephenson's medium containing sodium nitrite as the nitrogen source. For the control, add 1 ml of sterile water without inoculation. The experimental design was repeated three times for each measurement.

[0446] The culture was placed in a shaking incubator at a rotation speed of 180 rpm, a temperature of 28°C, a CO2 content of 5%, and a RH of 50%.

[0447] 1.4 Measurement indicators

[0448] Starting from the 8th day of culture, samples were taken every 2 days for analysis, with 3 parallel samples taken each time. 600 ), pH value and NH4 + -N, NO2 - -N, NO x - -N, TN content. NO X - -N content and NO2 - -The difference in N content is NO3 - -N.

[0449] After the culture was completed, the dry weight of the bacteria and its nitrogen content (N%) and carbon content (C%) were determined.

[0450] 2 Results

[0451] Figure 10a shows the growth of JD (left) and YT (right) under aerobic culture conditions with sodium nitrite as the nitrogen source and the changes in the pH of the culture medium. Both JD and YT can grow, and the pH of the culture medium changes accordingly with the growth of the strains. In the early stage of culture, the growth of both strains was extremely slow, especially around the 8th day of culture, when the OD 600 The changes are very small.

[0452] Although JD and YT strains grew very slowly in the early stage of culture, the NO2 - -N content is significantly reduced, NO3 - -N content increased significantly. As shown in Table 10a, after 8 days of cultivation, the NO2 - The average content of -N decreased from the initial 50mg / L to 15mg / L, and NO3 - -N appears and reaches about 50 mg / L, and nitrite is oxidized to nitrate.

[0453] On the 14th day of culture, NO2 - -N, NO3 - -N concentrations were 0.36 mg / L and 1.51 mg / L, respectively, and NO2 - -N, NO3 - -N concentrations were 0.30 mg / L and 1.54 mg / L, respectively, and both forms of nitrogen basically disappeared; at the same time, a certain amount of NH4 appeared in the culture medium. +-N but the concentration is not high. As for TN, it was determined that the TN content of the two culture systems did not change significantly in the early stage of culture (i.e., the stage of nitrite oxidation to nitrate), accompanied by the rapid growth of the bacteria and NO2 - -N, NO3 - The rapid decrease in -N concentration and TN concentration (Figure 10b) showed that the TN concentrations in the culture medium were 4.29 mg / L and 4.58 mg / L after 14 days of cultivation, indicating that nitrate reduction occurred during the above stages.

[0454] Table 10a NH4 in pure culture of JD and YT using sodium nitrite as nitrogen source + -N, NO2 - -N and NO3 - -N concentration changes

[0455] JD and YT bacterial cells grew during nitrate reduction. After 14 days of culture, the cultures were centrifuged, and the resulting cells were freeze-dried in a vacuum, weighed, and their nitrogen and carbon contents were determined. The nitrogen contents of JD and YT cells were approximately 6.5% and 7.4%, respectively (Table 10b). The amount of nitrogen converted to the cells was calculated based on the dry weight and nitrogen content. These results indicate that the organic nitrogen content in the cells at the end of culture was consistent with the amount of nitrogen source added to the culture medium, indicating that the nitrate reduction pathway is assimilation.

[0456] Table 10b JD and YT cell weight and nitrogen content

[0457] 3 Conclusion

[0458] The mycobacterium of the present invention oxidizes nitrite to nitrate under the condition of shaking culture with sodium nitrite as nitrogen source; during the culture process, the bacteria grow, inorganic nitrogen in the culture medium is basically synthesized into cells, and assimilated nitrate reduction occurs.

[0459] Example 10

[0460] Anaerobic denitrification (dissimilatory nitrate reduction) by mycobacteria of the present invention

[0461] 1 Materials and Methods

[0462] 1.1 Culture medium: PBS-JD bacterial solution.

[0463] 1.2 Culture medium:

[0464] On the basis of the modified Stephenson medium, the nitrogen source ammonium sulfate is replaced with an equal amount of sodium nitrite (content is 0.25g / L), and the other components and treatments remain unchanged, that is, the modified Stephenson medium with sodium nitrite as the nitrogen source.

[0465] The carbon source is sodium pyruvate. First, prepare a 0.4M sodium pyruvate solution and sterilize it separately. Before use, take 10ml of the 0.4M sodium pyruvate solution and add it to 100ml of modified Stephenson medium with sodium nitrite as the nitrogen source, so that the designed pyruvate concentration of the medium is about 0.04M.

[0466] 1.3 Training System

[0467] 1 ml of PBS-JD was inoculated into modified Stephenson's medium containing sodium nitrite as the nitrogen source. A control was left uninoculated and 1 ml of sterile water was added. The experimental design was repeated three times for each measurement.

[0468] 1.4 Anaerobic environment treatment and culture conditions: The culture system of this experiment needs to ensure that there is no O2 and N2 in the bottle. The specific operation is: Place the culture bottle containing the culture system in a vacuum device and evacuate for 10 minutes, then introduce helium for 15 seconds, and repeat the above operation 6 times; during the vacuuming process, in order to ensure pure culture, a 0.25μm filter membrane must be added to prevent contamination by miscellaneous bacteria.

[0469] After the air pressure is balanced, apply sealant on the air extraction hole and place the culture in a shaking incubator at a speed of 180 rpm and a temperature of 28°C.

[0470] 1.5 Measurement indicators

[0471] Starting from the 8th day of culture, samples were taken every 2 days for analysis, with 3 parallel samples taken each time. 600 ), pH value and NH4 + -N, NO2 - -N, NO3 - -N(NO X - -N content and NO2 - -N content difference), TN content, and determination of N2, CO2, and N2O content.

[0472] 2 Results

[0473] Culture system NO2 - The initial content of -N was about 50 mg / L. When JD was cultured to the 6th day, the NO2 - -N concentration dropped to 0.5mg / L, NH4 + -N, NO x - -N content was lower than 0.5 mg / L, and TN content was about 2.0 mg / L; at this time, the pH of the culture medium was 7.18, and OD 600When the concentration was 0.005, there was almost no bacterial growth (Figure 11). Analysis of the gas content in the culture system revealed accumulation of CO2, N2, and N2O in the culture flask (see table below). This indicates that JD is undergoing anaerobic denitrification and dissimilatory nitrate reduction.

[0474] Table 11 Changes of three gases in the culture system with culture time

[0475] 3 Conclusion

[0476] Using nitrite as nitrogen source, under anaerobic conditions, the culture medium NO2 - -N, NO3 - -N almost disappears, and N2, N2O, and CO2 gases are produced. The mycobacteria of the present invention undergo anaerobic denitrification, while no bacterial growth is observed, and dissimilatory nitrate reduction occurs.

[0477] Example 11

[0478] Ammonia oxidation direct denitrification by mycobacteria of the present invention

[0479] 1 Materials and Methods

[0480] 1.1 Culture medium: PBS-JD bacterial solution

[0481] 1.2 Culture medium: Modified Stephenson's medium formula, adjusted to pH 6.4, the volume of each bottle of culture medium is 50 ml, and sterilized at high temperature and high pressure for 20 minutes before use.

[0482] The carbon source is sodium pyruvate. First, prepare a 0.2M sodium pyruvate solution (concentration based on pyruvate), adjust the pH to 6.4, and sterilize it separately for later use. During culture, take 2.5ml of the 0.2M sodium pyruvate solution and add it to 50ml of modified Stephenson's medium to make the designed concentration of sodium pyruvate 0.01M.

[0483] 1.3 Training System

[0484] Inoculate 1 ml of the PBS-JD bacterial solution into the modified Stephenson's medium (pH 6.4) containing sodium pyruvate as the carbon source. For the control, add 1 ml of sterile water without inoculation. The experimental design was designed with three replicates per measurement.

[0485] The culture bottle needs to be covered with a sterilized rubber stopper and pressed tightly to maintain a closed environment.

[0486] 1.4 Closed environment culture conditions

[0487] The above culture systems were placed in a vacuum device and evacuated for 10 minutes. A mixture of 20% oxygen and 80% helium was then introduced for 15 seconds. This process was repeated six times. During the vacuuming process, a 0.25 μm filter was used to ensure pure culture to prevent contamination by other bacteria.

[0488] After the air pressure is balanced, apply sealant on the air extraction hole and place the culture in a shaking incubator at a temperature of 28°C and a rotation speed of 180 r / min.

[0489] 1.5 Index determination

[0490] Samples were taken every 2 days for analysis, with 3 parallel samples taken each time. 600 ), pH value, take the culture supernatant to determine NH4 + -N, NO2 - -N, NO x - -N and TN content, determine the content of four gases N2, CO2, O2, and N2O in the culture system, and determine the pyruvate concentration in the culture medium.

[0491] 2 Results

[0492] Figure 12a shows the growth of JD and the changes in the pH of the liquid culture medium during culture. JD grew rapidly after 7 days of culture, and the pH also increased. After 11 days of culture, the pH was 6.73.

[0493] As shown in Figure 12b, the qualitative observation of NO2 during the culture process - -N changes, no color was developed when titrated with Gliese reagent, indicating that there was no NO2 in the culture medium - -N accumulation; simultaneous quantitative determination of NO2 - -N, NO x - -N content did not increase compared with the control, and NH4 + The concentrations of -N and TN decreased, indicating that under this culture condition, JD oxidized ammonia but did not nitrify.

[0494] At the same time, the concentration changes of four gases, CO2, O2, N2, and N2O, in the culture system were measured. Table 12 summarizes the changes in the concentrations of CO2, O2, N2, and N2O in the culture system during JD cultivation compared to the control. Figures 12c and 12d show the changes in gas concentrations in the JD system over time. Analysis showed that as the culture time increased, the O2 concentration in the JD culture system decreased, while the CO2 concentration gradually increased. N2 and N2O were clearly present in the JD culture system, and N2O gas in particular showed an increasing cumulative amount with increasing culture days. This indicates that under these culture conditions, during JD metabolism, pyruvate is oxidized to CO2, and ammonia is oxidized to N2 and N2O.

[0495] Table 12 Changes in gas concentration in the culture system Note: (1) + indicates an increase compared to the control; - indicates a decrease compared to the control. (2) The data in the table are mean values, n = 3.

[0496] 3 Conclusion

[0497] The nitrogen metabolism of the mycobacterium of the present invention has a pathway of directly denitrifying by oxidizing ammonia.

[0498] Example 12

[0499] Inhibition of nitrification activity of mycobacteria of the present invention by nitrification inhibitors

[0500] 1 Materials and Methods

[0501] 1.1 Culture medium: PBS-YT bacterial solution.

[0502] 1.2 Culture medium

[0503] The modified Stephenson medium ((NH4)2SO4 content 2.0g / L) with (NH4)2SO4 as nitrogen source and L-arginine hydrochloride (C6H 14 N4O2·HCl) as the nitrogen source of the modified Stephenson medium (C6H 14 N4O2·HCl content is 1.6g / L), and the nitrogen amounts of the two nitrogen sources are equal.

[0504] When sodium pyruvate is added as a carbon source in the culture medium, first prepare a 1.0 M sodium pyruvate solution (concentration based on pyruvate), adjust the pH to 7.0-7.2, and sterilize it separately for later use. Before use, add 1 ml of 1.0 M sodium pyruvate solution to approximately 100 ml of modified Stephenson medium with (NH4)2SO4 as the nitrogen source or L-arginine hydrochloride (C6H 14 In the modified Stephenson medium with N4O2·HCl) as the nitrogen source, the pyruvate concentration of the culture system was designed to be 0.01M.

[0505] 1.3 Nitrification inhibitors

[0506] Four nitrification inhibitors were selected: 4-amino-1,2,4-triazole (ATC), dicyandiamide (DCD, C2H4N4), aminoguanidine hydrochloride (CH6N4·HCl), and guanylthiourea (C2H6N4S). Each of these inhibitors was prepared at a concentration of 500 mM. After filtration sterilization, 1 ml of each 500 mM solution was added to the culture media containing the two different nitrogen sources, ensuring a 5 mM concentration for each system.

[0507] 1.4 Training system setting

[0508] Two different nitrogen source cultures were used in five treatments (no inhibitor, ATC, DCD, aminoguanidine hydrochloride, and guanylthiourea) as shown in the table below. A YT culture without inhibitors and a YT culture without inhibitors served as controls. Each culture was inoculated with 1 ml of PBS-YT solution; when not inoculated, 1 ml of sterile water was used instead. The total volume of each culture was approximately 100 ml.

[0509] Table 13a

[0510] At the same time, we also designed culture media without any nitrogen source, but with four inhibitors as nitrogen sources and pyruvate as carbon source to observe its growth and nitrogen metabolism.

[0511] 1.5 Culture conditions: shaker at 180 rpm and 28°C.

[0512] 1.6 Index determination and calculation: Measure the bacterial growth OD of the culture medium every week 600 , pH value, determination of NH4 + -N, NO2 - -N, NO x - -N content, calculate and compare the inhibitory effects of nitric oxide synthase (nitrification) activity in different culture systems.

[0513] Nitric oxide synthase (nitrification) inhibition rate (%) = (NO2 - -N YT Concentration-NO2 - -N 添加抑制剂培养体系 concentration)×100 / NO2 - -N YT concentration

[0514] 2 Research results

[0515] 2.1 Effects of aminoguanidine hydrochloride and guanylthiourea on YT growth and nitrification activity

[0516] When YT was cultivated with ammonium sulfate and L-arginine hydrochloride as nitrogen sources, it could undergo nitrification, and the NO2 - -N accumulation gradually increased. The NO2 in the culture system with ammonium sulfate as the nitrogen source for 35 days without adding any nitrification inhibitors - -N concentration was 6.88 mg / L; after adding aminoguanidine hydrochloride, the NO2 - -N concentrations were lower than those in the system without adding inhibitors; after adding guanylthiourea, the NO2 - The -N concentrations in the culture medium were lower than those in the system without inhibitors. This indicates that aminoguanidine hydrochloride inhibits the nitrification activity of YT, and guanylthiourea also has an inhibitory effect on nitrification, but the inhibitory effect gradually weakens with the extension of the culture time. See the table below for details.

[0517] The NO2 in the culture system without adding any nitrification inhibitors after pure culture with L-arginine hydrochloride as nitrogen source for 7 days, 14 days, 21 days and 35 days was - The concentrations of -N were 0, 101.59, 113.72, and 113.65 mg / L, and the NO2 - -N concentrations were lower than those in the system without adding inhibitors. The NO2 - -N concentrations were lower than those in the system without inhibitors. This indicates that both aminoguanidine hydrochloride and guanylthiourea inhibited the nitrification activity of YT. The nitrification inhibitory effect of aminoguanidine hydrochloride remained significant over 35 days, while the nitrification inhibitory effect of guanylthiourea weakened with prolonged incubation. See the table below for details.

[0518] Table 13b Effects of aminoguanidine hydrochloride and guanylthiourea on YT nitration activity

[0519] As shown in Figures 13a and 13b, YT was pure cultured with ammonium sulfate (Figure 13a) and L-arginine hydrochloride (Figure 13b) as two nitrogen sources. When YT was pure cultured with L-arginine hydrochloride as the nitrogen source, its growth was faster than when it was pure cultured with ammonium sulfate as the nitrogen source. The addition of aminoguanidine hydrochloride inhibited the growth of YT under pure culture with both nitrogen sources, and the inhibitory effect remained obvious during the culture period. The addition of guanylthiourea significantly inhibited the growth of YT under pure culture with both nitrogen sources in the early stage of culture, and the growth inhibition with ammonium sulfate as the nitrogen source weakened after 14 days of culture.

[0520] 2.2 Effects of ATC and DCD on YT growth and nitrification activity

[0521] First, as shown in the table below, the growth and nitrification activity of YT were stronger when L-arginine hydrochloride was used as nitrogen source than when ammonium sulfate was used as nitrogen source; - -N concentration analysis showed that ATC consistently inhibited the nitrification activity of YT under both nitrogen sources, while DCD did not. On the other hand, both nitrification inhibitors, ATC and DCD, did not inhibit the growth of YT.

[0522] Table 13c Effects of ATC and DCD on the growth and nitrification activity of pure cultured YT on two nitrogen sources

[0523] 2.4 Comparison of the inhibitory effects of different inhibitors on YT nitric oxide synthase (nitrification) activity

[0524] As shown in the table below, aminoguanidine hydrochloride inhibited YT nitration activity by over 99% using L-arginine hydrochloride as the nitrogen source, and over 97% using ammonium sulfate as the nitrogen source. Guanylthiourea inhibited YT nitric oxide synthase (nitration) activity by over 95% before day 14 of culture using both nitrogen sources, but the inhibition rate decreased thereafter, dropping to 11% after 35 days of culture using ammonium sulfate and 72% after 35 days of culture using L-arginine hydrochloride as the nitrogen source.

[0525] Table 13d Inhibitory effect of aminoguanidine hydrochloride and guanylthiourea on YT nitric oxide synthase (nitration) activity Note: * in the table indicates that no nitrification occurred in the control group without inhibitors and the treatment group with inhibitors

[0526] ATC significantly inhibited YT nitric oxide synthase (nitrification) activity, almost completely inhibiting it when L-arginine hydrochloride was used as the nitrogen source. However, the inhibitory effect was weaker when ammonium sulfate was used as the nitrogen source, showing a decreasing trend with incubation time. DCD had little effect on YT nitric oxide synthase (nitrification) activity (see Table 13e).

[0527] Table 13e Inhibitory effect of ATC and DCD on YT nitric oxide synthase (nitrification) activity

[0528] 2.5 Growth and metabolism of YT in pure culture with four nitrification inhibitors as nitrogen source

[0529] When YT was cultured purely with the four nitrification inhibitors as nitrogen sources and pyruvate as carbon source, it was able to grow. At the same time, the TN content in the culture medium was reduced compared with the initial content of the culture medium before inoculation, indicating that the nitrification inhibitors were involved in nitrogen metabolism ( Figure 13c ).

[0530] 3 Conclusion

[0531] (1) The four selected nitrification inhibitors can all serve as alternative nitrogen source substrates, enabling YT to grow with arginine as the sole nitrogen source.

[0532] (2) Aminoguanidine hydrochloride inhibited the growth and nitrification activity of YT; guanylthiourea inhibited the nitrification activity of YT, and the effect was obvious in the early stage of culture. As the culture time increased, the inhibitory effect on nitrification activity weakened; ATC inhibited the nitrification activity of YT significantly, but did not inhibit its growth; DCD had no obvious inhibitory effect on the growth and nitrification activity of YT.

[0533] (3) The growth and nitrification activity of YT were stronger when L-arginine hydrochloride was used as the nitrogen source than when ammonium sulfate was used as the nitrogen source.

[0534] (4) Nitration inhibitors actually inhibit the activity of nitric oxide synthase.

[0535] Example 13

[0536] Antagonistic effect of the mycobacterium of the present invention on other mycobacteria

[0537] 1 Materials and Methods

[0538] 1.1 Test strains: YT obtained in Preparation Example 1; Mycobacterium austroafricanum (Deposit No. CMCC(B)95044), Mycobacterium vaccae (ATCC No. 15483 TM ), Mycolicibacterium vanbaalenii (DSM 7251) standard strain.

[0539] The above strains were all prepared into PBS solutions.

[0540] 1.2 LB plate: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, agar powder 20 g / L, pH 7.2.

[0541] 1.3 Experimental methods

[0542] There are two methods:

[0543] The first method is the LB plate streak method. Specific steps: Evenly streak two or three test standard strains onto the same LB plate, then streak YT between each pair. Place the plate in an incubator at 28°C and grow for one week. Observe the growth of the test strains on each plate.

[0544] The second method is the KB method, also known as the filter paper method. Specific steps: (1) Select filter paper with uniform texture and use a punch to make several discs of the same diameter (6 mm in diameter). After sterilization and drying, take a certain number of discs and soak them in PBS-YT solution for use; (2) Take a loop of each of the three standard strains and add them to three 100 ml PBS Erlenmeyer flasks, then take 200 μl and evenly spread them on different LB plates to form test plates; (3) Place the filter paper discs soaked in PBS-YT solution on the test plates, with 3 pieces of the filter paper placed on each test plate, and invert them after 5 minutes; (4) Use the test plate with filter paper soaked in physiological saline as a control; (5) Place the treated plates in an incubator and culture them at 28°C for 1 week. After taking them out, measure the size of the inhibition zone and photograph the morphology of the inhibition zone with a Nikon AZ100 stereoscope.

[0545] 2 Results

[0546] When Mycobacterium vaccae and Mycobacterium van Barensis were inoculated on an LB plate and YT was streaked between the two standard strains, both Mycobacterium vaccae and Mycobacterium van Barensis strains were clearly observed to spread and grow toward the side not inoculated with YT. When the three standard strains were streaked on an LB plate and YT was inoculated between Mycobacterium serrata and Mycobacterium van Barensis, and between Mycobacterium van Barensis and Mycobacterium vaccae, Mycobacterium serrata and Mycobacterium vaccae on both sides of the LB plate spread and grow toward the side not inoculated with YT, while Mycobacterium van Barensis in the middle did not spread and grow (see Figure 14a).

[0547] After one week of culture using the KB method, electron microscopic observation revealed a zone of inhibition exceeding 6 mm in diameter formed at the location on the LB plate where the YT-soaked filter paper had been applied. This zone showed no growth of the three standard strains of Mycobacterium spp. (M. vaccae, M. africanum, and M. vaccae). Furthermore, application of Gliese's reagent to the filter paper revealed a pink color, indicating nitrification, as shown in Figure 14b (with the YT-soaked filter paper still applied; three LB plates were coated with M. van Baren, M. vaccae, and M. spp., from left to right) and Figure 14c (with the YT-soaked filter paper removed). In contrast, the three standard strains grew uniformly on the control plate with the filter paper not soaked in YT. This demonstrates that YT has an antagonistic effect on other mycobacteria.

[0548] 3 Conclusion

[0549] The mycobacterium of the present invention has an antagonistic effect on mycobacteria including van Baren mycobacterium, vaccae mycobacterium and south africa mycobacterium.

[0550] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

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Claims

1. Mycobacterium sp., characterized in that The deposit number of the mycobacterium is CGMCC No. 21272 or CGMCC No. 21273.

2. A bacterial agent, characterized in that The bacterial agent contains the mycobacterium according to claim 1.

3. A denitrification method, characterized in that: The method comprises: contacting the mycobacterium according to claim 1 or the bacterial agent according to claim 2 with a system to be denitrified.

4. The method according to claim 3, wherein: In the system to be denitrified, N is at least one of molecular nitrogen, ammonia nitrogen, nitrate nitrogen and nitrite nitrogen; And / or, the system to be denitrified is wastewater, waste gas or waste residue.

5. A method for controlling nitrogen pollution, characterized in that: The method comprises: contacting the mycobacterium according to claim 1 or the bacterial agent according to claim 2 with a nitrogen-containing pollutant.

6. A method for capturing CO2, characterized in that: The method comprises: contacting the mycobacterium according to claim 1 or the bacterial agent according to claim 2 with a system containing CO2.

7. A method for screening agricultural chemicals, characterized in that: The method comprises: inoculating the mycobacterium according to claim 1 into an ammonia nitrogen culture medium containing the agricultural chemical to be screened for culturing, and determining whether the agricultural chemical to be screened has the function of inhibiting ammonia nitrogen loss based on changes in N and / or the growth status of the mycobacterium during the culturing process.

8. Use of the mycobacterium according to claim 1 or the bacterial agent according to claim 2 in nitrogen pollution control, carbon capture, screening of agricultural chemicals, treatment and / or prevention of skin disorders, treatment and / or prevention of diseases associated with low nitrite levels, treatment and / or prevention of body odor, treatment for supplying nitric oxide to a subject, or inhibition of microbial growth.

9. The use according to claim 8, wherein: The agricultural chemical is a nitrification inhibitor and / or a nitric oxide synthase inhibitor; and / or, the inhibition is in vitro inhibition; and / or, the use is for preparing a medicament or preparation for the treatment and / or prevention of skin disorders, the treatment and / or prevention of diseases associated with low nitrite levels, the treatment and / or prevention of body odor, the supply of nitric oxide to a subject, or the inhibition of microbial growth; And / or, the microorganism is a Mycobacterium microorganism, more preferably Mycobacterium vaccae and / or Mycobacterium van Barensis.

10. A method for isolating mycobacteria having nitrification activity, characterized in that: The method comprises: (1) preliminarily screening mycobacteria with nitrification activity; and (2) further enriching mycobacteria with high nitrification activity using a culture medium supplemented with antibiotics.

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