A strain of low-temperature-resistant Halomonas expectans LJ2 with complete denitrification function and its application, product and method

Abstract

The present invention "a strain of Halomonas desiderata LJ2 that is resistant to low temperatures and has complete denitrification function and its application, product and method" belongs to the field of microbial technology. The present invention provides a strain of Halomonas desiderata LJ2, with a preservation number of CGMCC NO: 24345. The present invention also provides the application of the strain LJ2 in denitrification, as well as a bacterial agent and a denitrification method based on the strain LJ2. The LJ2 strain of the present invention has good salt tolerance, low temperature tolerance, complete denitrification ability and high nitrate and nitrite removal ability.

Description

Technical Field

[0001] The invention belongs to the field of microbial technology, and particularly relates to a strain of low-temperature-resistant Halomonas spp. LJ2 with complete denitrification function and an application, product and method thereof. Background Art

[0002] Nitrogen pollution is one of the most serious environmental problems in the 21st century. In recent decades, the global input of active nitrogen has been increasing. Excessive nitrite in water bodies has a strong toxic effect on aquatic organisms and humans. Wastewater discharged from industries such as petroleum, chemical industry, fertilizer, and marine aquaculture often contains a large amount of nitrite and has high salt characteristics. Traditional biological methods have advantages in treating low-salinity nitrogen-containing wastewater, but when the salt concentration of wastewater is too high, it will inhibit the metabolism of denitrifying microorganisms. In coastal wetland ecosystems, the microenvironment provided by the rhizosphere effect of plants has a positive effect on the enrichment and activity of microorganisms. Active microorganisms gather in large numbers in the rhizosphere of plants, which plays an important role in improving denitrification efficiency and preventing eutrophication of water bodies. Greenhouse gas nitrous oxide is produced during denitrification, so it is currently very important to screen microorganisms with complete denitrification functions, promote the complete denitrification process, and reduce greenhouse gas emissions in the intermediate process.

[0003] Halomonas desiderata is a microorganism of the genus Halomonas. The bacteria are rod-shaped and motile. When grown on an M2 plate at 25°C for 1 week, the colonies are milky white. The surface is smooth and moist, opaque, slightly convex, with neat edges and no halos, and a diameter of 0.5-1mm. The main use is research, and the specific use is potential organic pollutant degrading bacteria / isolated from petroleum-enriched bacterial flora.

[0004] There is no report on the desired Halomonas with complete denitrification ability under high salt and low temperature conditions in the prior art. At the same time, the art is in urgent need of developing a desired Halomonas with high salt tolerance, low temperature tolerance and complete denitrification ability. Summary of the invention

[0005] Based on the above-mentioned gaps and needs in the prior art in the art, the present invention provides a cold-resistant Halomonas desiderata strain LJ2 having both complete denitrification function and salt and cold-resistant characteristics, and its applications, products and methods.

[0006] The technical solution of the present invention is as follows:

[0007] A low-temperature-resistant Halomonas desiderata strain LJ2 with complete denitrification function, characterized in that its deposit number is CGMCC NO:24345.

[0008] The cold-resistant Halomonas desiderata strain LJ2 with complete denitrification function contains the gene encoding membrane-bound nitrate reductase narG, the gene encoding periplasmic nitrate reductase napA, the gene encoding Cu-type nitrite reductase nirK, the gene encoding cytochrome cd1-type nitrite reductase nirS, the gene encoding nitric oxide reductase qnorB and the gene encoding nitrous oxide reductase nosZ;

[0009] Preferably, the complete denitrification includes: the membrane-bound nitrate reductase and periplasmic nitrate reductase of strain LJ2 reduce nitrate to nitrite; the Cu-type nitrite reductase and cytochrome cd1-type nitrite reductase of strain LJ2 reduce nitrite to nitric oxide; the nitric oxide reductase of strain LJ2 reduces nitric oxide to nitrous oxide; and the nitrous oxide reductase of strain LJ2 reduces nitrous oxide to nitrogen gas.

[0010] Preferably, the low temperature resistance means that the Halomonas desiderata strain LJ2 is expected to have complete denitrification function in an environment below 15°C.

[0011] The invention discloses an application of a cold-resistant Halomonas desiderata strain LJ2 with complete denitrification function and the deposit number is CGMCC NO: 24345 in complete denitrification.

[0012] The complete denitrification comprises: the membrane-bound nitrate reductase narG and the periplasmic nitrate reductase napA of the strain LJ2 reduce nitrate to nitrite; the Cu-type nitrite reductase nirK and the cytochrome cd1-type nitrite reductase nirS of the strain LJ2 reduce nitrite to nitric oxide; the nitric oxide reductase qnorB of the strain LJ2 reduces nitric oxide to nitrous oxide; and the nitrous oxide reductase nosZ of the strain LJ2 reduces nitrous oxide to nitrogen gas.

[0013] The complete denitrification means that under high salt conditions, the membrane-bound nitrate reductase, periplasmic nitrate reductase, Cu-type nitrite reductase, cytochrome cd1-type nitrite reductase, nitric oxide reductase, and nitrous oxide reductase of strain LJ2 sequentially carry out the following reactions:

[0014] Membrane-bound nitrate reductase, periplasmic nitrate reductase reduces nitrate to nitrite;

[0015] Cu-type nitrite reductase and cytochrome cd1-type nitrite reductase reduce nitrite to nitric oxide;

[0016] Nitric oxide reductase reduces nitric oxide to nitrous oxide;

[0017] Nitrous oxide reductase reduces nitrous oxide to nitrogen gas;

[0018] Preferably, the high salt condition refers to a NaCl concentration of 3%-15%.

[0019] Preferably, the temperature of the complete denitrification is 10-25°C.

[0020] A bacterial agent, characterized in that it comprises: a strain of Halomonas desiderata LJ2 with a deposit number of CGMCC NO: 24345.

[0021] The bacterial agent also includes auxiliary materials.

[0022] A complete denitrification method, characterized in that a cold-resistant Halomonas desiderata strain LJ2 with a complete denitrification function and a preservation number of CGMCC NO: 24345 is used to perform denitrification treatment on a sample to be treated.

[0023] The complete denitrification comprises: under high salt conditions, the membrane-bound nitrate reductase narG and the periplasmic nitrate reductase napA of the strain LJ2 reduce nitrate to nitrite; the Cu-type nitrite reductase nirK and the cytochrome cd1-type nitrite reductase nirS of the strain LJ2 reduce nitrite to nitric oxide; the nitric oxide reductase qnorB of the strain LJ2 reduces nitric oxide to nitrous oxide; the nitrous oxide reductase nosZ of the strain LJ2 reduces nitrous oxide to nitrogen gas;

[0024] Preferably, the complete denitrification means that under high salt conditions, the membrane-bound nitrate reductase, periplasmic nitrate reductase, Cu-type nitrite reductase, cytochrome cd1-type nitrite reductase, nitric oxide reductase, and nitrous oxide reductase of strain LJ2 sequentially perform the following reactions:

[0025] Membrane-bound nitrate reductase, periplasmic nitrate reductase reduces nitrate to nitrite;

[0026] Cu-type nitrite reductase and cytochrome cd1-type nitrite reductase reduce nitrite to nitric oxide;

[0027] Nitric oxide reductase reduces nitric oxide to nitrous oxide;

[0028] Nitrous oxide reductase reduces nitrous oxide to nitrogen gas;

[0029] Preferably, the denitrification treatment time is 1-5 days, and the temperature is 10-25°C;

[0030] Preferably, the high salt condition refers to a NaCl concentration of 3%-15%.

[0031] The inoculation amount of the strain LJ2 in the sample to be treated is 10%;

[0032] Preferably, the sample to be processed contains NO3 - and / or NO2 - of samples.

[0033] The present invention isolates a highly efficient denitrifying strain LJ2 with salt tolerance and low temperature tolerance from the rhizosphere of coastal plants, which uses nitrite as a nitrogen source. The strain belongs to Halomonas desiderata in taxonomy. Experiments have confirmed that the LJ2 strain of the present invention simultaneously has a membrane-bound nitrate reductase encoding gene narG, a periplasmic nitrate reductase encoding gene napA, a Cu-type nitrite reductase encoding gene nirK, a cytochrome cd1-type nitrite reductase encoding gene nirS, a nitric oxide reductase encoding gene qnorB and a nitrous oxide reductase encoding gene nosZ, so that it has complete denitrification ability. The bacterium has all the genes required for the complete denitrification process and can carry out the complete denitrification process, including membrane-bound nitrate reductase and periplasmic nitrate reductase for reducing nitrate to nitrite; Cu-type nitrite reductase and cytochrome cd1-type nitrite reductase for reducing nitrite to nitric oxide; nitric oxide reductase for reducing nitric oxide to nitrous oxide; and nitrous oxide reductase for reducing nitrous oxide to nitrogen gas. The denitrification process from nitrate to nitrogen gas can be completed by carrying out denitrification with the LJ2 strain of the present invention. At the same time, the LJ2 strain of the present invention has good salt tolerance, low temperature tolerance and nitrate and nitrite removal capabilities, has great potential in treating high-salt sewage under various temperature environments, can provide the possibility of removing nitrate and nitrite in high-salt wastewater, thoroughly carry out the denitrification process, and reduce greenhouse gas emissions (nitrous oxide).

[0034] The deposit information of the desired Halomonas desiderata strain LJ2 of the present invention is as follows:

[0035] Deposit number: CGMCC NO:24345;

[0036] Taxonomic nomenclature: Halomonas desiderata;

[0037] Deposit date: January 19, 2022;

[0038] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0039] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a line graph of the nitrate removal rate of the LJ2 strain under different salinity conditions in Experimental Example 2 of the present invention.

[0041] Figure 2 It is a line graph of the nitrite removal rate of the LJ2 strain under different salinity conditions in Experimental Example 3 of the present invention.

[0042] Figure 3 This is a line graph of the analysis of the gas component content in the complete denitrification process of the LJ2 strain in Experimental Example 4 of the present invention.

[0043] Figure 4 This is the growth curve of the LJ2 strain in Experimental Example 5 of the present invention under low temperature conditions.

[0044] Figure 5 This is the denitrification ability of the LJ2 strain in Experimental Example 5 of the present invention under low temperature conditions.

[0045] Figure 6 This is the amplification curve of the napA gene of the LJ2 strain in the sixth part of Experimental Example 1 of the present invention.

[0046] Figure 7 This is the amplification curve of the narG gene of the LJ2 strain in the sixth part of Experimental Example 1 of the present invention.

[0047] Figure 8 This is the amplification curve of the nirK gene of the LJ2 strain in the sixth part of Experimental Example 1 of the present invention.

[0048] Fig. 9 This is the amplification curve of the nirS gene of the LJ2 strain in the sixth part of Experimental Example 1 of the present invention.

[0049] Fig.10 This is the amplification curve of the qnorB gene of the LJ2 strain in the sixth part of Experimental Example 1 of the present invention.

[0050] Fig.11 This is the amplification curve of the nosZ gene of the LJ2 strain in the sixth part of Experimental Example 1 of the present invention.

[0051] Fig.12 Schematic diagram of the complete denitrification process of the LJ2 strain of the present invention. DETAILED DESCRIPTION

[0052] The content and technical effects of the present invention are further described in detail below in conjunction with specific embodiments and experimental examples, but the protection scope of the present invention is not limited thereto.

[0053] The first group of examples, strain LJ2 of the present invention

[0054] This group of embodiments provides a cold-resistant Halomonasdesiderata strain LJ2 with complete denitrification function. All embodiments in this group have the following common features: the deposit number of the strain LJ2 is CGMCC NO:24345.

[0055] In a specific embodiment, the strain LJ2 simultaneously contains the membrane-bound nitrate reductase encoding gene narG, the periplasmic nitrate reductase encoding gene napA, the Cu-type nitrite reductase encoding gene nirK, the cytochrome cd1-type nitrite reductase encoding gene nirS, the nitric oxide reductase encoding gene qnorB and the nitrous oxide reductase encoding gene nosZ.

[0056] In a preferred embodiment, the complete denitrification includes: the membrane-bound nitrate reductase and periplasmic nitrate reductase of strain LJ2 reduce nitrate to nitrite; the Cu-type nitrite reductase and cytochrome cd1-type nitrite reductase of strain LJ2 reduce nitrite to nitric oxide; the nitric oxide reductase of strain LJ2 reduces nitric oxide to nitrous oxide; and the nitrous oxide reductase of strain LJ2 reduces nitrous oxide to nitrogen gas.

[0057] In a preferred embodiment, the complete denitrification comprises: under high salt conditions, the membrane-bound nitrate reductase and periplasmic nitrate reductase of strain LJ2 reduce nitrate to nitrite; the Cu-type nitrite reductase and cytochrome cd1-type nitrite reductase of strain LJ2 reduce nitrite to nitric oxide; the nitric oxide reductase of strain LJ2 reduces nitric oxide to nitrous oxide; the nitrous oxide reductase of strain LJ2 reduces nitrous oxide to nitrogen gas;

[0058] Preferably, the complete denitrification means that under high salt conditions, the membrane-bound nitrate reductase, periplasmic nitrate reductase, Cu-type nitrite reductase, cytochrome cd1-type nitrite reductase, nitric oxide reductase, and nitrous oxide reductase of strain LJ2 sequentially perform the following reactions:

[0059] Membrane-bound nitrate reductase, periplasmic nitrate reductase reduces nitrate to nitrite;

[0060] Cu-type nitrite reductase and cytochrome cd1-type nitrite reductase reduce nitrite to nitric oxide;

[0061] Nitric oxide reductase reduces nitric oxide to nitrous oxide;

[0062] Nitrous oxide reductase reduces nitrous oxide to nitrogen gas.

[0063] Any use, use, sale, promise to sell, production, preparation, culture, propagation, cloning, fermentation of the desired Halomonas desiderata strain LJ2 with a preservation number of CGMCC NO: 24345 falls within the scope of protection of the present invention.

[0064] According to the teachings and inspiration of the present invention, those skilled in the art can select appropriate auxiliary materials for actual production needs in combination with commonly used technical means in the field of microbial technology, and prepare the desired Halomonas desiderata strain LJ2 with a deposit number of CGMCC NO: 24345 of the present invention into various dosage forms that meet various process production requirements, such as powders, tablets, liquids, etc.

[0065] Herein, the biocontrol bacteria, Halomonas desiderata LJ2, LJ2, strain LJ2, LJ2 strain, and Halomonas desiderata described in the content of the invention and the specific implementation methods all refer to: Halomonas desiderata strain LJ2 with the deposit number of CGMCC NO: 24345 of the present invention.

[0066] In a specific embodiment, the low temperature resistance means that the Halomonas desiderata strain LJ2 is expected to have complete denitrification function in an environment below 15°C.

[0067] The second group of examples, application of strain LJ2 of the present invention

[0068] This group of embodiments provides the application of a cold-resistant Halomonas desiderata strain LJ2 with a complete denitrification function and a deposit number of CGMCC NO: 24345 in complete denitrification.

[0069] In a preferred embodiment, the complete denitrification includes: the membrane-bound nitrate reductase and periplasmic nitrate reductase of strain LJ2 reduce nitrate to nitrite; the Cu-type nitrite reductase and cytochrome cd1-type nitrite reductase of strain LJ2 reduce nitrite to nitric oxide; the nitric oxide reductase of strain LJ2 reduces nitric oxide to nitrous oxide; and the nitrous oxide reductase of strain LJ2 reduces nitrous oxide to nitrogen gas.

[0070] In a more preferred embodiment, the complete denitrification refers to: Fig.10As shown, under high salt conditions, the membrane-bound nitrate reductase, periplasmic nitrate reductase, Cu-type nitrite reductase, cytochrome cd1-type nitrite reductase, nitric oxide reductase, and nitrous oxide reductase of strain LJ2 sequentially carried out the following reactions:

[0071] Membrane-bound nitrate reductase narG and periplasmic nitrate reductase napA reduce nitrate to nitrite;

[0072] Cu-type nitrite reductase nirK and cytochrome cd1-type nitrite reductase nirS reduce nitrite to nitric oxide;

[0073] Nitric oxide reductase qnorB reduces nitric oxide to nitrous oxide;

[0074] Nitrous oxide reductase nosZ reduces nitrous oxide to nitrogen gas.

[0075] In a specific embodiment, the complete denitrification refers to the removal of nitrate, nitrite, nitric oxide, and nitrous oxide under high salt conditions until they are finally completely converted into nitrogen gas;

[0076] Preferably, the high salt condition refers to a NaCl concentration of 3%-15%.

[0077] In a preferred embodiment, the temperature of complete denitrification is 10-25°C.

[0078] The third group of embodiments, the bacterial agent of the present invention

[0079] This group of embodiments provides a bacterial agent. All embodiments of this group have the following common features: the bacterial agent comprises: a cold-resistant Halomonas desiderata strain LJ2 with a deposit number of CGMCC NO: 24345 and having a complete denitrification function.

[0080] In a further embodiment, the bacterial agent further includes: auxiliary materials.

[0081] In a more specific embodiment, the pharmaceutical excipient is selected from: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculating agents, filter aids, release retardants, etc.

[0082] The dosage form of the bacterial agent of the present invention is not limited. According to the teachings and inspiration of the present invention, those skilled in the art select suitable excipients for preparation based on actual production needs, in combination with commonly used technical means in the field of microbial technology (for example, "Encyclopedia of Preparation Technology", "Pharmaceutical Preparation Technology", etc.), and prepare the desired Halomonas desiderata strain LJ2 with a deposit number of CGMCC NO: 24345 of the present invention into various other dosage forms that meet various process production requirements, such as powders, tablets, liquids, sprays, granules, capsules, etc.

[0083] The fourth group of embodiments, the complete denitrification method of the present invention

[0084] This group of embodiments provides a complete denitrification method. All embodiments in this group have the following common features: a strain of Halomonas desiderata LJ2 with a deposit number of CGMCC NO: 24345 is used to perform complete denitrification treatment on the treated sample.

[0085] In a specific embodiment, the complete denitrification includes: the membrane-bound nitrate reductase and periplasmic nitrate reductase of strain LJ2 reduce nitrate to nitrite; the Cu-type nitrite reductase and cytochrome cd1-type nitrite reductase of strain LJ2 reduce nitrite to nitric oxide; the nitric oxide reductase of strain LJ2 reduces nitric oxide to nitrous oxide; and the nitrous oxide reductase of strain LJ2 reduces nitrous oxide to nitrogen gas.

[0086] In a further embodiment, the complete denitrification means: under high salt conditions, the membrane-bound nitrate reductase and periplasmic nitrate reductase of strain LJ2 reduce nitrate to nitrite; the Cu-type nitrite reductase and cytochrome cd1-type nitrite reductase of strain LJ2 reduce nitrite to nitric oxide; the nitric oxide reductase of strain LJ2 reduces nitric oxide to nitrous oxide; the nitrous oxide reductase of strain LJ2 reduces nitrous oxide to nitrogen gas;

[0087] Preferably, the complete denitrification means that under high salt conditions, the membrane-bound nitrate reductase, periplasmic nitrate reductase, Cu-type nitrite reductase, cytochrome cd1-type nitrite reductase, nitric oxide reductase, and nitrous oxide reductase of strain LJ2 sequentially perform the following reactions:

[0088] Membrane-bound nitrate reductase, periplasmic nitrate reductase reduces nitrate to nitrite;

[0089] Cu-type nitrite reductase and cytochrome cd1-type nitrite reductase reduce nitrite to nitric oxide;

[0090] Nitric oxide reductase reduces nitric oxide to nitrous oxide;

[0091] Nitrous oxide reductase reduces nitrous oxide to nitrogen gas.

[0092] In some embodiments, the complete denitrification treatment lasts for 1-5 days at a temperature of 10-25°C.

[0093] In other embodiments, the inoculation amount of the strain LJ2 in the sample to be treated is 10%;

[0094] Preferably, the sample to be processed contains NO3 - and / or NO2 - of samples.

[0095] Experimental Example 1. Acquisition of LJ2 strain of the present invention

[0096] 1. Sample collection: Take 2 g of rhizosphere soil sample of Phragmites australis from Hangzhou Bay, Cixi City, Zhejiang Province, China (30°22'8"N, 121°8'27"E") and put it into a 250 ml conical bottle, add 200 ml of denitrification liquid culture medium; seal the bottle mouth with sealing film to create an anaerobic environment; and culture it at a constant temperature of 25°C for 5 days to obtain the enrichment culture solution.

[0097] 2. Preparation of denitrification medium: 1L denitrification liquid medium contains the following components: sodium acetate: 5g, dipotassium hydrogen phosphate: 1g, sodium nitrite: 0.8g, calcium chloride: 0.03g, sodium carbonate: 1g, ferrous sulfate heptahydrate: 0.06g, magnesium sulfate heptahydrate: 0.2g, deionized water: 1000ml, sterilized at 121℃ high pressure for 30min. On this basis, 20g agar is added per liter to the solid plate medium.

[0098] 3. Dilute the enriched culture fluid obtained in step 1 in multiples: take each dilution (10 -4 , 10 -5 , 10 -6 , 10 -7 ) suspension 0.5 ml on the flat culture medium, spread the enrichment solution evenly with a sterilized triangular spreading rod, and then pour into a second layer of sterilized culture medium at a temperature not higher than 40°C to create an anaerobic environment; seal the plate with a sealing film; wait for the second layer of culture medium to solidify, turn the plate upside down, put it in a constant temperature incubator at a temperature of 25°C, and culture it until obvious colonies grow, select the separated strains, separate them by streaking on the double-layer flat culture medium, and culture them in a 15°C incubator, and repeat several times until no foreign bacteria are observed under a microscope.

[0099] 4. Salt tolerance screening of strains: The strain obtained in step 3 was inoculated (10% inoculation amount) into 250 ml of liquid denitrification medium (NaCl concentration was 0%, 3%, 5%, 10%, 15%, 20%), and cultured anaerobically at 25°C for 5 days. The growth of the strain is shown in Table 1 below.

[0100] Table 1

[0101]

[0102] “-” No obvious colonies appeared on the surface of the culture medium, indicating that this salt concentration was not suitable for the growth of Halomonas desiderata LJ2.

[0103] The presence of trace colonies on the surface of the “+” culture medium indicated that Halomonas desiderata LJ2 grew slowly under this salinity.

[0104] “++” Obvious colonies appeared on the surface of the culture medium, indicating that Halomonas desiderata LJ2 grew normally under this salinity

[0105] 5. Identification of strains by 16S rRNA sequencing

[0106] The 16SrDNA sequence of the strain was compared and analyzed using universal primers as shown in SEQ ID NO.1 and SEQ ID NO.2. The homology with the 16SrDNA sequence of the expected Halomonas desiderata was 99.72%. Combined with the morphological characteristics and physiological and biochemical identification results, it was determined that the strain of the present invention belongs to Halomonas desiderata. It was named LJ2 and deposited. The deposit information is as follows:

[0107] Deposit number: CGMCC NO:24345;

[0108] Taxonomic nomenclature: Halomonas desiderata;

[0109] Deposit date: January 19, 2022;

[0110] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0111] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0112] VI. Identification of denitrification functional genes

[0113] The specific amplification primers of each gene in Table 2 were used to obtain the ct values ​​of the denitrification functional genes narG, napA, nirK, nirS, qnorB, and nosZ in the strain by real-time fluorescence quantitative PCR, which were 27.44, 34.2, 24.35, 28.34, 27.71, and 25.2, respectively. This shows that the strain has the coding genes of membrane-bound nitrate reductase (narG), periplasmic nitrate reductase (napA), nitrite reductase (nirK, nirS), nitric oxide reductase (qnorB), and nitrous oxide reductase (nosZ) involved in the denitrification process, and can convert nitrate nitrogen into nitrite nitrogen, nitric oxide, nitrous oxide, and nitrogen gas in sequence. Therefore, Halomonas desiderata LJ2 is a denitrifying bacterium with complete denitrification.

[0114] Table 2

[0115]

[0116]

[0117] Experimental Example 2: Performance test of strains in removing nitrate nitrogen

[0118] Halomonas desiderata LJ2 on the storage medium was inoculated into the liquid denitrification medium and cultured in a 25°C incubator under anaerobic conditions for 5 days to obtain the enriched bacterial solution as the seed solution. The seed solution (bacterial solution concentration of 1×10 7 CFU / mL) were inoculated with NO3 - -N was used as the nitrogen source in the denitrification liquid culture medium, and the NaCl concentrations were 0%, 3%, 5%, 10%, 15%, and 20%, respectively. The culture medium was anaerobically cultured in a constant temperature incubator at 25°C for 5 days, and samples were taken on the 1st, 2nd, 3rd, 4th, and 5th day to measure the NO3 - -N content, the results show that within 5% NaCl concentration, the nitrate removal capacity can reach more than 98%, and within 10% NaCl concentration, the nitrate removal capacity can reach more than 90% ( Figure 1 ).

[0119] Experimental Example 3: Performance test of strains in removing nitrite nitrogen

[0120] The seed solution (bacterial solution concentration is 1×10 7 CFU / mL) were inoculated with NO2 - -N was used as the nitrogen source in the culture medium, and the culture medium was anaerobically cultured in a constant temperature incubator at 25°C for 5 days. The NO2 in the culture medium was sampled and measured on the 1st, 2nd, 3rd, 4th and 5th day. --N content, the results show that within 5% NaCl concentration, the nitrite removal capacity can reach more than 90%, and within 10% NaCl concentration, the nitrite removal capacity can reach more than 85% ( Figure 2 )

[0121] Experimental Example 4: Analysis of gas components produced by denitrification of bacterial strains

[0122] NO3 - -N as the nitrogen source was placed in a 100 mL serum bottle, sealed with a rubber stopper and an aluminum cap, and the air in the serum bottle was replaced with helium using an aeration cleaning system. 7 CFU / mL) were inoculated into serum bottles, and the contents of nitric oxide, nitrous oxide, and nitrogen in the system were measured every 2 h at 25 °C ( Figure 3 ).

[0123] Experimental Example 5: Low temperature growth curve and denitrification capacity of strains

[0124] Add 100 ml of liquid culture medium with a salinity of 10% to a 150 ml conical flask and inoculate 10 ml of bacterial solution (concentration of 1×10 7 CFU / mL), set 10 replicates, cultured at 10℃ and 15℃ respectively, took out one replicate every 12h, measured using a spectrophotometer at a wavelength of 600nm, and drew a growth curve using the measured OD value. The results are shown in Figure 4 As shown. The concentration is 1×10 7 CFU / mL of bacterial solution was added at a ratio of 1% to a polluted water body with a TN concentration of 8.21 mg / L and a salinity of 12%. After 5 days, the TN removal rates were measured at 10℃ and 15℃, and the results were as follows: Figure 5 shown.

Claims

1. A strain of low-temperature-tolerant Halomonas with complete denitrification function ( Halomonas desiderata ) strain LJ2, characterized in that Its deposit number is CGMCC NO: 24345.

2. A strain of cold-resistant Halomonas with complete denitrification function (CGMCC NO: 24345) Halomonas desiderata ) Application of strain LJ2 in complete denitrification, characterized in that: The complete denitrification comprises: the membrane-bound nitrate reductase and the periplasmic nitrate reductase of the strain LJ2 reduce nitrate to nitrite; the Cu-type nitrite reductase and the cytochrome cd1-type nitrite reductase of the strain LJ2 reduce nitrite to nitric oxide; the nitric oxide reductase of the strain LJ2 reduces nitric oxide to nitrous oxide; and the nitrous oxide reductase of the strain LJ2 reduces nitrous oxide to nitrogen gas.

3. A strain of cold-resistant desirable halomonas bacteria (CGMCC NO: 24345) having complete denitrification function according to claim 2. Halomonas desiderata ) Application of strain LJ2 in complete denitrification, characterized in that: The complete denitrification means that under high salt conditions, the membrane-bound nitrate reductase, periplasmic nitrate reductase, Cu-type nitrite reductase, cytochrome cd1-type nitrite reductase, nitric oxide reductase, and nitrous oxide reductase of strain LJ2 sequentially carry out the following reactions: Membrane-bound nitrate reductase, periplasmic nitrate reductase reduces nitrate to nitrite; Cu-type nitrite reductase and cytochrome cd1-type nitrite reductase reduce nitrite to nitric oxide; Nitric oxide reductase reduces nitric oxide to nitrous oxide; Nitrous oxide reductase reduces nitrous oxide to nitrogen gas; The high salt condition refers to a NaCl concentration of 3%-15%.

4. A strain of cold-resistant desired halomonas bacteria with complete denitrification function (CGMCC NO: 24345) according to any one of claims 2-3 Halomonas desiderata ) Application of strain LJ2 in complete denitrification, characterized in that: The temperature of the complete denitrification is 10-25°C.

5. A bacterial agent, characterized in that: include: A strain of expected Halomonas (CGMCC NO: 24345) Halomonas desiderata ) strain LJ2.

6. A bacterial agent according to claim 5, characterized in that: Also includes: Auxiliary materials.

7. A complete denitrification method, characterized in that: A strain of cold-resistant Halomonas spp. with complete denitrification function and a preservation number of CGMCC NO: 24345 was used. Halomonas desiderata ) strain LJ2 carried out denitrification treatment on the samples to be treated; The complete denitrification includes: the membrane-bound nitrate reductase and the periplasmic nitrate reductase of the strain LJ2 reduce nitrate to nitrite; The Cu-type nitrite reductase and cytochrome cd1-type nitrite reductase of strain LJ2 reduce nitrite to nitric oxide; the nitric oxide reductase of strain LJ2 reduces nitric oxide to nitrous oxide; the nitrous oxide reductase of strain LJ2 reduces nitrous oxide to nitrogen gas; the sample to be treated for complete denitrification is NO3 - and / or NO2 - of samples.

8. A complete denitrification method according to claim 7, characterized in that: Under high salt conditions, the membrane-bound nitrate reductase, periplasmic nitrate reductase, Cu-type nitrite reductase, cytochrome cd1-type nitrite reductase, nitric oxide reductase, and nitrous oxide reductase of strain LJ2 performed the following reactions in sequence: Membrane-bound nitrate reductase, periplasmic nitrate reductase reduces nitrate to nitrite; Cu-type nitrite reductase and cytochrome cd1-type nitrite reductase reduce nitrite to nitric oxide; Nitric oxide reductase reduces nitric oxide to nitrous oxide; Nitrous oxide reductase reduces nitrous oxide to nitrogen gas; high salt conditions refer to NaCl concentrations of 3%-15%.

9. A complete denitrification method according to claim 7 or 8, characterized in that: The denitrification treatment time is 1-5 days, and the temperature is 10-25℃.

10. A complete denitrification method according to claim 7, characterized in that: The inoculation amount of the strain LJ2 in the sample to be treated is 10%.

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