Low-temperature-resistant and high-salt-resistant aerobic pseudomonas kirschner kirschner K2 as well as preparation method and application of pseudomonas kirschner kirschner K2

Abstract

The invention discloses low-temperature-resistant and high-salt-resistant aerobic pseudomonas kirschner kirschner K2 as well as a preparation method and application thereof. The preparation method comprises the following steps: carrying out low-temperature and high-salt enrichment culture; screening and domesticating, and monitoring the concentration of ammonia nitrogen; and after 3-5 times of enrichment culture, diluting the enrichment culture, further culturing until a macroscopic bacterial colony is obtained, and then picking and carrying out plate streaking to separate a pure strain. The invention also relates to application of the strain in preparation or prevention of nitrification and denitrification nitrogen removal drugs in treatment of high-salt nitrogen-containing wastewater. The strain is low-temperature-resistant and high-salt-resistant heterotrophic-aerobic denitrifying bacteria, has the characteristic of low temperature resistance, grows well at low temperature and high salt salinity, has higher heterotrophic nitrification-aerobic denitrification nitrogen removal capacity, is suitable for nitrogen removal treatment of high-salt wastewater such as food processing wastewater and landfill leachate, and has wide application prospects. The superiority is particularly obvious under a low-temperature condition.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a low-temperature-resistant, high-salt-resistant Pseudomonas kluyveri K2, a preparation method of the low-temperature-resistant, high-salt-resistant, aerobic Pseudomonas kluyveri K2, and a use of the low-temperature-resistant, high-salt-resistant, aerobic Pseudomonas kluyveri K2. Background Art

[0002] According to data from the National Bureau of Statistics, the annual emissions of ammonia nitrogen in 2020 were 984,000 tons, and total nitrogen emissions were 3,223,400 tons. In 2021, the annual emissions of ammonia nitrogen were 867,500 tons, and the total nitrogen emissions were 3,166,600 tons. Water pollution has improved somewhat in recent years, but large amounts of high-salt nitrogen-containing wastewater are generated in industrial processes such as oil production, food processing, aquaculture, landfills, pesticide production, textiles, and papermaking. High salinity can cause microbial dehydration, cell rupture, and death, thereby affecting the operation of sewage treatment systems. Therefore, it is necessary to select and breed salt-tolerant denitrifying bacteria to maintain the activity of high-salt wastewater biochemical treatment systems and achieve standard emissions.

[0003] Furthermore, most regions of my country experience cold winters lasting three to six months each year. Low temperatures (5°C-15°C) affect most sewage treatment plants, leading to reduced biological denitrification performance and impacting effluent quality and operating costs. my country's wastewater discharge standards even include separate winter ammonia nitrogen concentration standards. Breeding strains that demonstrate high denitrification efficiency in low-temperature environments is crucial for optimizing existing biological denitrification processes and improving their efficiency.

[0004] Traditional biological denitrification is divided into two relatively independent processes: nitrification and denitrification. Due to the different dissolved oxygen requirements of (aerobic) nitrification and (anoxic) denitrification, synchronization and unification of the entire denitrification process are difficult to achieve. This results in lengthy wastewater treatment processes, significant investment in treatment facilities and equipment, and high energy consumption and operation and maintenance costs. This significantly hinders improving the efficiency of biological denitrification. The discovery of heterotrophic nitrification and aerobic denitrification has transformed the traditional understanding of biological denitrification theory and provided potential for the development of new biological denitrification technologies. The application of aerobic denitrification allows the temporal and spatial unification of the nitrification and denitrification processes. Aerobic denitrification also offers advantages such as simultaneous nitrification and denitrification, compensation for alkalinity consumed by nitrification, and strong bacterial adaptability. Aerobic denitrifying bacteria are widely distributed, primarily isolated from natural environments such as activated sludge, soil and sediment, sewage and sewage treatment systems, and lakes.

[0005] Since Robertson isolated the first heterotrophic nitrifying and aerobic denitrifying bacteria, Thiosphaera pantotropha, from activated sludge in 1984, numerous researchers have identified numerous strains capable of heterotrophic nitrification and aerobic denitrification from soil, wastewater, and wastewater treatment systems, primarily including Paracoccus sp., Pseudomonas sp., Alcaligenes sp., and Acinetobacter sp. Heterotrophic nitrifying and aerobic denitrifying bacteria have the advantages of high growth rates, strong environmental adaptability, and high organic matter metabolism efficiency. Heterotrophic nitrifying and aerobic denitrifying bacteria can remove nitrogen through various pathways, including homogenization, dissimilatory, nitrification, and denitrification.

[0006] At present, the breeding and application research of heterotrophic nitrification-aerobic denitrifying bacteria are mostly concentrated under single adaptation conditions, either cold-resistant, such as: a kind of cold-loving efficient denitrifying bacteria and screening, application, preparation method of microbial agent (CN117305148A), or high salt resistance, such as: a kind of salt-tolerant heterotrophic nitrification and denitrification bacteria and its culture method and application (CN116925965A). The related patents and reports of strain breeding and application with cold-resistant and high-salt environment are also less common. The related patents about the application of Pseudomonas kluyveri in sewage treatment currently only have one: a strain of Pseudomonas kluyveri and its application in sewage purification field (CN115975874A), which mainly utilizes Pseudomonas kluyveri to degrade benzene pollutants in sewage. There are no patents and documents about Pseudomonas kluyveri in sewage denitrification field at present. The heterotrophic nitrifying-aerobic denitrifying bacteria K2 in this study belongs to Pseudomonas kluyveri, which was obtained through enrichment and selection in harsh environments of low temperature and high salt. It has excellent denitrification performance under low temperature and high salt conditions. At the same time, it also has good denitrification performance under normal temperature and low salt conditions, and has broad application prospects. Summary of the Invention

[0007] The present invention provides a low-temperature, high-salt, and aerobic strain, Pseudomonas kluyveri K2. This strain can achieve an ammonia nitrogen removal rate of 99.83%, a nitrate nitrogen removal rate of 96%, and a nitrite nitrogen removal rate of over 90% in a high-salt environment of 5%. Furthermore, the strain can achieve an ammonia nitrogen removal rate of over 98% at a salinity of 0.2%-6% and a temperature of 10°C.

[0008] Another object of the present invention is to provide a method for preparing low-temperature-resistant, high-salt-resistant, aerobic Pseudomonas kluyveri K2. The strain K2 is inoculated into LB culture medium and cultured to the logarithmic growth phase. The obtained bacterial suspension is centrifuged to remove the supernatant, and the suspension is washed with sterile water to obtain the strain K2 mother liquor. The method is easy to implement and simple to operate.

[0009] Another object of the present invention is to provide an application of a low-temperature-resistant, high-salt-resistant, aerobic Pseudomonas kluyveri K2 in the denitrification treatment of high-salt nitrogen-containing wastewater, and the application of this strain in nitrification and denitrification denitrification in the treatment of high-salt nitrogen-containing wastewater. The present invention selects saline wastewater generated in the production of kimchi by a certain food company in Wuhan and leachate from a certain landfill. The ammonia nitrogen removal rate of the group adding K2 bacterial agent to the saline wastewater reaches 91.17%, and the ammonia nitrogen removal rate of the group adding K2 bacterial agent to the leachate reaches 39.16%, while the control group has almost no ammonia nitrogen removal. The denitrification efficiency of the strain in saline wastewater and leachate is very significant.

[0010] In order to achieve the above-mentioned purpose, the present invention adopts the following technical measures:

[0011] A method for preparing low-temperature-resistant, high-salt-resistant, and aerobic Pseudomonas kluyveri K2 comprises the following steps:

[0012] A. Transfer activated sludge from a domestic sewage treatment plant to a 100 mL conical flask and add liquid heterotrophic nitrification medium to the flask. Incubate the flask at 9-11°C and a shaker at 150 rpm for 24-48 hours. Monitor the ammonia nitrogen concentration during the incubation process. If no ammonia nitrogen is detected, transfer 1% of the culture to fresh liquid heterotrophic nitrification medium for the next round of enrichment culture. The liquid heterotrophic nitrification medium contains the following ingredients: 0.1-6.0 g / L of ammonium chloride (NH4Cl), 0.18-0.42 g / L of magnesium sulfate (MgSO4), 0.08-0.8 g / L of dipotassium hydrogen phosphate (K2HPO4), 0.4-1.2 g / L of sodium bicarbonate (NaHCO3), 2-10 g / L of anhydrous sodium acetate (CH3COONa anhydrous), 0.02-0.08 g / 10 mL of ferrous sulfate heptahydrate (FeSO4·7H2O) (sterilized separately and added proportionally to 1 L of the medium), 30-100 g / L of sodium chloride (NaCl), 1 mL of trace elements, and distilled water to 1 L. A solid heterotrophic nitrification medium can be prepared by adding 1%-2% by weight of agar powder to the liquid heterotrophic nitrification medium. The heterotrophic nitrification medium is sterilized in an autoclave at 103.4 kPa and 121°C for 20 minutes and used after cooling to room temperature (20-25°C). The trace elements mentioned above contain the following components: copper sulfate (CuSO4) 0.025-0.13 g / L, zinc sulfate (ZnSO4) 0.15-0.85 g / L, cobalt chloride (CoCl2) 0.25-0.869 g / L, manganese chloride (MnCl2) 0.10-0.90 g / L, ethylenediaminetetraacetic acid (EDTA) 0. 10 H 16N2O8) 0.1-1.3g / L, sodium molybdate (NaMoO4) 0.02-0.30g / L, boric acid (H3BO4) 0.01-0.10g / L, calcium carbonate (CaCO3) 0.02-0.50g / L.

[0013] B. After repeating 3-5 rounds of enrichment culture, dilute the enrichment culture obtained in step A and spread it on a new solid culture medium similar to that described in step A. After culturing until colonies are visible to the naked eye, pick a single colony and streak it on a plate to isolate it. After 3-5 streaks, a pure strain is isolated.

[0014] A low-temperature, high-salt, aerobic strain obtained by the above technical measures was named Pseudomonas knackmussii, with the strain number K2. The strain was deposited in the China Center for Type Culture Collection on October 7, 2023, address: Wuhan, China, with the deposit number: CCTCC M 20231838.

[0015] The morphological characteristics of the K2 colony are as follows: the K2 colony on the agar plate is milky white, opaque, protruding around and sunken in the middle, with a smooth surface and irregular edges.

[0016] The physiological and biochemical characteristics are as follows: strain K2 is a Gram-negative bacterium that can survive under aerobic and anaerobic conditions. It is positive in oxidase, catalase, starch hydrolysis, citrate, and nitrate reduction tests, but negative in acetic acid oxidation, pectin hydrolysis, gelatin liquefaction, and lactose oxidation and fermentation.

[0017] On the basis of the above technical solution, the Pseudomonas knackmussii K2 strain contains the 16SrDNA nucleotide sequence shown in SEQ ID NO: 1.

[0018] A method for applying a low-temperature-resistant, high-salt-resistant, aerobic Pseudomonas kluyveri K2 strain to denitrification treatment of high-salt nitrogen-containing wastewater comprises the following steps:

[0019] A. The low-temperature-resistant, high-salt-resistant, aerobic Pseudomonas kluyveri K2 strain was inoculated into the above-mentioned heterotrophic nitrification medium and cultured to the logarithmic growth phase. The obtained bacterial suspension was centrifuged to remove the supernatant, and washed with sterile saline to obtain the Pseudomonas kluyveri K2 strain stock solution.

[0020] B. Add the Pseudomonas kluyveri K2 bacterial stock solution prepared in step A to a liquid, preferably heterotrophic nitrification culture medium, and perform fermentation culture to obtain a Pseudomonas kluyveri K2 liquid bacterial agent.

[0021] C. Adding the liquid bacterial agent obtained by fermentation in step B to different types of high-salinity nitrogen-containing wastewater with various salinities, various C / N ratios, and various temperatures to be treated can effectively remove nitrogen.

[0022] Based on the above technical solutions,

[0023] Preferably, the heterotrophic nitrification medium contains the following ingredients: ammonium chloride (NH4Cl) 0.38g / L, magnesium sulfate (MgSO4) 0.2g / L, potassium dihydrogen phosphate (K2HPO4) 0.1g / L, sodium bicarbonate (NaHCO3) 1g / L, anhydrous sodium acetate (CH3COONa) 3.4g / L, ferrous sulfate heptahydrate (FeSO4·7H2O) 0.050g / 10mL (sterilized separately and added to 1L of culture medium), sodium chloride (NaCl) 50g / L, trace elements 1m, distilled water added to 1000mL, pH 7-8.

[0024] Preferably, the trace elements include the following components: copper sulfate (CuSO4) 0.075g / L, zinc sulfate (ZnSO4) 0.3g / L, cobalt chloride (CoCl2) 0.375g / L, manganese chloride (MnCl2) 0.3g / L, ethylenediaminetetraacetic acid (EDTA) 10 H 16 N2O8) 0.5g / L, sodium molybdate (NaMoO4) 0.22g / L, boric acid (H3BO4) 0.014g / L.

[0025] Compared with the prior art, the present invention has the following advantages and effects:

[0026] The low-temperature-resistant, high-salinity-tolerant, aerobic Pseudomonas knackmussii K2 strain of the present invention is cold-resistant and grows well at temperatures as low as 10°C and at a high salinity of 5%. It also exhibits high heterotrophic nitrification-aerobic denitrification denitrification capacity, with virtually no accumulation of nitrite-nitrogen intermediates during the heterotrophic nitrification process. This strain exhibits highly efficient heterotrophic nitrification-aerobic denitrification performance in low-temperature, high-salinity environments, simultaneously reducing the concentrations of ammonia, nitrite, and nitrate in wastewater and efficiently removing total nitrogen. It has broad application prospects in the field of denitrification of low-temperature, high-salinity wastewater. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the colony morphology of Pseudomonas kluyveri K2 on agar medium;

[0028] Figure 2 This is a schematic diagram of the denitrification efficiency of Pseudomonas kluyveri K2 at different salinities;

[0029] Figure 3This is a schematic diagram of the denitrification efficiency of Pseudomonas kluyveri K2 at different temperatures;

[0030] Figure 4 This is a graph of denitrification efficiency of Pseudomonas kluyveri K2 at different C / N ratios;

[0031] Figure 5 This is a graph showing the denitrification efficiency of Pseudomonas kluyveri K2 at different pH values;

[0032] Figure 6 This is a graph showing the denitrification efficiency of Pseudomonas kluyveri K2 at different revolutions;

[0033] Figure 7 This is a graph showing the denitrification efficiency of Pseudomonas kluyveri K2 under different carbon sources;

[0034] Figure 8 This is a graph showing the denitrification efficiency of Pseudomonas kluyveri K2 in different nitrogen sources;

[0035] Figure 9 This is a graph showing the denitrification test results of Pseudomonas kluyveri K2 in saline wastewater (10°C);

[0036] Figure 10 This is a graph showing the denitrification effect of Pseudomonas kluyveri K2 in saline wastewater (30°C); S-0 and CK-0 are the starting test results of the bacterial group and the control group, respectively; S-24 and CK-24 are the test results of the bacterial group and the control group after 24 hours of cultivation, respectively.

[0037] Figure 11 This is a graph showing the denitrification test results of Pseudomonas kluyveri K2 in landfill leachate (10°C);

[0038] Figure 12 This is a graph showing the denitrification test results of Pseudomonas kluyveri K2 in landfill leachate (30°C);

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work. DETAILED DESCRIPTION

[0040] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0041] Example 1: Isolation and performance testing of K2 strains

[0042] The present invention provides a low-temperature-resistant, high-salt-resistant, aerobic denitrifying bacterium that denitrifies via a heterotrophic nitrification-aerobic denitrification pathway. The strain is classified as Pseudomonas knackmussii, with the strain number K2. The strain was deposited with the China Center for Type Culture Collection on October 7, 2023, with the deposit number: CCTCC M20231838. The strain was isolated by the inventors in 2022 from a sample of activated sludge from domestic sewage.

[0043] A method for preparing Pseudomonas kluyveri K2, comprising the following steps:

[0044] 1. Isolation and identification of bacterial species

[0045] (1) Culture medium:

[0046] Liquid heterotrophic nitrification medium contains the following components in mass-to-volume ratios (g / L): ammonium chloride (NH4Cl) 0.38 g / L, magnesium sulfate (MgSO4) 0.2 g / L, potassium dihydrogen phosphate (K2HPO4) 0.1 g / L, sodium bicarbonate (NaHCO3) 1 g / L, anhydrous sodium acetate (CH3COONa) 3.4 g / L, ferrous sulfate heptahydrate (FeSO4·7H2O) 0.050 g / 10 mL (sterilized separately and added to 1 L of medium), sodium chloride (NaCl) 50 g / L, trace elements 1 m, and distilled water added to 1000 mL, pH 7-8.

[0047] The solid culture medium is prepared by adding 1-2% by mass of agar powder to the aforementioned liquid heterotrophic nitrification culture medium.

[0048] The trace elements include the following components: copper sulfate (CuSO4) 0.075g / L, zinc sulfate (ZnSO4) 0.3g / L, cobalt chloride (CoCl2) 0.375g / L, manganese chloride (MnCl2) 0.3g / L, ethylenediaminetetraacetic acid (EDTA) 10 H 16 N2O8) 0.5g / L, sodium molybdate (NaMoO4) 0.22g / L, boric acid (H3BO4) 0.014g / L.

[0049] (2) Separation method:

[0050] Activated sludge from a domestic sewage treatment plant was placed in a 100 mL conical flask, and heterotrophic nitrification medium was added to the conical flask. The sludge was cultured at a low temperature of 10°C, a high salt content of 5% (calculated as NaCl), and a speed of 150 r / min for 24-48 hours for screening and acclimation, and the concentration of ammonia nitrogen was monitored. When the concentration of ammonia nitrogen could not be monitored, a sample was inoculated in a heterotrophic nitrification medium for enrichment culture. After 3-5 enrichment cultures, the enrichment culture was diluted and spread on a solid culture medium. After culture until colonies were visible to the naked eye, a single colony was picked up for plate streak isolation. After 3-5 streaks, a pure strain of Pseudomonas kluyveri K2 was isolated.

[0051] (2) Identification of bacterial species:

[0052] 1. Bacterial morphology observation:

[0053] Pseudomonas kluyveri K2 strain was cultured to the logarithmic phase under conditions of low temperature of 10°C, high salt of 5% (calculated as NaCl), and 150 r / min. The bacterial solution was diluted and evenly spread on a solid plate and cultured in a biochemical incubator at 10°C. After a single colony was formed on the plate, it was observed and subjected to Gram staining microscopy.

[0054] 2. Colony morphology characteristics:

[0055] The colonies of K2 strain on agar plates are milky white, opaque, with protruding edges and sunken centers, smooth surfaces, and irregular edges. Figure 1 shown.

[0056] 3. Molecular identification of bacterial species:

[0057] 16S rRNA amplification was performed using bacterial genomic DNA as a template, using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') as upstream and downstream primers. PCR reaction conditions were: initial denaturation at 94°C for 4 minutes, followed by 25 cycles of denaturation at 94°C for 25 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds; followed by a final extension at 72°C for 5 minutes. After amplification, the PCR product was subjected to 1% agarose gel electrophoresis to verify its integrity. The PCR product was then sent to the sequencing department of Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0058] The amplified 16S rDNA gene of the strain contained approximately 1443 nucleotide sequences, as shown in SEQ ID NO: 1. This sequence was entered into GenBank. Comparison analysis using Blast software with database sequences revealed a high similarity of 99.72% with the 16S rDNA sequence of Pseudomonas knackmussii. Based on phylogenetic analysis of the 16S rDNA gene and its physiological and biochemical properties, the strain was identified as a new strain of Pseudomonas knackmussii and named K2. The strain was deposited with the China Center for Type Culture Collection under the accession number CCTCC M 20231838.

[0059] 2. Bacteria test:

[0060] (1) Denitrification effect of K2 bacteria at different salinities:

[0061] At 10°C, a K2 suspension was inoculated at a 5% inoculum ratio into a 250 mL Erlenmeyer flask containing 150 mL of the heterotrophic nitrification medium. The culture was then incubated at different salinities (0.2%, 2%, 3%, 6%, 8%, and 10%), with the shaker set at 150 rpm. Furthermore, samples were taken periodically during the culture process to determine the ammonia nitrogen concentration in the culture medium.

[0062] At a salinity of 0.2%, K2 bacteria can remove 99.45% of ammonia nitrogen and 37.22% of total nitrogen; at a salinity of 3% and 5%, it can also remove more than 99% of ammonia nitrogen and more than 47% of total nitrogen; at a salinity of 6%, the ammonia nitrogen removal rate can reach 98.45%, and the total nitrogen removal rate can reach 79.84%; at a salinity of 8%, it can remove 51.39% of ammonia nitrogen; at a salinity of 10%, K2 bacteria can grow but the ammonia nitrogen removal rate is only 19.65%. The experimental results are as follows Figure 2 shown.

[0063] (2) Denitrification effect of K2 bacteria at different temperatures:

[0064] A K2 bacterial suspension was inoculated at a 5% inoculum ratio into 150 mL of the heterotrophic nitrification medium in a 250 mL Erlenmeyer flask in a high-salt, 5% (as NaCl) atmosphere. The culture was incubated at various temperatures (4°C, 10°C, 20°C, 30°C, and 40°C) with the shaker set at 150 rpm. Furthermore, samples were taken periodically during the incubation process to monitor the ammonia nitrogen concentration in the culture medium.

[0065] The K2 strain can remove 44.75% of ammonia nitrogen in a 4℃ environment, and the removal rate of ammonia nitrogen can reach 99.71% in a 10℃ environment. At 20℃, 30℃, and 40℃, the removal rate of ammonia nitrogen can reach more than 99%. Figure 3 shown.

[0066] (3) Denitrification effect of K2 bacteria under different C / N ratios:

[0067] A 250 mL Erlenmeyer flask containing 150 mL of the heterotrophic nitrification medium was inoculated with a K2 suspension at a 5% inoculum ratio at a low temperature of 10°C and a high salt concentration of 5% (calculated as NaCl). The culture was incubated at different C / N ratios (3, 6, 9, 12, 15, and 18) with a shaker speed of 150 rpm. Furthermore, samples were taken periodically during the incubation process to monitor the ammonia nitrogen concentration of the culture medium.

[0068] In the environment of C / N ratio of 3, the removal rate of ammonia nitrogen can reach 48.79%, in the environment of C / N ratio of 6, the removal rate of ammonia nitrogen can reach 86.36%, and in the environment of C / N ratio of 12, 15, and 18, the removal rate of ammonia nitrogen can reach more than 99%. Figure 4 shown.

[0069] (4) Denitrification effect of K2 bacteria at different pH:

[0070] A 250 mL Erlenmeyer flask containing 150 mL of the heterotrophic nitrification medium was inoculated with a K2 suspension at a 5% inoculum ratio at a low temperature of 10°C and a high salt concentration of 5% (calculated as NaCl). The culture was incubated at different pH values (4, 6, 8, 10, and 12) with a shaker speed of 150 rpm. Furthermore, samples were taken periodically during the incubation process to monitor the ammonia nitrogen concentration of the culture medium.

[0071] The removal rate of ammonia nitrogen in the environment of pH 6 and 8 is more than 99.00%, the removal rate of nitrogen in the environment of pH 10 can reach about 85%, and the removal rate of ammonia nitrogen in the strong alkaline environment of pH 12 is still 63.36%. Figure 5 shown.

[0072] (5) Denitrification effect of K2 bacteria at different rotation speeds:

[0073] At a low temperature of 10°C and a high salt content of 5% (calculated as NaCl), a K2 bacterial suspension was inoculated into a 250 mL Erlenmeyer flask containing 150 mL of the above-mentioned heterotrophic nitrification medium at a 5% inoculation ratio, and cultured at different rotation speeds (0 r / min, 50 r / min, 100 r / min, 150 r / min, 200 r / min). In addition, samples were taken regularly during the culture process to detect the ammonia nitrogen concentration of the culture solution.

[0074] At a speed of 0 r / min, the ammonia nitrogen removal rate can reach 20.28%. As the speed increases, the ammonia nitrogen removal rate of K2 bacteria also increases greatly. When the speed is 150 r / min, the removal rate can reach 100%. However, when the speed increases to 200 r / min, the ammonia nitrogen removal rate is only 86.24%. The experimental results are as follows: Figure 6 shown.

[0075] (6) Denitrification effect of K2 strains under different carbon sources:

[0076] A 250 mL Erlenmeyer flask containing 150 mL of the aforementioned heterotrophic nitrification medium was inoculated with a K2 suspension at a 5% inoculum ratio at a low temperature of 10°C and a high salt concentration of 5% (calculated as NaCl). The culture was incubated with different carbon sources (anhydrous sodium acetate, sodium citrate, sodium succinate, sucrose, and glucose) at a shaking speed of 150 rpm. Furthermore, samples were taken periodically during the culture process to monitor the ammonia nitrogen concentration of the culture medium.

[0077] The removal rate of ammonia nitrogen using anhydrous sodium acetate, sodium citrate and sodium succinate as carbon sources can reach more than 98%, and the removal rate of total nitrogen can reach more than 50%. Figure 7 shown.

[0078] (7) Denitrification effect of K2 bacteria under different nitrogen sources:

[0079] Based on the heterotrophic nitrification medium without ammonium chloride, different nitrogen sources (NH4 + -N, NO3 - -N, NO2 - -N), test the denitrification effect of K2 strains on different nitrogen sources. At a low temperature of 10 ° C and high salt of 5% (calculated as NaCl), K2 bacterial suspension was inoculated into a 250mL triangular flask containing 150mL of culture medium at a 5% inoculation ratio, and cultured in groups under different nitrogen sources. The shaker speed was set to 150r / min during the culture process. In addition, samples were taken regularly during the culture process to detect the concentrations of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and total nitrogen in the culture solution. With ammonia nitrogen as the only nitrogen source, the removal rate of ammonia nitrogen can reach 100%; with nitrate nitrogen as the only nitrogen source, the strain can remove 96.84% of NO3 - -N; Using nitrite as the only nitrogen source, it can remove 90.63% of NO2 - -N. The experimental results are as follows Figure 8 shown.

[0080] Example 2: (Application of K2 strain in saline wastewater)

[0081] A preparation method of Pseudomonas kluyveri K2 and its application in saline wastewater, comprising the following steps:

[0082] A. Inoculate Pseudomonas kluyveri K2 into the above-mentioned heterotrophic nitrification medium and culture until the logarithmic growth phase. Centrifuge the resulting bacterial suspension to remove the supernatant, and wash with sterile saline to obtain a K2 bacterial stock solution.

[0083] B. Prepare liquid heterotrophic nitrification culture medium. The culture medium needs to be sterilized in an autoclave at 103.4 kPa and 121°C for 20 minutes and then cooled to room temperature (20-25°C).

[0084] C. Add K2 bacterial strain mother solution to the cooled heterotrophic nitrification medium prepared in step B and culture in a shaking incubator until the OD 600 When it reaches 1.2, K2 liquid bacterial agent is obtained.

[0085] D. Add the K2 liquid bacterial agent obtained by culturing in step C to the corresponding saline wastewater.

[0086] 50L of wastewater was collected from the saline wastewater generated during the production of kimchi by a food company in Wuhan, Hubei, and the denitrification effect of the K2 strain was tested at 10°C and 30°C respectively. The average initial water quality parameters of the wastewater were: ammonia nitrogen 100.36mg / L, nitrite nitrogen 0mg / L, total nitrogen 411.08mg / L, and salinity 5.96%. The sewage was divided into 12 triangular flasks with a volume of 3L, 6 of which were added with 5% bacterial suspension as the bacteria-added group (S), and the other 6 were not added with any substance as the control group (CK). Three samples each from the bacteria-added group and the control group were cultured at 10°C to test the actual application effect of the strain K2 at low temperature; the remaining 6 triangular flasks were cultured at 30°C to test the actual application effect of the strain K2 at room temperature. During the test, the system ammonia nitrogen (NH4 + -N), nitrate (NO3 - -N), nitrite (NO2 - -N), total nitrogen (TN), and dissolved total nitrogen (DTN) were measured to analyze the actual denitrification ability of the bacteria in saline wastewater.

[0087] The denitrification effect of strain K2 in 10℃ saline wastewater is shown in Figure 9 , Figure 9 -A is the test effect of the bacteria-added group, Figure 9 -B is the test effect of the control group without bacteria; the denitrification effect of strain K2 in 30℃ saline wastewater is shown in Figure 10; S-0 and CK-0 are the starting test results of the bacterial group and the control group, respectively. S-24 and CK-24 are the test results of the bacterial group and the control group after 24 hours of cultivation, respectively. The ammonia nitrogen removal rate of the bacterial group reached 91.17% (10℃) and 91.62% (30℃), and the nitrate nitrogen removal rate could reach 84.20% (10℃) and 80.36% (30℃), respectively. The control group without bacterial addition had almost no ammonia nitrogen removal. The denitrification efficiency of the strain in saline wastewater at two temperatures was very significant. This shows that: whether under low temperature or normal temperature conditions, the strain K2 has very good adaptability in actual high-salt wastewater, high denitrification efficiency, and extremely high application value.

[0088] Example 3: (Application of K2 strain in landfill leachate)

[0089] Steps AC are the same as in Example 2;

[0090] D. Add the cultured K2 liquid bacterial agent into the corresponding landfill leachate.

[0091] 5L of leachate was collected from a landfill in Wuhan, Hubei. The average initial water quality parameters were 2800-3200mg / L ammonia nitrogen and 3000-3400mg / L total nitrogen.

[0092] Landfill leachate was diluted to an ammonia nitrogen concentration of approximately 1000 mg / L and then divided into 12 3L Erlenmeyer flasks. Six of these were dosed with a 5% bacterial suspension as the "supplemented" group (S), while the remaining six were left untreated as the "control" group (CK). Three samples from each of the "supplemented" and "control" groups were incubated at 10°C to test the effectiveness of strain K2 at low temperatures. The remaining six flasks were incubated at 30°C to test the effectiveness of strain K2 at room temperature. During the test, system ammonia nitrogen and total nitrogen concentrations were regularly monitored to analyze the strain's denitrification capabilities in landfill leachate.

[0093] Inoculate the bacterial solution into the landfill leachate and place it at 10℃ for 10 days. Figure 11 . Figure 11 -A is the treatment effect of the bacteria-added group, and the removal rates of ammonia nitrogen, total nitrogen, and dissolved total nitrogen can reach 16.23%, 19.21%, and 14.95%, respectively; Figure 11 -B is the treatment effect of the control group without bacteria, and the removal rates of ammonia nitrogen, total nitrogen and dissolved total nitrogen are only 0.76%, 0.31% and 1.01% respectively.

[0094] The bacteria were inoculated into the landfill leachate and placed in a 30℃ environment to detect the changes in ammonia nitrogen, total nitrogen and dissolved total nitrogen. After 10 days of treatment, the effect was Figure 12 . Figure 12-A is the treatment effect of the bacteria-added group, which can remove 39.16% of ammonia nitrogen, 37.57% of total nitrogen and 35.43% of dissolved total nitrogen. Figure 12 -B is the treatment effect of the control group without bacteria. The removal rates of ammonia nitrogen, total nitrogen and dissolved total nitrogen in the control group were only 1.73%, 0.28% and 1.01% respectively.

[0095] This shows that the K2 strain showed good tolerance in high-salt and high-ammonia nitrogen landfill leachate, and exhibited good denitrification ability both at low and normal temperatures, and has the value of being promoted and applied in the denitrification treatment of landfill leachate.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A low-temperature-resistant, high-salt-resistant, aerobic Pseudomonas kluyveri K2, characterized by: The Pseudomonas kluyveri K2 is deposited in the China Center for Type Culture Collection with the deposit number CCTCC M 20231838.

2. The low-temperature-resistant, high-salt-resistant, aerobic Pseudomonas kluyveri K2 according to claim 1, characterized in that: The Pseudomonas knackmussii K2 strain contains the 16SrDNA nucleotide sequence shown in SEQ ID NO:

1.

3. A low-temperature-resistant, high-salt-resistant, aerobic bacterial culture of Pseudomonas kluyveri, characterized by: The bacterial culture contains the Pseudomonas kluyveri K2 as described in claims 1 and 2.

4. The method for preparing a low-temperature-resistant, high-salt-resistant, aerobic Pseudomonas kluyveri K2 according to claim 1, characterized in that: The steps include: Activated sludge from domestic sewage is placed in a 100 mL conical flask, and a heterotrophic nitrification medium is added to the conical flask to adjust the ammonia nitrogen concentration to 100-120 mg / L. The sludge is cultured at a low temperature of 9-11° C., a high salt concentration of 4.5-5.5%, and a speed of 150 r / min for 24-48 hours for screening and acclimation, and the ammonia nitrogen concentration is monitored. When the ammonia nitrogen concentration cannot be monitored, a sample is inoculated in the heterotrophic nitrification medium for enrichment culture to obtain an enriched culture. After repeating the above enrichment culture process 3-5 times, the obtained enriched culture is diluted and spread on a solid culture medium. After culturing until colonies are visible to the naked eye, a single colony is picked for plate streaking isolation. After 3-5 streaking cycles, a pure strain of Pseudomonas kluyveri K2 is isolated.

5. The preparation method according to claim 4, characterized in that The heterotrophic nitrification medium contains the following components in mass-to-volume ratios: ammonium chloride (NH4Cl) 0.1-6.0 g / L, magnesium sulfate (MgSO4) 0.18-0.42 g / L, potassium dihydrogen phosphate (K2HPO4) 0.08-0.8 g / L, sodium bicarbonate (NaHCO3) 0.4-1.2 g / L, anhydrous sodium acetate (CH3COONa anhydrous) 2-10 g / L, ferrous sulfate heptahydrate (FeSO4·7H2O) 0.02-0.08 g / 10 mL added separately after sterilization, sodium chloride (NaCl) 30-100 g / L, and trace elements 1 mL, and distilled water is added to 1000 mL, with a pH of 7-8; The trace elements include: copper sulfate (CuSO4) 0.025-0.13g / L, zinc sulfate (ZnSO4) 0.15-0.85g / L, cobalt chloride (CoCl2) 0.25-0.869g / L, manganese chloride (MnCl2) 0.10-0.90g / L, ethylenediaminetetraacetic acid (EDTA) 10 H 16 N2O8) 0.1-1.3g / L, sodium molybdate (NaMoO4) 0.02-0.30g / L, boric acid (H3BO4) 0.01-0.10g / L, calcium carbonate (CaCO3) 0.02-0.50g / L; The solid culture medium is obtained by adding 1% to 2% by mass of agar powder into a heterotrophic nitrification culture medium and sterilizing the culture medium under the conditions of 103.4 kPa and 121° C.

6. Application of a low-temperature-resistant, high-salt-resistant, aerobic Pseudomonas kluyveri K2 strain in nitrification and denitrification in the treatment of high-salt nitrogen-containing wastewater, characterized in that: The steps include: A. Inoculate Pseudomonas kluyveri K2 into heterotrophic nitrification medium and culture until the logarithmic growth phase. Centrifuge the resulting bacterial suspension to remove the supernatant, and wash with sterile saline to obtain a stock solution of Pseudomonas kluyveri K2. B. Prepare heterotrophic nitrification culture medium, sterilize the culture medium in an autoclave at 103.4 kPa and 121° C. for 20 min, and cool to room temperature; C. adding the mother liquor of Pseudomonas kluyveri strain K2 to the heterotrophic nitrifying base cooled after treatment in step B, and performing fermentation culture to obtain a liquid bacterial solution of Pseudomonas kluyveri K2; D. Add the liquid bacterial agent obtained by fermentation in step C to the corresponding high-salt wastewater.

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