Salt-tolerant composite denitrifying bacteria, application thereof and salt-tolerant denitrifying composite microbial agent

Abstract

The invention relates to the technical field of microbial denitrification, in particular to a salt-tolerant composite denitrifying bacterium, application thereof and a salt-tolerant denitrification composite microbial agent. The salt-tolerant composite denitrifying bacteria comprise klebsiella pneumoniae TYF-CJJ-P06 and klebsiella pneumoniae TYF-CJJ-P07, and the salt-tolerant composite denitrifying bacteria comprise klebsiella pneumoniae TYF-CJJ-P06 and The salt-tolerant composite denitrifying bacteria can simultaneously remove nitrate nitrogen and ammoniacal nitrogen in a water body, has relatively strong adaptability to a high-salinity environment, can be used for removing organic nitrogen in some high-salinity wastewater such as pharmaceutical wastewater, printing and dyeing wastewater, food wastewater and kitchen waste wastewater, has the advantages of environmental friendliness and strong applicability, and can remove phenol at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial denitrification, and particularly to a salt-tolerant composite denitrifying bacterium, its application, and a salt-tolerant denitrifying composite microbial agent. Background Art

[0002] In recent years, with the continuous expansion of China's industrial scale, industrial water consumption has increased sharply. At the same time, the amount of wastewater generated has also increased rapidly, posing a huge challenge to current wastewater treatment and recycling technologies. Among the discharged wastewater, high-salt wastewater has become a category that cannot be ignored and has attracted increasing attention. High-salt wastewater refers to industrial or domestic wastewater with a salt concentration significantly higher than that of ordinary water bodies, usually measured by total dissolved solids (TDS). A salt concentration exceeding 1% (about 10,000 mg / L) can be classified as such, but the specific standards vary by industry. For example, the salt content of the concentrated brine produced by seawater desalination can reach 3.5%, and some chemical wastewater even exceeds 10%. This type of wastewater not only has a high salt content but may also contain complex pollutants such as organic matter, heavy metals, or chemical additives, making it difficult to treat.

[0003] Currently, the methods for treating high-salt wastewater are mainly divided into physicochemical methods and biological methods. Among the physicochemical methods, evaporation crystallization (such as multi-effect evaporation, mechanical vapor recompression MVR) evaporates water by heating and recovers salts, but has high energy consumption; membrane separation technologies such as reverse osmosis (RO) and electrodialysis (ED) use semi-permeable membranes or electric fields to separate salts, but have strict requirements for the quality of the influent water, and the problem of membrane fouling is prominent. The biological method relies on salt-tolerant bacteria (such as halophilic bacillus) or biofilm processes, but requires long-term domestication of the bacteria, and the stability may be affected by water quality fluctuations. In practical applications, combined processes are often used. For example, the salt content is first reduced by evaporation and concentration, and then the biological method is combined to treat organic matter, or the salt concentration is reduced by dilution and then enters the conventional treatment process, but the cost and feasibility need to be weighed.

[0004] The existing biological methods for treating high-salt wastewater are not satisfactory. Although SBR, activated sludge method, biofilm method, and anaerobic treatment have low costs, excessive salinity will inhibit the growth of ordinary microorganisms; traditional biotechnology can generally only tolerate wastewater with a salt content of less than 1% in practical applications. When the salt content is greater than 1%, the efficiency of the system is greatly affected due to microbial cell dehydration, plasmolysis, and the reduction of various enzymatic reactions required for the growth of microorganisms, resulting in a significant reduction in the degradation rate of the system. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a salt-tolerant composite denitrifying bacterium, its application, and a salt-tolerant denitrifying composite microbial agent. The salt-tolerant composite denitrifying bacterium provided by the present invention can efficiently remove organic nitrogen in high-salt wastewater.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a salt-tolerant composite denitrifying bacterium, including Klebsiella pneumoniae TYF-CJJ-P06 and Klebsiella sp. TYF-CJJ-P07;

[0008] The Klebsiella pneumoniae TYF-CJJ-P06 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 17, 2024, with the deposit number CGMCC No. 29832;

[0009] The Klebsiella sp. TYF-CJJ-P07 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 17, 2024, with the deposit number CGMCC No. 29833.

[0010] Preferably, the viable cell number ratio of the Klebsiella pneumoniae TYF-CJJ-P06 to the Klebsiella sp. TYF-CJJ-P07 is 1:1.

[0011] The present invention also provides the application of the salt-tolerant composite denitrifying bacterium described in the above technical solution in removing organic nitrogen.

[0012] Preferably, the organic nitrogen includes ammonia nitrogen and / or nitrate nitrogen.

[0013] The present invention also provides the application of the salt-tolerant composite denitrifying bacterium described in the above technical solution in removing phenol.

[0014] The present invention also provides a salt-tolerant denitrifying composite microbial agent, including the Klebsiella pneumoniae TYF-CJJ-P06 and the Klebsiella sp. TYF-CJJ-P07 in the salt-tolerant composite denitrifying bacterium described in the above technical solution.

[0015] Preferably, the bacterial content of the Klebsiella pneumoniae TYF-CJJ-P06 in the salt-tolerant denitrifying composite microbial agent is 5×10 9 CFU / ml;

[0016] The bacterial content of the Klebsiella sp. TYF-CJJ-P07 in the salt-tolerant denitrifying composite microbial agent is 5×10 9 CFU / ml.

[0017] The present invention also provides the application of the salt-tolerant denitrifying composite microbial agent described in the above technical solution in removing organic nitrogen in high-salt wastewater.

[0018] Preferably, the application includes: mixing the salt-tolerant denitrifying composite microbial agent with the high-salt wastewater, and degrading the organic nitrogen in the high-salt wastewater at a temperature of 20-35 °C, a pH value of 5.0-9.0, and a rotation speed of 120 rpm;

[0019] The organic nitrogen includes ammonia nitrogen and / or nitrate nitrogen;

[0020] The salinity of the high-salt wastewater is 0.1-70 g / L;

[0021] The volume ratio of the salt-tolerant denitrifying composite microbial agent to the high-salt wastewater is 1:20.

[0022] The present invention also provides an application of the salt-tolerant denitrifying composite microbial agent described in the above technical solution in removing phenol.

[0023] Advantages of the present invention:

[0024] (1) The salt-tolerant composite denitrifying bacteria of the present invention can simultaneously remove ammonia nitrogen and nitrate nitrogen in water bodies with high removal efficiency;

[0025] (2) The composite bacteria of the present invention still maintain good denitrifying activity under high salinity and can be used for removing organic nitrogen in some high-salt wastewaters such as pharmaceutical wastewater, printing and dyeing wastewater, food wastewater, and kitchen waste wastewater.

[0026] (3) The culture medium components required for activation and expansion of the composite bacteria of the present invention are simple, and the preparation process of the bacterial liquid is relatively easy, which is beneficial to industrial production and subsequent applications. Description of the drawings

[0027] Figure 1 It is an agarose gel electrophoresis map of the 16S rRNA gene amplification products of strains TYF-CJJ-P06 and TYF-CJJ-P07;

[0028] Figure 2 It is a phylogenetic tree of strain TYF-CJJ-P06;

[0029] Figure 3 It is a phylogenetic tree of strain TYF-CJJ-P07;

[0030] Figure 4 It is a denitrification performance test map of the composite denitrifying bacteria under different salinity conditions;

[0031] Figure 5 It is a denitrification performance test map of the composite denitrifying bacteria under different temperature conditions;

[0032] Figure 6 It is a denitrification performance test map of the composite denitrifying bacteria under different pH conditions;

[0033] Figure 7It is a test chart of the denitrification performance of the composite denitrifying bacteria when the phenol concentration is 200 mg / L.

[0034] Biological preservation description

[0035] Klebsiella pneumoniae TYF-CJJ-P06, with the Latin name Klebsiella pneumoniae, is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the preservation number CGMCC No. 29832, and the address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0036] Klebsiella sp. TYF-CJJ-P07, with the Latin name Klebsiella sp., is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the preservation number CGMCC No. 29833, and the address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Specific implementation manners

[0037] The present invention provides a salt-tolerant composite denitrifying bacteria, including Klebsiella pneumoniae TYF-CJJ-P06 and Klebsiella sp. TYF-CJJ-P07; the Klebsiella pneumoniae TYF-CJJ-P06 was preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on January 17, 2024, with the preservation number CGMCC No. 29832; the Klebsiella sp. TYF-CJJ-P07 was preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on January 17, 2024, with the preservation number CGMCC No. 29833. In the present invention, the viable bacteria number ratio of the Klebsiella pneumoniae TYF-CJJ-P06 and the Klebsiella sp. TYF-CJJ-P07 is preferably 1:1.

[0038] The present invention also provides the application of the salt-tolerant composite denitrifying bacteria described in the above technical solution in removing organic nitrogen. In the present invention, the organic nitrogen preferably includes ammonia nitrogen and / or nitrate nitrogen.

[0039] The present invention also provides the application of the salt-tolerant composite denitrifying bacteria described in the above technical solution in removing phenol.

[0040] The present invention also provides a salt-tolerant denitrifying composite microbial agent, including Klebsiella pneumoniae TYF-CJJ-P06 and Klebsiella sp. TYF-CJJ-P07 in the salt-tolerant composite denitrifying bacteria described in the above technical solution. In the present invention, the bacterial content of Klebsiella pneumoniae TYF-CJJ-P06 in the salt-tolerant denitrifying composite microbial agent is preferably 5×10 9CFU / ml; the bacterial content of Klebsiella sp. TYF-CJJ-P07 in the salt-tolerant denitrifying composite microbial agent is preferably 5×10 9 CFU / ml. The present invention has no special limitation on the preparation method of the salt-tolerant denitrifying composite microbial agent, and those skilled in the art can prepare it according to the conventional preparation methods of Klebsiella pneumoniae and Klebsiella sp. agents.

[0041] The present invention also provides the application of the salt-tolerant denitrifying composite microbial agent described in the above technical solution in removing organic nitrogen in high-salt wastewater. In the present invention, the application preferably includes: mixing the salt-tolerant denitrifying composite microbial agent with high-salt wastewater, and degrading the organic nitrogen in the high-salt wastewater at a temperature of 20-35°C, a pH value of 5.0-9.0, and a rotation speed of 120 rpm; the organic nitrogen includes ammonia nitrogen and / or nitrate nitrogen; the salinity of the high-salt wastewater is 0.1-70 g / L; the volume ratio of the salt-tolerant denitrifying composite microbial agent to the high-salt wastewater is 1:20. In the present invention, the temperature is preferably 30°C. In the present invention, the pH value is preferably 7.0.

[0042] The present invention also provides the application of the salt-tolerant denitrifying composite microbial agent described in the above technical solution in removing phenol.

[0043] In order to further illustrate the present invention, the following examples are used to describe the present invention in detail, but they should not be construed as limiting the protection scope of the present invention.

[0044] The materials and instruments used in the following examples are as follows:

[0045] (1) Strain source:

[0046] The strains screened in the experiment were derived from the activated sludge in the aerobic tank of Qingxu Hongbo Sewage Treatment Plant.

[0047] (2) Culture medium:

[0048] Beef extract peptone medium: beef extract 5 g / L, peptone 10 g / L, NaCl 5 g / L, pH 7.0±0.2.

[0049] Denitrifying medium: C6H5Na3O7·2H2O 5.719 g / L, (NH4)2SO4 0.472 g / L, KNO3 0.722 g / L, NaNO2 0.246 g / L, K2HPO4 0.200 g / L, MgSO4·7H2O 0.050 g / L, MnSO4·4H2O 0.010 g / L, FeSO4 0.010 g / L, NaCl 0.120 g / L, pH 7.0±0.2.

[0050] The solid medium is supplemented with 2%-2.5% agar on the basis of the above medium. All media need to be sterilized by moist heat under high pressure at 121 °C for 30 min before use, and then cooled to room temperature for subsequent experiments.

[0051] (3) Main experimental instruments:

[0052] Constant temperature biochemical incubator, high-speed refrigerated centrifuge, constant temperature shaker, ultra-clean workbench, vertical pressure steam sterilizer, full-wavelength microplate reader, PCR instrument, electrophoresis instrument, etc.

[0053] Example 1

[0054] Screening of strains

[0055] (1) Enrichment of strains

[0056] Inoculate 10 mL of the retrieved activated sludge sample into a conical flask containing 90 mL of sterilized beef extract peptone medium, and culture it at 120 r / min and 30 °C for 5 d.

[0057] (2) Isolation and preservation of strains

[0058] In the ultra-clean workbench, dilute the enriched culture solution with sterile water in gradient and coat it on the denitrifying solid medium. After standing for 30 min, invert the plate and place it in a constant temperature incubator at 30 °C for more than 24 h. Pick single colonies with different morphological characteristics and inoculate them into the denitrifying liquid medium. After culturing at 120 r / min and 30 °C for 24 h, continue to streak and purify on the plate. After repeating 3 times, inoculate the grown single colonies into the denitrifying liquid medium and culture for 24 h under the same conditions. Take samples to measure the NH4 + -N and NO3 - -N content, screen out the strains that can degrade ammonia nitrogen and nitrate nitrogen simultaneously for the next experiment. Inoculate the screened strains into the paraffin slant medium and store them refrigerated in a 4 °C refrigerator. At the same time, take 500 μL of the bacterial solution and mix it with 50% glycerol at a ratio of 1:1, and then store it frozen in an -80 °C refrigerator.

[0059] (3) Re-screening of strains

[0060] Pre-culture each strain to the logarithmic growth phase, and measure its OD 600 value, centrifuge to remove the influence of the initial medium, rinse it three times with sterile water, and use PBS buffer to adjust the initial OD of each strain 600 to the same level, and inoculate it into the denitrifying medium at an inoculation amount of 5%. Add NaCl to simulate high-salinity wastewater, and set the salinity to 1% (10 g / L). Culture it at 30 °C and 120 r / min for 48 h, and take samples every 12 h to measure NH4+ -N and NO3 - -N concentration, with three parallels set for each group. Finally, strains with high NH4 + -N and NO3 - -N removal rates both higher than 95% were selected, and the strain numbers were TYF-CJJ-P06 and TYF-CJJ-P07.

[0061] Molecular biological identification of the strains in Example 2

[0062] The purified strains were inoculated into the basal medium and cultured at 120 r / min and 30 °C for more than 12 h. The DNA of the bacterial liquid was extracted as the template, and the universal primer pair was used (27F (SEQ ID No. 3): 5'-AGAGTTTGATCCTGGCTCAG-3', and the downstream primer was 1492R (SEQ ID No. 4): 5'-TACGGCTACCTTGTACGACTT-3'). The PCR reaction system was: 10X PCR Buffer, dNTP (10 mM), Taq DNA polymerase (5 U / μL), 50 mM MgSO4, a total of 12.5 μL, 1 μL of each primer (10 μM), 1 μL of template DNA, 9.5 μL of ddH2O, and the total system was 25 μL. The reaction conditions for PCR amplification were: pre-denaturation at 95 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s, extension at 72 °C for 90 s, for 30 cycles; extension at 72 °C for 5 min to 10 min; preservation at 4 °C for 15 min.

[0063] The PCR amplification products were subjected to agarose gel electrophoresis. The results shown by the agarose gel electrophoresis indicated that for strains TYF-CJJ-P06 and TYF-CJJ-P07, the amplified product bands were relatively bright at about 1500 bp, and there were no other miscellaneous bands, as Figure 1 shown.

[0064] The 16S rDNA products obtained by PCR amplification were entrusted to Sangon Biotech Co., Ltd. for first-generation sequencing. The 16S rRNA gene sequences of strains TYF-CJJ-P06 and TYF-CJJ-P07 are shown as SEQ ID No.1 and SEQ ID No.2 respectively. The obtained sequences were submitted to the NCBI website and compared with the existing strain data in the GenBank database. It was preliminarily determined that strain TYF-CJJ-P06 has the closest genetic relationship with the genus Klebsiella pneumoniae, and TYF-CJJ-P07 has the closest genetic relationship with the genus Klebsiella. Then, BLAST (http: / / www.ncbi.nlm.nih.gov / blast / ) was used to search for strains with higher similarity. Using the MEGA11.0 software, the phylogenetic tree was constructed by the Neighbor Joining method, as Figure 2 and Figure 3 shown, to further analyze the genus and species of the strains.

[0065] SEQ ID No.1:

[0066] GGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGCGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAATGTCGCAAGACCAAAGTGGGGGACCTTCGGGCCTCATGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGATAAGGTTAATAACCTCGTCGATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGATTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACAGAACTTTCCAGAGATGGTTTGGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTATGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGAAAA;

[0067] SEQ ID No.2:

[0068] GCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGCAGTAAGGTTAATAACCTTGTTCATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTGGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGACTTGGAGGTTGTTCCCTTGAGGAGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTAGCAGAGATGCTTTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTATGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCT。

[0069] Example 3

[0070] Denitrification performance test of composite denitrifying bacteria under different salinity conditions

[0071] Inoculate the TYF-CJJ-P06 and TYF-CJJ-P07 strains stored in the -80°C refrigerator in Example 1 into the nitrification medium, and then place them in a shaker at 30°C for activation. After they grow to the logarithmic phase, measure their OD 600 values respectively, centrifuge to remove the influence of the initial medium, rinse three times with sterile water, and adjust the initial OD 600 of the two strains to the same level. Prepare a composite bacterial solution according to a volume ratio of 1:1, with the bacterial content of TYF-CJJ-P06 and TYF-CJJ-P07 both being 5×10 9 CFU / ml.

[0072] Take the compound bacterial liquid and inoculate it into the denitrification medium at an inoculation amount of 5%. The concentrations of nitrate nitrogen and ammonia nitrogen in the denitrification medium are both 100 mg / L. Set the salinity gradients of 1%, 3%, 5%, and 7% (mass percentage), and then incubate at a constant temperature of 30 °C and 120 r / min for 2 days. After the incubation period, take samples, centrifuge, and measure the concentrations of ammonia nitrogen and nitrate nitrogen in the supernatant, and calculate their degradation rates. The results are as Figure 4 shown.

[0073] Among them, the method used to test the content of NH4 + -N is the Nessler's reagent spectrophotometry method, and the method used to test the content of NO3 - -N is the ultraviolet spectrophotometer method.

[0074] It can be seen from Figure 4 that when the salinity is 1%, 3%, and 5%, the degradation rates of NH4 + -N and NO3 - -N by the compound denitrifying bacteria are both above 90%. Among them, when the salinity is 1%, the degradation rate of NH4 + -N is 98.4%, and the degradation rate of NO3 - -N is 97.1%. When the salinity is 7%, the degradation rates of NH4 + -N and NO3 - -N decrease significantly, and the degradation rates are 56.2% and 42.1% respectively. This may be because the excessive salinity causes severe dehydration stress to the bacteria, leading to their death and thus unable to exert their denitrification ability.

[0075] It can be seen from this that the denitrifying bacteria have strong adaptability to high-salinity environments and can be used for the removal of organic nitrogen in some high-salinity wastewaters such as pharmaceutical wastewater, printing and dyeing wastewater, food wastewater, and kitchen waste wastewater.

[0076] Example 4

[0077] Denitrification performance test of compound denitrifying bacteria under different temperature conditions

[0078] Take the compound bacterial liquid in Example 3 and inoculate it into the denitrification medium at an inoculation amount of 5%. The concentrations of nitrate nitrogen and ammonia nitrogen in the denitrification medium are both 100 mg / L. Set the temperature gradients of 10 °C, 20 °C, 30 °C, and 35 °C. Then incubate at a constant temperature of 120 r / min for 2 days. After the incubation period, take samples, centrifuge, and measure the concentrations of ammonia nitrogen and nitrate nitrogen in the supernatant, and calculate their degradation rates. The results are as Figure 5 shown.

[0079] Among them, the method used to test the content of NH4 + -N is the Nessler's reagent spectrophotometry method, and the method used to test the content of NO3 - -N is the ultraviolet spectrophotometer method.

[0080] It can be seen from Figure 5 that when the temperature is 20 °C, 30 °C, and 35 °C, the degradation rates of NH4 + -N and NO3 - -N by the composite denitrifying bacteria are both above 93%. Among them, when the temperature is 30 °C, the degradation rate of NH4 + -N is 99.2%, and the degradation rate of NO3 - -N is 98.1%. When the temperature is 10 °C, the degradation rates of NH4 + -N and NO3 - -N slightly decrease, and the degradation rates are 80.6% and 71.7% respectively. It can be seen that the optimal temperature of this denitrifying bacteria is 30 °C, but its temperature tolerance range is relatively wide.

[0081] Example 5

[0082] Denitrification performance test of composite denitrifying bacteria under different pH values

[0083] Take the composite bacteria solution in Example 3 and inoculate it into the denitrification medium at an inoculation amount of 5%. The concentrations of nitrate nitrogen and ammonia nitrogen in the denitrification medium are both 100 mg / L. Set the pH value gradients of 5.0, 7.0, and 9.0. Then incubate at a constant temperature of 30 °C and 120 r / min for 2 days. After the incubation period ends, take samples, centrifuge, and measure the concentrations of ammonia nitrogen and nitrate nitrogen in the supernatant, and calculate their degradation rates. The results are as Figure 6 shown.

[0084] Among them, the method used to test the content of NH4 + -N is the Nessler reagent spectrophotometry method, and the method used to test the content of NO3 - -N is the ultraviolet spectrophotometer method.

[0085] It can be seen from Figure 6 that when the pH value is 7.0, the denitrification effect of the composite bacteria is the best. The degradation rate of NH4 + -N is 99.2%, and the degradation rate of NO3 - -N is 98.1%. However, the degradation rates of the composite bacteria under other pH value conditions also reach 90%. It can be seen that the optimal pH value of this denitrifying bacteria is 7.0, but its pH tolerance range is relatively wide.

[0086] Example 7

[0087] Denitrification performance test of composite denitrifying bacteria when the phenol concentration is 200 mg / L

[0088] The compound bacterial solution in Example 3 was inoculated into the denitrification medium at an inoculation amount of 5%, and the concentrations of nitrate nitrogen and ammonia nitrogen in the denitrification medium were both 100 mg / L, and 200 mg / L of phenol was added thereto. Then, it was incubated at a constant temperature of 30 °C and 120 r / min. Samples were taken every 12 h, centrifuged, and the concentrations of ammonia nitrogen, nitrate nitrogen, and phenol in the supernatant were measured, and their degradation rates were calculated. The results are as Figure 7 shown.

[0089] Among them, the method used to test the content of NH4 + -N is the Nessler's reagent spectrophotometry method, and the method used to test the content of NO3 - -N is the ultraviolet spectrophotometer method. The phenol concentration was determined by high performance liquid chromatography Waters e2695 (Waters, USA). Chromatographic conditions: C18 reversed-phase silica gel column, injection volume 10 μL, mobile phase acetonitrile to water volume ratio 6:4, flow rate 1.0 mL / min, wavelength 280 nm.

[0090] It can be seen from Figure 7 that at 48 h, the degradation rates of NH4 + -N and NO3 - -N both reached more than 98%. Phenol basically had no effect on the degradation of organic nitrogen by the compound bacteria, and after 36 hours, the compound bacteria showed an obvious degradation effect on phenol, and also reached 99.6% at 60 h. It can be seen that the compound bacteria also have a certain tolerance to phenol, and it also has great significance for treating some phenol-containing wastewater, such as coking wastewater and pharmaceutical wastewater.

[0091] Although the above embodiments have made a detailed description of the present invention, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A salt-tolerant composite denitrifying bacterium, characterized in that, It includes Klebsiella pneumoniae TYF-CJJ-P06 and Klebsiella sp. TYF-CJJ-P07; The Klebsiella pneumoniae TYF-CJJ-P06 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 17, 2024, with the deposit number CGMCC No. 29832; The Klebsiella sp. TYF-CJJ-P07 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 17, 2024, with the deposit number CGMCC No. 29833.

2. The salt-tolerant composite denitrifying bacteria according to claim 1, characterized in that, The viable cell number ratio of the Klebsiella pneumoniae TYF-CJJ-P06 and Klebsiella sp. TYF-CJJ-P07 is 1:

1.

3. Application of the salt-tolerant composite denitrifying bacteria described in claim 1 or 2 in removing organic nitrogen.

4. The application according to claim 3, characterized in that, The organic nitrogen includes ammonium nitrogen and / or nitrate nitrogen.

5. Application of the salt-tolerant composite denitrifying bacteria described in claim 1 or 2 in removing phenol.

6. A salt-tolerant denitrifying composite microbial inoculant, characterized in that, It includes Klebsiella pneumoniae TYF-CJJ-P06 and Klebsiella sp. TYF-CJJ-P07 in the salt-tolerant composite denitrifying bacteria described in claim 1.

7. The salt-tolerant denitrifying composite microbial inoculant according to claim 6, wherein The bacterial content of Klebsiella pneumoniae TYF-CJJ-P06 in the salt-tolerant denitrifying complex microbial inoculant is 5×10 9 CFU / ml; The bacterial content of Klebsiella sp. TYF-CJJ-P07 in the salt-tolerant denitrifying complex microbial inoculum is 5×10 9 CFU / ml.

8. Application of the salt-tolerant denitrifying composite microbial agent described in claim 7 in removing organic nitrogen in high-salt wastewater.

9. The application according to claim 7, characterized in that The application includes: mixing the salt-tolerant denitrifying composite microbial agent with high-salt wastewater, and degrading the organic nitrogen in the high-salt wastewater at a temperature of 20 - 35 °C, a pH value of 5.0 - 9.0, and a rotation speed of 120 rpm; The organic nitrogen includes ammonium nitrogen and / or nitrate nitrogen; The salinity of the high-salt wastewater is 0.1 - 70 g / L; The volume ratio of the salt-tolerant denitrifying composite microbial agent to the high-salt wastewater is 1:

20.

10. Application of the salt-tolerant denitrifying composite microbial agent described in claim 7 in removing phenol.

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