A microbial inoculant capable of withstanding low temperatures and efficiently removing nitrogen and its application

Abstract

The present invention provides a microbial inoculum capable of withstanding low temperatures and efficiently removing nitrogen, and its application, belonging to the field of microbial technology. The microbial inoculum provided by the present invention comprises Klebsiella pneumoniae TYF-CJJ-P07 and Klebsiella pneumoniae TYF-CJJ-A11; the preservation number of TYF-CJJ-P07 is CGMCC NO.29833, and the preservation number of TYF-CJJ-A11 is CGMCC NO.29831. The culture medium components required for activation and amplification of the microbial inoculum of the present invention are simple, and the process for preparing the bacterial liquid is relatively easy, which is conducive to industrial production and subsequent application; it can simultaneously remove ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in water, and has a high removal efficiency; it still maintains good nitrogen removal activity at low temperatures and can be used for nitrogen removal treatment of sewage in geographical regions with large annual temperature differences in northern China.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a microbial inoculum that can tolerate low temperatures and efficiently denitrify, and its application. Background Art

[0002] The problem of nitrogen pollution in water bodies in China is relatively severe, mainly manifested as an increase in the nitrogen content in water bodies, which is closely related to industrial development, urbanization processes, and agricultural activities. Nitrogen pollution mainly comes from industrial and domestic sewage discharges, excessive use of agricultural fertilizers and pesticides, and discharges of livestock and poultry breeding wastes. These nitrogenous substances exist in the forms of ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen, leading to eutrophication of water bodies, causing algal blooms and excessive algal reproduction, and in severe cases, resulting in oxygen deficiency in water bodies, damaging the aquatic ecosystem.

[0003] Coking wastewater, food waste digestate, landfill leachate, semiconductor wastewater, feces of the breeding and livestock industries, etc. all contain relatively high nitrogen concentrations. During the denitrification treatment process of these wastewaters, they are not only affected by the water quality but also by external environments such as temperature. Due to geographical regions and seasonal changes, the temperature of domestic sewage in winter in high-altitude regions or northern regions can drop below 5°C, and most microorganisms in sewage biological treatment systems are mesophilic microorganisms, with their optimal growth temperature being 25°C - 37°C. When the temperature is lower than 15°C, the denitrification performance significantly decreases, and when it is lower than 5°C, the denitrification ability of the system is severely inhibited. How to improve the sewage biological denitrification performance under lower temperature conditions has become one of the research hotspots in water treatment. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a microbial inoculum that can tolerate low temperatures and efficiently denitrify, and its application. The microbial inoculum provided by the present invention can efficiently remove ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in water bodies, and still has good denitrification performance when the temperature is lower than 15°C.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] A microbial inoculum that can tolerate low temperatures and efficiently denitrify, the microbial inoculum includes Klebsiella sp. TYF-CJJ-P07 and Klebsiella sp. TYF-CJJ-A11; the preservation number of TYF-CJJ-P07 is CGMCC NO.29833, and the preservation number of TYF-CJJ-A11 is CGMCC NO.29831; both TYF-CJJ-P07 and TYF-CJJ-A11 are preserved in the China General Microbiological Culture Collection Center, the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation date is January 17, 2024.

[0007] In the present invention, the viable count ratio of TYF-CJJ-P07 and TYF-CJJ-A11 in the microbial inoculum is preferably 1:1.

[0008] In the present invention, the activation culture conditions of TYF-CJJ-P07 and TYF-CJJ-A11 are preferably a rotation speed of 80 r / min to 180 r / min, a temperature of 20 °C to 35 °C, an initial pH of the culture medium of 7.0 to 8.0, and constant temperature shaking culture for 24 h to 48 h.

[0009] In the present invention, the low temperature is preferably 6 °C to 20 °C.

[0010] In the present invention, the use temperature of the microbial inoculum is preferably 6 °C to 35 °C.

[0011] The present invention also provides the application of the microbial inoculum described in the above technical solution in sewage treatment.

[0012] The present invention also provides the application of the microbial inoculum described in the above technical solution in water body restoration.

[0013] In the present invention, the microbial inoculum can convert ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen into gaseous nitrogen.

[0014] In the present invention, the pH value of the sewage or water body is preferably 5 to 9.

[0015] In the present invention, the concentration of dissolved oxygen in the sewage or water body is preferably ≥ 3 mg / L; calculated by the NaCl concentration, the salinity is preferably 0.1 g / L to 30 g / L.

[0016] In the present invention, the residence time of the microbial inoculum in the sewage or water body is preferably ≥ 2 d.

[0017] Beneficial technical effects: The present invention provides a microbial inoculum that can tolerate low temperature and has high-efficiency denitrification. The microbial inoculum includes Klebsiella sp. TYF-CJJ-P07 and Klebsiella sp. TYF-CJJ-A11; the preservation number of TYF-CJJ-P07 is CGMCC NO.29833, and the preservation number of TYF-CJJ-A11 is CGMCC NO.29831. The components of the culture medium required for activation and expansion of the microbial inoculum of the present invention are simple, and the process for preparing the bacterial liquid is relatively easy, which is conducive to industrial production and subsequent applications; it can simultaneously remove ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in the water body, and has a high removal efficiency; it still maintains good denitrification activity at low temperature and can be used for sewage denitrification treatment in geographical regions with large annual temperature differences in northern China. Description of the Drawings

[0018] Figure 1 Phylogenetic trees of strains TYF-CJJ-P07 and TYF-CJJ-A11;

[0019] Figure 2 Agarose gel electrophoresis pattern of the 16S rRNA gene amplification products of strains TYF-CJJ-P07 and TYF-CJJ-A11;

[0020] Figure 3 Graph showing the denitrification performance test of the composite denitrifying bacteria under different initial pH conditions. Detailed implementation manners

[0021] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with embodiments. However, the content of the present invention is not limited to the following embodiments. The materials, reagents, etc. used in the embodiments and experimental examples of the present invention can be obtained from commercial channels without special instructions; the methods used in the embodiments and experimental examples of the present invention are conventional methods without special instructions.

[0022] Materials and instruments

[0023] (1) Sample source:

[0024] Collect the activated sludge from the aerobic tank of Qingxu Hongbo Sewage Treatment Plant and the water samples from the wetland in the middle and lower reaches of the Fenhe River (September 2021).

[0025] (2) Culture media:

[0026] Beef extract peptone medium: Beef extract 5 g / L, peptone 10 g / L, NaCl 5 g / L, pH = 7.0 ± 0.2.

[0027] Denitrification medium: C6H5Na3O7·2H2O 5.719 g / L, KNO3 0.722 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.

[0028] Nitrification medium: C6H5Na3O7·2H2O 5.719 g / L, (NH4)2SO4 0.472 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.

[0029] Denitrification medium: 5.719 g / L of C6H5Na3O7·2H2O, 0.472 g / L of (NH4)2SO4, 0.722 g / L of KNO3, 0.246 g / L of NaNO2, 0.200 g / L of K2HPO4, 0.050 g / L of MgSO4·7H2O, 0.010 g / L of MnSO4·4H2O, 0.010 g / L of FeSO4, 0.120 g / L of NaCl, pH = 7.0 ± 0.2.

[0030] Solid medium: Add 2% - 2.5% agar to 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.

[0031] (3) Main experimental instruments:

[0032] Constant temperature biochemical incubator, high-speed refrigerated centrifuge, constant temperature shaker, laminar flow hood, vertical pressure steam sterilizer, full-wavelength microplate reader, PCR instrument, electrophoresis instrument, etc.

[0033] Example 1

[0034] (1) Enrichment of strains

[0035] Inoculate 10 mL each of the retrieved activated sludge and river water samples into a conical flask containing 90 mL of sterilized beef extract peptone medium, and culture at 120 r / min and 30 °C for 5 d.

[0036] (2) Isolation and preservation of strains

[0037] In the laminar flow hood, dilute the enriched culture solution with sterile water in gradient and spread it on the nitrification 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 nitrification 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 nitrification liquid medium, culture for 24 h under the same conditions, inoculate them 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 in a ratio of 1:1, and then store it frozen in an -80 °C refrigerator. Take samples to measure the NH4 + -N content, and further screen out the strains that can efficiently degrade ammonia nitrogen for the next experiment.

[0038] (3) Re-screening of strains under low temperature conditions

[0039] Pre-culture each strain to the logarithmic growth phase and measure its OD 600The initial OD values of each strain were calculated by centrifugation to remove the influence of the initial culture medium, and the culture medium was washed three times with sterile water. 600 Adjust to the same level and inoculate 5% of the inoculum into the nitrification medium and denitrification medium. Blank medium plus sludge as a control, culture at 10 ° C, 120 r / min conditions, culture for 48 hours, every 12 hours to measure NH4 + -N and NO3 - -N concentration, three replicates were set for each group. Finally, NH4 + -N and NO3 - The strains with the highest -N removal rates were numbered TYF-CJJ-P07 and TYF-CJJ-A11. TYF-CJJ-P07 was isolated from the activated sludge in the aerobic tank of the Qingxu Hongbo Wastewater Treatment Plant, and TYF-CJJ-A11 was isolated from water samples collected from the Fenhe River wetland.

[0040] (4) Molecular biological identification of strains

[0041] The purified strain was inoculated into basal culture medium and cultured at 120 rpm and 30°C for at least 12 hours. DNA was extracted from the bacterial suspension and used as a template. The universal primer pair (27F (SEQ ID NO. 3): 5'-AGAGTTTGATCCTGGCTCAG-3'; downstream primer 1492R (SEQ ID NO. 4): 5'-TACGGCTACCTTGTACGACTT-3') was used. The PCR reaction system consisted of 12.5 μL of 10X PCR buffer, 10 mM dNTPs, 5 U / μL of Taq DNA polymerase, and 50 mM MgSO₄. The reaction mixture was 1 μL of each primer (10 μM), 1 μL of template DNA, and 9.5 μL of ddH₂O for a total of 25 μL. The reaction conditions for PCR amplification were as follows: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 90 s, for 30 cycles; extension at 72°C for 5–10 min; and storage at 4°C for 15 min.

[0042] The PCR amplification products were subjected to agarose gel electrophoresis. The results of agarose gel electrophoresis showed that the amplification product bands of strains TYF-CJJ-P07 and TYF-CJJ-A11 were bright at about 1500bp, and there were no other miscellaneous bands, such as Figure 2 shown.

[0043] 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-P07 and TYF-CJJ-A11 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 strains TYF-CJJ-P07 and TYF-CJJ-A11 had 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 MEGA 11.0 software, the phylogenetic tree was constructed by the Neighbor Joining method, as Figure 1 shown, to further analyze the genus and species of the strains.

[0044] SEQ ID NO.1 (TYF-CJJ-P07):

[0045]

[0046] SEQ ID NO.2 (TYF-CJJ-A11):

[0047] GGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGA

[0048] ACGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGTGGCGGACGGGT

[0049] GAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAA

[0050] CGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTC

[0051] GGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGG

[0052] GGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGA

[0053] CCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCA

[0054] GCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCC

[0055] GCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAG

[0056] GAAGGCAGTAAGGTTAATAACCTTGTTCATTGACGTTACCCGCAGAAG

[0057] AAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTG

[0058] CAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTG

[0059] TCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGA

[0060] AACTGGCAGGCTGGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAG

[0061] CGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGC

[0062] CCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAA

[0063] CAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGACTTGGA

[0064] GGTTGTTCCCTTGAGGAGTGGCTTCCGGAGCTAACGCGTTAAGTCGAC

[0065] CGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGG

[0066] GGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGA

[0067] AGAACCTTACCTACTCTTGACATCCAGAGAACTTAGCAGAGATGCTTT

[0068] GGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCT

[0069] CGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTAT

[0070] CCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGT

[0071] GATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTA

[0072] CGAGTAGGGCTACACACGTGCTACAATGGCATATACAAAGAGAAGCGA

[0073] CCTCGCGAGAGCAAGCGGACCTCATAAAGTATGTCGTAGTCCGGATTG

[0074] GAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGA

[0075] TCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCG

[0076] TCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCG

[0077] GGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGAAAAGGGGGGAAAACCA.

[0078] Example 2: Denitrification performance test of composite denitrifying bacteria at 6°C

[0079] The TYF-CJJ-P07 and TYF-CJJ-A11 strains stored in a -80°C refrigerator in Example 1 were inoculated into nitrification medium and then placed in a shaker at 30°C for activation. After they grew to the logarithmic phase, their OD values were measured. 600 The initial OD values of the two strains were calculated by centrifugation to remove the influence of the initial culture medium, and the culture medium was washed three times with sterile water. 600 Adjust to the same level and prepare a composite bacterial solution in a volume ratio of 1:1.

[0080] The composite bacterial solution was then inoculated at a 5% inoculum into a denitrified medium containing 100 mg / L nitrate and ammonia nitrogen. The culture was then incubated at 6°C and 120 rpm for 2 days. A control group was also inoculated with an equal volume of the composite bacterial solution sterilized by high-temperature steam, using the same culture conditions as the inoculated group. After the incubation period, samples were collected and centrifuged to determine the concentrations of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in the supernatant. The results are shown in Table 1.

[0081] Among them, NH4 + -N content is detected by Nessler's reagent spectrophotometry, testing NO3 - -N content is detected by UV spectrophotometry, testing NO2 - The -N content was determined by N-(1-naphthyl)-ethylenediamine spectrophotometry.

[0082] Table 1 Denitrification performance test of composite denitrifying bacteria at 6°C

[0083]

[0084] As can be seen from Table 1, after 2 days, the degradation rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in the treatment group inoculated with the composite bacterial solution were 56.3%, 49.5% and 37.5% respectively; the degradation rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in the control group water sample inoculated with the sterilized composite bacterial solution were only 0.1%, 0.2% and 0.2% respectively. This result shows that the denitrification effect of inoculating the composite bacteria is better under the low temperature condition of 6°C.

[0085] Example 3 Denitrification performance test of composite denitrifying bacteria at 10°C

[0086] Take the composite bacterial solution in Example 2 and inoculate it into the denitrification medium at an inoculation amount of 5%, and then incubate it at a constant temperature of 10°C and 120 r / min for 2 days. Another control group was inoculated with an equal volume of the composite bacterial solution sterilized by high-temperature steam, and other culture conditions were the same as those of the inoculation group. After the incubation period ended, the samples were centrifuged and the concentrations of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in the supernatant were measured. The test method was the same as that in Example 2. The results are shown in Table 2.

[0087] Table 2 Denitrification performance test of composite denitrifying bacteria at 10°C

[0088]

[0089]

[0090] As can be seen from Table 2, after 2 days, the degradation rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in the treatment group inoculated with the composite bacterial solution were 81.3%, 73.7% and 74.5% respectively; the degradation rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in the control group water sample inoculated with the sterilized composite bacterial solution were only 0.2%, 0.4% and 0.3% respectively. This result shows that after inoculating the composite bacteria, the denitrification effect under the low temperature condition of 10°C is improved compared with that under the condition of 6°C, and the effect is obvious.

[0091] Example 4 Denitrification performance test of composite denitrifying bacteria at 20°C)

[0092] Take the composite bacterial solution in Example 2 and inoculate it into the denitrification medium at an inoculation amount of 5%, and then incubate it at a constant temperature of 20°C and 120 r / min for 2 days. Another control group was inoculated with an equal volume of the composite bacterial solution sterilized by high-temperature steam, and other culture conditions were the same as those of the inoculation group. After the incubation period ended, the samples were centrifuged and the concentrations of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in the supernatant were measured. The test method was the same as that in Example 2. The results are shown in Table 3.

[0093] Table 3 Denitrification performance test of composite denitrifying bacteria at 20 °C

[0094]

[0095] As can be seen from Table 3, after 2 days, the degradation rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in the treatment group inoculated with the composite bacterial solution were 94.5%, 93.7% and 88.6% respectively; the degradation rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in the control group water sample inoculated with the sterilized composite bacterial solution were only 0.1%, 0.2% and 0.1% respectively. This result shows that the composite denitrifying bacteria have good denitrification performance at 20 °C.

[0096] Example 5 Denitrification performance test of composite denitrifying bacteria at 35 °C

[0097] Take the composite bacterial solution in Example 2 and inoculate it into the denitrification medium at an inoculation amount of 5%. Then, incubate it at a constant temperature of 35 °C and 120 r / min for 2 days. Another control group inoculated with an equal volume of the composite bacterial solution sterilized by high-temperature steam was set, and other culture conditions were the same as those of the inoculation group. After the end of the culture period, samples were taken and centrifuged, and then the concentrations of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in the supernatant were measured. The test method was the same as that in Example 2. The results are shown in Table 4.

[0098] Table 4 Denitrification performance test of composite denitrifying bacteria at 35 °C

[0099]

[0100]

[0101] As can be seen from Table 4, after 2 days, the degradation rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in the treatment group inoculated with the composite bacterial solution were 99.2%, 98.1% and 92.4% respectively; the degradation rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in the control group water sample inoculated with the sterilized composite bacterial solution were only 0.5%, 0.3% and 0.2% respectively. This result shows that the composite denitrifying bacteria have excellent denitrification performance at 35 °C.

[0102] Example 6 Denitrification performance test of composite denitrifying bacteria under different initial pH conditions

[0103] Take the composite bacterial solution in Example 2 and inoculate it into the denitrification media with different initial pH values at an inoculation amount of 5%. Set the pH gradient to 5, 7, and 9. Incubate it at a constant temperature of 25 °C and 120 r / min for 2 days. After the end of the culture period, samples were taken and centrifuged, and then the concentrations of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in the supernatant were measured. The test method was the same as that in Example 2. The results are as Figure 3 shown.

[0104] From Figure 3It can be seen that when the pH value is 7, the denitrification effect of the composite bacteria is the best, but the degradation rates of the composite bacteria under other pH value conditions also reach about 90%. It can be seen from this that the optimal pH of this bacterial community is 7.0, but its tolerated pH range is relatively wide.

[0105] Comparative Example 1

[0106] The invention patent with the application number 202211635729.0 discloses a strain of low-temperature aerobic synchronous denitrifying and phosphorus-removing bacteria and its application. This patent involves a Klebsiella oxytoca D1 bacterial agent, and under the low-temperature conditions of 10°C to 15°C in sewage, the degradation rates of NO3-N and TP are 85% and 78% respectively. [[ID=⑧]]

[0107] A comparative experiment was carried out with Experiment 2 in this invention patent, and the effects were compared under the same experimental conditions.

[0108] Take the composite bacterial liquid in Example 2 and inoculate it into two test tubes a and b containing 20 mL of denitrifying phosphorus-deficient medium respectively at an inoculation amount of 5%, and additionally add 1.0 g / L of (NH4)2SO4 to this medium to measure the ammonia nitrogen removal ability of this composite bacteria. Place test tubes a and b in a forced-air constant-temperature incubator, and at the same time place a test tube c containing only 20 mL of denitrifying phosphorus-deficient medium without inoculating the bacterial liquid in this incubator as a reference for abiotic denitrification. After culturing at 20°C to 25°C for 36 hours, take samples and centrifuge them, and then measure the contents of ammonia nitrogen and nitrate nitrogen in the supernatant. The test method is the same as that in Example 2.

[0109] The composition of the above denitrifying phosphorus-deficient medium is: KNO3 2.0 g / L, sodium citrate 5.0 g / L, K2HPO4 0.05 g / L, MgSO4·7H2O 0.2 g / L, CaCl2 0.5 g / L, trace elements 2 mL / L; the composition of the trace elements is: FeCl3·6H2O 1.5 g / L, H3BO3 0.15 g / L, CuSO4·5H2O 0.03 g / L, KI 0.03 g / L, Na2MoO4·2H2O 0.06 g / L, MnCl2·4H2O 0.12 g / L, ZnSO4·7H2O 0.12 g / L, CoCl2·2H2O 0.12 g / L.

[0110] The results of 3 groups of parallel experiments show that at the original NO3 - -N concentration of 277 mg / L, NH4 +Under the condition that the -N concentration is 212 mg / L, the nitrate nitrogen removal rate of this complex bacterium is about 91.3%. Compared with the removal rate of Klebsiella oxytoca D, which is 85.7% - 90.5%, there is an improvement. Moreover, this complex bacterial community can simultaneously remove ammonium nitrogen, and the ammonium nitrogen removal rate reaches about 93.4%. It has a broader application prospect compared with Klebsiella oxytoca D1.

[0111] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A microbial inoculant that can withstand low temperatures and has high-efficiency denitrification, characterized in that, The microbial inoculum includes Klebsiella sp. TYF-CJJ-P07 and Klebsiella sp. TYF-CJJ-A11; the preservation number of TYF-CJJ-P07 is CGMCC NO.29833, and the preservation number of TYF-CJJ-A11 is CGMCC NO.29831; both TYF-CJJ-P07 and TYF-CJJ-A11 are preserved in the China General Microbiological Culture Collection Center, with the preservation address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation date being January 17, 2024; The ratio of viable counts of TYF-CJJ-P07 and TYF-CJJ-A11 in the microbial inoculum is 1:1; The low temperature is 6°C to 20°C; The application temperature of the microbial inoculum is 6°C to 35°C.

2. Application of the microbial inoculum according to claim 1 in sewage treatment.

3. Application of the microbial inoculum according to claim 1 in water body restoration.

4. The application according to claim 2 or 3, characterized in that, The microbial inoculum can convert ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen into gaseous nitrogen.

5. The application according to claim 2 or 3, characterized in that, The pH of the sewage or water body is 5 to 9.

6. The application according to claim 2 or 3, characterized in that, The residence time of the microbial inoculum in the sewage or water body is ≥2 d.

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