Microbial agent capable of resisting low temperature and efficiently denitrifying and application of microbial agent

Abstract

The invention provides a low-temperature-resistant efficient denitrification microbial agent and application thereof, and belongs to the technical field of microorganisms. The microbial agent provided by the invention comprises klebsiella sp. TYF-CJJ-P07 and klebsiella sp. TYF-CJJ-A11, and is characterized in that the klebsiella sp. TYF-CJJ-P07 and the klebsiella sp. The preservation number of the TYF-CJJ-P07 is CGMCC (China General Microbiological Culture Collection Center) NO.29833, and the preservation number of the TYF-CJJ-A11 is CGMCC NO.29831. The invention further discloses a preparation method of the strain. The components of the culture medium required by activation and propagation of the microbial agent are simple, the preparation process of the bacterial liquid is relatively easy, and industrial production and subsequent application are facilitated; ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in the water body can be removed at the same time, and the removal efficiency is high; and the catalyst can still maintain good denitrification activity at low temperature, and can be used for sewage denitrification treatment in geographic areas with large annual average temperature difference change in northern China.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a low-temperature-resistant and highly efficient denitrification microbial agent and application thereof. Background Art

[0002] The nitrogen pollution problem in my country's water bodies is quite serious, mainly manifested in the increase of nitrogen content in water bodies, which is closely related to industrial development, urbanization and agricultural activities. Nitrogen pollution mainly comes from industrial and domestic sewage discharge, excessive use of agricultural fertilizers and pesticides, and discharge of livestock and poultry breeding waste. These nitrogen exist in the form of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen, leading to eutrophication of water bodies, causing algal blooms and excessive algae growth. In severe cases, it can also cause water hypoxia and damage aquatic ecosystems.

[0003] Coking wastewater, food waste digestion liquid, landfill leachate, semiconductor wastewater, livestock manure, etc. all contain high nitrogen concentrations. These wastewaters are not only affected by water quality during the denitrification process, but also by the external environment such as temperature. Due to geographical and seasonal changes, the temperature of domestic sewage in high altitude areas or northern areas can drop below 5°C in winter, while most microorganisms in sewage biological treatment systems are mesophilic microorganisms, and their optimal growth temperature is 25°C to 37°C. When the temperature is below 15°C, the denitrification performance decreases significantly, and when it is below 5°C, the system's denitrification capacity is severely inhibited. How to improve the biological denitrification performance of sewage under lower temperature conditions has become one of the hot topics in water treatment research. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a low-temperature resistant and highly efficient denitrification microbial agent and its application. The microbial agent 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 below 15°C.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A low-temperature-resistant and highly efficient denitrifying microbial agent, the microbial agent comprising Klebsiella sp. TYF-CJJ-P07 and Klebsiella sp. TYF-CJJ-A11; the deposit number of TYF-CJJ-P07 is CGMCC NO.29833, and the deposit number of TYF-CJJ-A11 is CGMCC NO.29831; TYF-CJJ-P07 and TYF-CJJ-A11 are both deposited in the General Microbiology Center of China Microbiological Culture Management Committee, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit date is January 17, 2024.

[0007] In the present invention, the ratio of the number of viable bacteria of TYF-CJJ-P07 and TYF-CJJ-A11 in the microbial agent 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, 20° C. to 35° C., an initial culture medium pH 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 microbial agent is preferably used at a temperature of 6°C to 35°C.

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

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

[0013] In the present invention, the microbial agent 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 5-9.

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

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

[0017] Beneficial technical effects: The present invention provides a low-temperature-resistant and highly efficient denitrification microbial agent, the microbial agent comprising Klebsiella sp. TYF-CJJ-P07 and Klebsiella sp. TYF-CJJ-A11; the deposit number of TYF-CJJ-P07 is CGMCC NO.29833, and the deposit number of TYF-CJJ-A11 is CGMCC NO.29831. The microbial agent of the present invention has simple culture medium components required for activation and expansion, and the bacterial liquid preparation process is relatively easy, which is conducive to industrial production and subsequent applications; it can simultaneously remove ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in water bodies, and has high removal efficiency; it still maintains good denitrification activity at low temperatures, and can be used for sewage denitrification treatment in geographical areas in northern my country with large annual average temperature differences. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0019] Figure 2 It is the agarose gel electrophoresis diagram of the 16S rRNA gene amplification products of strains TYF-CJJ-P07 and TYF-CJJ-A11;

[0020] Figure 3 This is a test chart of the denitrification performance of composite denitrifying bacteria under different initial pH conditions. DETAILED DESCRIPTION

[0021] In order to better understand the present invention, the content of the present invention is further explained in conjunction with the following examples, but the content of the present invention is not limited to the following examples. The materials, reagents, etc. used in the examples and test examples of the present invention, unless otherwise specified, can be obtained from commercial channels; the methods used in the examples and test examples of the present invention, unless otherwise specified, are conventional methods.

[0022] Materials and instruments

[0023] (1) Sample source:

[0024] Activated sludge samples were collected from the aerobic tank of Qingxu Hongbo Sewage Treatment Plant and water samples from the middle and lower reaches of the Fenhe River wetlands (September 2021).

[0025] (2) Culture medium:

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

[0027] Denitrification medium: C 6 H 5 Na 3 O 7 ·2H 2 O 5.719g / L, KNO 3 0.722g / L, K 2 HPO 4 0.200g / L, MgSO 4 7H 2 O 0.050g / L, MnSO 4 ·4H 2 O 0.010g / L, FeSO 4 0.010g / L, NaCl 0.120g / L, pH=7.0±0.2.

[0028] Nitration medium: C 6 H5 Na 3 O 7 ·2H 2 O 5.719 g / L, (NH 4 ) 2 SO 4 0.472g / L, K 2 HPO 4 0.200g / L, MgSO 4 7H 2 O 0.050g / L, MnSO 4 ·4H 2 O 0.010g / L, FeSO 4 0.010g / L, NaCl 0.120g / L, pH=7.0±0.2.

[0029] Denitrification medium: C 6 H 5 Na 3 O 7 ·2H 2 O 5.719 g / L, (NH 4 ) 2 SO 4 0.472g / L, KNO 3 0.722 g / L, NaNO 2 0.246g / L, K 2 HPO 4 0.200g / L, MgSO 4 7H 2 O 0.050g / L, MnSO 4 ·4H 2 O 0.010g / L, FeSO 4 0.010g / L, NaCl 0.120g / L, pH=7.0±0.2.

[0030] Solid culture medium is based on the above culture medium with 2% to 2.5% agar added. All culture media need to be sterilized under high pressure at 121°C for 30 minutes before use, and then cooled to room temperature before subsequent experiments.

[0031] (3) Main experimental instruments:

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

[0033] Example 1

[0034] (1) Enrichment of bacterial strains

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

[0036] (2) Isolation and preservation of bacterial strains

[0037] In the clean bench, the enriched culture fluid was gradiently diluted with sterile water and spread on the nitrification solid culture medium. After standing for 30 minutes, the plate was inverted and placed in a 30°C constant temperature incubator for more than 24 hours. Single colonies with different morphological characteristics were picked and inoculated into the nitrification liquid culture medium. After culturing at 120r / min and 30°C for 24 hours, they were streaked and purified on the plate. After repeating 3 times, the grown single colonies were inoculated into the nitrification liquid culture medium, cultured under the same conditions for 24 hours, inoculated into the paraffin slant culture medium, and stored in a 4°C refrigerator. At the same time, 500μL of bacterial solution was mixed with 50% glycerol in a 1:1 ratio and stored in a -80°C refrigerator. Sampling and determination of NH in the culture fluid 4 + -N content, and further screen out strains that can efficiently degrade ammonia nitrogen for the next experiment.

[0038] (3) Rescreening of strains under low temperature conditions

[0039] Each strain was pre-cultured to the logarithmic growth phase, and its OD was measured. 600 The 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 The same level was adjusted and inoculated in nitrification medium and denitrification medium at a 5% inoculation rate. The blank medium plus sludge was used as a control and cultured at 10°C and 120 r / min for 48 hours. Samples were taken every 12 hours to measure NH 4 + -N and NO 3 - -N concentration, three replicates were set for each group. 4 + -N and NO 3 - The strains with the highest -N removal rate were numbered TYF-CJJ-P07 and TYF-CJJ-A11. Among them, strain TYF-CJJ-P07 was isolated from the activated sludge in the aerobic pool of Qingxu Hongbo Wastewater Treatment Plant, and TYF-CJJ-A11 was isolated from water samples collected from Fenhe Wetland.

[0040] (4) Molecular biological identification of strains

[0041] The purified strain was inoculated into the basic culture medium, cultured at 120 r / min and 30°C for more than 12 h, and the bacterial liquid DNA was extracted as a template, and 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 was: 10X PCR Buffer, dNTP (10 mM), Taq DNA polymerase (5 U / μL), 50 mM MgSO 4 , 12.5 μL in total, 1 μL of each primer (10 μM), 1 μL of template DNA, ddH 2 O 9.5μL, total system 25μL. PCR amplification reaction conditions: 95℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 57℃ annealing for 30s, 72℃ extension for 90s, 30 cycles; 72℃ extension for 5min~10min; 4℃ storage for 15min.

[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 1500 bp, and there were no other mixed bands, such as Figure 2 shown.

[0043] The 16S rDNA product obtained by PCR amplification was 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 in 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 were most closely related to the genus Klebsiella. BLAST (http: / / www.ncbi.nlm.nih.gov / blast / ) was then used to search for strains with higher similarity. The phylogenetic tree was constructed using MEGA 11.0 software by selecting the Neighbor Joining method, as shown in the figure. Figure 1 The strains were further analyzed to their genus and species as shown.

[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 the nitrification medium and then placed in a 30°C shaker 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] Then take the composite bacterial solution and inoculate it into the denitrification medium at an inoculation volume of 5%, and the concentrations of nitrate nitrogen and ammonia nitrogen in the denitrification medium are both 100 mg / L. Then, culture at a constant temperature of 6°C and 120r / min for 2 days. Set up another control group with an equal volume of composite bacterial solution sterilized by high-temperature steam, and the other culture conditions are the same as those of the inoculation group. After the culture period, take a sample and centrifuge it 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, NH 4 + The detection method of -N content is Nessler's reagent spectrophotometry, testing NO 3 - -N content is detected by UV spectrophotometer, testing NO 2- The -N content was determined by N-(1-naphthyl)-ethylenediamine spectrophotometry.

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

[0083]

[0084] As shown in 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 inoculated with the sterilized composite bacterial solution were only 0.1%, 0.2% and 0.2% respectively. The results show that the denitrification effect of inoculating composite bacteria is better under low temperature conditions of 6℃.

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

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

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

[0088]

[0089]

[0090] As shown in 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 inoculated with the sterilized composite bacterial solution were only 0.2%, 0.4% and 0.3% respectively. The results show that after inoculation with the composite bacteria, the denitrification effect at low temperature of 10℃ is better than that at 6℃, and the effect is obvious.

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

[0092] The composite bacterial solution in Example 2 was inoculated into the denitrification medium at an inoculum volume of 5%, and then cultured at a constant temperature of 20°C and 120r / min for 2 days. An equal volume of composite bacterial solution sterilized by high-temperature steam was inoculated as a control group, and other culture conditions were the same as those of the inoculation group. After the culture period, the concentrations of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in the supernatant were determined after sampling and centrifugation. 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℃

[0094]

[0095] As shown in 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 inoculated with the sterilized composite bacterial solution were only 0.1%, 0.2% and 0.1% respectively. The results show that the composite denitrification bacteria have good denitrification performance at 20℃.

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

[0097] The composite bacterial solution in Example 2 was inoculated into a denitrification medium at an inoculum volume of 5%, and then cultured at a constant temperature of 35°C and 120r / min for 2 days. An equal volume of composite bacterial solution sterilized by high-temperature steam was inoculated as a control group, and other culture conditions were the same as those of the inoculation group. After the culture period, the concentrations of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in the supernatant were determined after sampling and centrifugation. The test method was the same as in Example 2. The results are shown in Table 4.

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

[0099]

[0100]

[0101] As shown in 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 inoculated with the sterilized composite bacterial solution were only 0.5%, 0.3% and 0.2% respectively. The results show that the composite denitrification 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 denitrification medium with different initial pH at an inoculum of 5%, and set the pH gradient to 5, 7, and 9. Culture at 25°C and 120r / min for 2 days. After the culture period, take a sample and centrifuge it to determine the concentrations of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in the supernatant. The test method is the same as in Example 2. The results are as follows Figure 3 shown.

[0104] from Figure 3 It can be seen that the composite bacteria has the best denitrification effect at pH 7, but the degradation rate of the composite bacteria under other pH conditions also reached about 90%. It can be seen that the optimal pH of the bacterial community is 7.0, but its tolerance range of pH is relatively wide.

[0105] Comparative Example 1

[0106] The invention patent with application number 202211635729.0 discloses a strain of low-temperature aerobic synchronous nitrogen and phosphorus removal bacteria and its application. The patent involves an acid-producing Klebsiella D1 bacterial agent, which has the degradation rates of NO3-N and TP of 85% and 78% respectively under low temperature conditions of 10℃ to 15℃ in sewage.

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

[0108] The composite bacterial solution in Example 2 was inoculated with 5% inoculum into two test tubes a and b, each containing 20 mL of denitrification phosphorus-deficient culture medium, and 1.0 g / L of (NH 4 ) 2 SO 4 , in order to determine the removal capacity of the composite bacteria for ammonia nitrogen. Test tubes a and b were placed in a constant temperature incubator with forced draft, and a test tube c containing only 20 mL of denitrification phosphorus-deficient culture medium without inoculation of bacterial solution was placed in the incubator as a reference for abiotic denitrification. After culturing at 20°C to 25°C for 36 hours, the contents of ammonia nitrogen and nitrate nitrogen in the supernatant were determined after sampling and centrifugation. The test method was the same as in Example 2.

[0109] The above denitrification phosphorus-deficient culture medium is composed of: KNO 3 2.0g / L, sodium citrate 5.0g / L, K 2 HPO 4 0.05g / L, MgSO 4 7H 2 O 0.2g / L, CaCl 2 0.5g / L, trace elements 2mL / L; trace element composition: FeCl 3 6H 2 O 1.5 g / L, H3 BO 3 0.15g / L,CuSO 4 ·5H 2 O 0.03 g / L, KI 0.03 g / L, Na 2 MoO 4 ·2H 2 O 0.06g / L, MnCl 2 ·4H 2 O0.12g / L, ZnSO 4 7H 2 O 0.12g / L, CoCl 2 ·2H 2 O 0.12g / L.

[0110] The results of three parallel experiments showed that in the original NO 3 - -N concentration is 277mg / L, NH 4 + Under the condition of -N concentration of 212 mg / L, the removal rate of nitrate nitrogen by the composite bacteria is about 91.3%, which is significantly improved compared with the removal rate of 85.7% to 90.5% of Klebsiella oxytoca D. In addition, the composite bacterial community can also remove ammonia nitrogen at the same time, with an ammonia nitrogen removal rate of about 93.4%, which has broader application prospects than Klebsiella oxytoca D1.

[0111] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A microbial agent that is resistant to low temperatures and has high efficiency in denitrification, characterized in that: The microbial agent 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; TYF-CJJ-P07 and TYF-CJJ-A11 are both preserved in the General Microbiology Center of China Microbiological Culture Management Committee, and the preservation address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the preservation date is January 17, 2024.

2. The low-temperature resistant denitrification bacterial agent according to claim 1, characterized in that The ratio of the number of live bacteria of TYF-CJJ-P07 and TYF-CJJ-A11 in the microbial agent is 1:

1.

3. The low-temperature resistant denitrification bacterial agent according to claim 1, characterized in that The low temperature is 6°C to 20°C.

4. The low-temperature resistant denitrification bacterial agent according to claim 1, characterized in that The microbial agent is used at a temperature of 6°C to 35°C.

5. Use of the microbial agent according to any one of claims 1 to 4 in sewage treatment.

6. Use of the microbial agent according to any one of claims 1 to 4 in water body remediation.

7. The use according to claim 5 or 6, characterized in that: The microbial agent can convert ammonia nitrogen, nitrate nitrogen and nitrite nitrogen into gaseous nitrogen.

8. The use according to claim 5 or 6, characterized in that: The pH of the sewage or water body is 5-9.

9. The use according to claim 5 or 6, characterized in that: The residence time of the microbial agent in sewage or water body is ≥2 days.

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