Coupling nitrogen catabolism complex microbial inoculant and application thereof

Abstract

The application relates to a coupling nitrogen catabolism composite microbial agent and application thereof, and belongs to the technical field of biology. The application constructs an 'ammoniation-Dirammox' coupling system by screening high-efficiency ammoniation strains (lysine bacillus L11) and direct ammonia oxidation strains (alkaligenes M8), and optimizes the co-culturing time sequence (L11 is inoculated 48 hours in advance), so that organic nitrogen is directly converted into N2. The total nitrogen removal rate in the synchronous ammoniation and nitrification medium reaches 96.84%, and the ammonia nitrogen removal rate reaches 99.37%. In actual application, the agent dosing makes the effluent ammonia nitrogen of the sewage treatment system stable at 9.44 mg / L, and the COD is reduced to 455.98 mg / L, which is significantly better than that of the traditional process. The application solves the problems of low treatment efficiency of high-ammonia-nitrogen wastewater and serious secondary pollution, and is suitable for the collaborative treatment of aged leachate and perishable garbage leachate.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a coupling nitrogen catabolism composite microbial inoculant and its application in the denitrification of landfill leachate wastewater. BACKGROUND

[0002] With the rapid growth of municipal solid waste, the traditional sanitary landfill technology is gradually replaced due to its high pollution and low efficiency. Under the background of "zero landfill", the treatment and management of landfill leachate face new challenges and opportunities. In view of the collaborative disposal mode demand of aged leachate and perishable garbage leachate under the background of "zero landfill", considering the complexity of leachate composition, especially the biochemical parameter characteristics of high ammonia nitrogen concentration of aged leachate and high organic nitrogen concentration of perishable garbage leachate, how to develop the collaborative treatment mode of the two, realize low carbon, high efficiency and green treatment effect, adapt to the treatment demand of harmless treatment and resource utilization of municipal solid waste, has important social value and economic benefit.

[0003] The leachate accumulated in the landfill for more than 5 years has the biochemical characteristics of near neutral pH value, decreased chemical oxygen demand (COD) and biochemical oxygen demand (BOD5) concentration, reduced BOD5 / COD ratio, and increased ammonia nitrogen concentration, resulting in poor biodegradability, high treatment cost and great difficulty. With the closure of the landfill sites in the first-line cities, the disposal problem of the existing aged leachate becomes particularly prominent. The waste of these leachates gradually rots, resulting in the degradation of most COD into humus, and it is also very difficult to proliferate sludge. Patent CN116179423A designs a simultaneous nitrification and denitrification artificial denitrification cell inoculant according to the physicochemical characteristics of high ammonia nitrogen concentration of landfill leachate, which significantly improves the removal efficiency of high concentration ammonia nitrogen of aged landfill leachate.

[0004] With the widespread implementation of "zero landfill" policy in many cities in China, the current perishable garbage in cities is mainly disposed by centralized anaerobic fermentation, and the landfill leachate containing high organic matter concentration produced in the process also faces problems such as complex and variable composition and great difficulty in disposal. Strengthening the comprehensive management of landfill leachate, especially developing low-carbon collaborative treatment technology of landfill leachate under the background of "zero landfill", has important social significance for ensuring the healthy and sustainable development of cities.

[0005] In the traditional simultaneous nitrification and denitrification process, the subsequent conversion of NH4 + -N depends on the multi-stage nitrification-denitrification path, which is easy to cause secondary pollution due to the accumulation of intermediate products (NO2 - -N and NO3 - -N). Recent studies have found a new microbial nitrogen conversion pathway, direct ammonia oxidation (Dirammox), whose metabolic pathway is NH4 +-N→NH2OH→N2. Compared with the traditional nitrification-denitrification path, the accumulation of activated sludge can be significantly reduced, and a new treatment idea is provided for high ammonia-nitrogen wastewater treatment. On the basis of previous studies, the present application isolates and obtains ammonia and direct ammonia oxidation efficient strains, and constructs a co-culture system that can convert organic nitrogen to NH4 + -N directly to N2, forming a closed nitrogen cycle path. In order to realize the synergistic and efficient removal of organic nitrogen-ammonia-nitrogen-nitrogen, it is of important application value to develop a biological green purification process for landfill leachate. SUMMARY

[0006] In view of the problems existing in the prior art, the purpose of the present application is to design a technical scheme of a coupled nitrogen catabolism composite microbial inoculant and its application.

[0007] The present application specifically adopts the following technical scheme:

[0008] The present application provides a kind of ammonia and direct ammonia oxidation coupled system in the first aspect, and the system includes lysine Bacillus ( Lysinibacillus boronitolerans )ZJB24343 and Alcaligenes faecalis ( Alcaligenes faecalis )ZJB24344, the preservation number of the lysine Bacillus ( Lysinibacillus boronitolerans )ZJB24343 is CCTCC NO: M20251029, and the preservation number of the Alcaligenes faecalis ( Alcaligenes faecalis )ZJB24344 is CCTCC NO: M20251030.

[0009] The second aspect of the present application provides the application of the above-mentioned ammonia and direct ammonia oxidation coupled system in wastewater denitrification treatment.

[0010] Further, in the application, lysine Bacillus ( Lysinibacillus boronitolerans )ZJB24343 and Alcaligenes faecalis ( Alcaligenes faecalis )ZJB24344 are inoculated into nitrogen-containing wastewater in turn, wherein lysine Bacillus ( Lysinibacillus boronitolerans )ZJB24343 is inoculated at 0h, and Alcaligenes faecalis ( Alcaligenes faecalis )ZJB24344 is inoculated with a delay of 48h.

[0011] Further, in the application, the inoculation amount of the lysine Bacillus ( Lysinibacillus boronitolerans )ZJB24343 and Alcaligenes faecalis ( Alcaligenes faecalis )ZJB24344 is 1:1.

[0012] The third aspect of the present application provides a coupled nitrogen catabolism composite microbial inoculant containing the above-mentioned ammonia and direct ammonia oxidation coupled system.

[0013] Further, the coupling nitrogen catabolism complex microbial agent also includes Bacillus vallismortis (Bacillus vallismortis) Bacillus haynesii ), Bacillus subtilis (Bacillus subtilis) Bacillus subtilis ) and Rhodopseudomonas palustris (Rhodopseudomonas palustris) Stenotrophomonas riizophila ).

[0014] Further, the coupling nitrogen catabolism complex microbial agent also includes a preservative and a protective agent, the preservative is sodium benzoate, and the protective agent is magnesium chloride.

[0015] Further, the wet bacteria in the coupling nitrogen catabolism complex microbial agent are mixed with 0.5% preservative and 0.1% protective agent at a ratio of 3g / 1000 mL.

[0016] The fourth aspect of the present application provides the application of the above-mentioned coupling nitrogen catabolism complex microbial agent in sewage denitrification treatment.

[0017] The fifth aspect of the present application provides the application of the above-mentioned coupling nitrogen catabolism complex microbial agent in landfill leachate denitrification treatment.

[0018] The present application selects high-efficiency ammoniating strains (lysine bacillus L11) and direct ammonia-oxidizing strains (alkaligenes M8), constructs an "ammoniation-Dirammox" coupling system, optimizes the co-culturing time sequence (L11 is inoculated 48 hours in advance), and realizes the direct conversion of organic nitrogen into N2. In the simultaneous ammoniation and nitrification medium, the total nitrogen removal rate reaches 96.84%, and the ammonia nitrogen removal rate reaches 99.37%. In practical application, the sewage treatment system effluent ammonia nitrogen is stabilized at 9.44 mg / L, and the COD is reduced to 455.98 mg / L, which is significantly better than the traditional process. The present application solves the problems of low treatment efficiency of high-ammonia-nitrogen wastewater and serious secondary pollution, and is suitable for the collaborative disposal of aged leachate and perishable landfill leachate. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is the nitrogen removal ability verification diagram of the ammoniating bacteria of the present application;

[0020] Figure 2 It is the nitrogen removal ability verification diagram of the nitrifying bacteria of the present application;

[0021] Figure 3 It is the phylogenetic tree of 16S rDNA of lysine bacillus of the present application;

[0022] Figure 4 It is the phylogenetic tree of 16S rDNA of alkaligenes of the present application;

[0023] Figure 5 It is the ammoniation capacity determination diagram of lysine bacillus of the present application;

[0024] Figure 6 Figure for determination of direct ammonia oxidation ability of Alcaligenes faecalis in the present application;

[0025] Figure 7 Figure for nitrogen removal ability of the co-culture system in the present application;

[0026] Figure 8 Figure for nitrogen removal ability of the co-culture system under different inoculation time in the present application;

[0027] Figure 9 Figure for combination of the coupled nitrogen catabolism complex microbial inoculant in the present application;

[0028] Figure 10 Figure for NH4 + concentration change of the water body after application of the coupled nitrogen catabolism complex microbial inoculant in the aerobic section of the actual sewage treatment in the present application;

[0029] Figure 11 Figure for COD concentration change of the water body after application of the coupled nitrogen catabolism complex microbial inoculant in the aerobic section of the actual sewage treatment in the present application;

[0030] Figure 12 Figure for TN concentration change of the water body after application of the coupled nitrogen catabolism complex microbial inoculant in the aerobic section of the actual sewage treatment in the present application. DETAILED DESCRIPTION

[0031] The present application will be further described in conjunction with specific embodiments, but the protection scope of the present application is not limited to this.

[0032] Example 1: Screening and identification of ammonifying bacteria and nitrifying bacteria

[0033] The ammonifying bacteria screening method is as follows: 3ml sample is taken from sewage and directly inoculated in a 250ml flask, 100ml ammonification culture medium is added in the flask, 160rpm, 30℃, and cultured for 72h, then 10ml of the suspension is added into 100ml fresh ammonification culture medium, and cultured for 72h under the same conditions, and the process is repeated for three times, and the dominant species will be enriched. The final culture is diluted 10 -7 times on LB solid culture medium, and cultured at 30℃ for 2-3 days, and different strains are separated according to the size and color of the morphology to obtain pure strain.

[0034] Further, the nitrifying bacteria screening method is the same as above, only the ammonification culture medium is replaced by nitrification culture medium.

[0035] The composition of the ammonification medium in this example is: tryptone 5 g, K2HPO4 6.5 g, KH2PO4 1.5 g, trace elements 1 mL, KCl 0.3 g, NaCl 0.25 g, MgSO4·7H2O 2 g, H2O 1 L. The composition of the nitrification medium is: (NH4)2SO4 2.36 g, sodium succinate 16.88 g, K2HPO4·3H2O 6.5 g, KH2PO4 1.5 g, trace elements 1 mL, EDTA-2Na 0.1 g, MgSO4·7H2O 0.2 g, H2O 1 L. The above trace element composition is: EDTA-2Na 50 g, ZnSO4·7H2O 3.92 g, CaCl2 5.55 g, MnCl2·4H2O 5.06 g, FeSO4·7H2O 5 g, (NH4)6Mo7O 24 ·4H2O 10 g, CuSO4·5H2O 1.57 g, CoCl2·6H2O 1.57 g, H2O 1 L. The above LB medium: tryptone 10 g, yeast powder 5 g, sodium chloride 10 g, distilled water to 1000 mL, natural pH.

[0036] A plurality of ammonification bacteria and nitrification bacteria screened from the LB solid medium, the ammonification bacteria screened are verified using the ammonification medium, and the specific method is to inoculate 3 mL of pre-cultured bacteria into 100 mL of medium and cultivate at a rotation speed of 160 rpm at 30°C. Ammonium nitrogen (NH4 + -N) and total nitrogen (TN) are detected regularly during the cultivation process. The nitrification bacteria screened are verified using the nitrification medium, and the method is the same as above. Ammonium nitrogen (NH4 + -N) is detected regularly during the cultivation process. All experiments are repeated three times.

[0037] ,

[0038] Among them, A is the concentration of nitrogen element in the initial medium (mg / L), and B is the concentration of nitrogen element in the medium at the end (mg / L). The results are shown in Figure 1 Table 2, which are the amounts of ammonia nitrogen generated by each ammonification strain in 72 h. The higher the amount of ammonia nitrogen generated, the higher the efficiency of organic nitrogen conversion to ammonia nitrogen and the higher the efficiency of organic nitrogen decomposition. Among them, L11 has the highest ammonia nitrogen generation amount of 234.12 mg / L, and the ammonia nitrogen generation rate in 72 h can reach 3.25 mg / (L·h).

[0039] The nitrification bacteria are verified using the nitrification medium, Figure 2 which is the ammonia nitrogen removal efficiency of each nitrification strain in 72 h. Among them, M8 has the highest ammonia nitrogen removal rate of 100% (500 mg / L).

[0040] For the two strains mentioned above, PCR amplification was performed using the universal bacterial primers 27F: 5′-AGAGTTTGATCMTGGCTCAG-3′ and 1492R: 5′-TACGGYTACCTTGTTACGACTT-3′, and the results were verified by agarose gel electrophoresis (1%). Electrophoresis detection, gel excision purification and sequencing were performed, and the sequences were determined by Qingke Biotechnology.

[0041] The above PCR reaction system (50 μL) consisted of: 25 μL of Phusion High-Fidelity PCR Master Mix with HF Buffer; 3 μL (10 μM) of F / R primers; 10 μL of DNA template; and 6 μL of ddH2O. The prepared PCR system was subjected to PCR amplification under the following conditions: pre-denaturation at 98℃ for 30 s, followed by 30 cycles; denaturation at 98℃ for 15 s; annealing at 58℃ for 15 s; extension at 72℃ for 15 s; and final extension at 72℃ for 1 min.

[0042] The 16S rDNA sequence of strain L11 is 1473 bp in length, and its gene sequence is shown in SEQ ID NO.1. The 16S rDNA sequence of strain M8 is 1433 bp in length, and its gene sequence is shown in SEQ ID NO.2. The strain sequence results were uploaded to the NCBI database and compared with existing bacterial 16S rDNA gene sequences in the database. A phylogenetic tree was constructed using the neighbor-joining method in Mega 7.0 software. The results are as follows: Figure 3 and 4 As shown, the ammonifying bacteria screened were Bacillus lysine-containing bacteria (Bacillus). Lysinibacillus boronitolerans The bacteria were named L11, and the nitrifying bacteria selected were *Alcaligenes faecalis* (Femtobacter). Alcaligenes faecalis ), named M8.

[0043] The above-mentioned strains were preserved.

[0044] Strain L11 was named Bacillus lysine ( Lysinibacillus boronitolerans The specimen, with serial number ZJB24343, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20251029 and deposit date May 12, 2025.

[0045] Strain M8 was named Alcaligenes faecalis ( Alcaligenes faecalis The specimen, with serial number ZJB24344, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20251030 and deposit date May 12, 2025.

[0046] Example 2: Analysis of the ammonification ability of Bacillus lysine-containing bacteria

[0047] Strain L11 was inoculated in LB medium for activation culture. When the bacteria entered the logarithmic growth phase, the seed liquid was inoculated into a 100 mL shake flask containing ammoniation medium with 3% inoculation amount, and the medium used organic nitrogen (500 mg / L) as the sole nitrogen source. The inoculated shake flask was placed in a constant temperature shaking incubator at 30°C and 160 rpm for aerobic culture. The bacterial growth (OD 600 ), NH4 + -N content was measured every 24 h.

[0048] The results are shown in Figure 5 . The NH4 + -N concentration showed significant accumulation with the culture time, and its concentration increased from undetectable at the beginning to 226.288±6.428 mg / L within 96 h of culture, with an accumulation conversion rate of 45.3%. The results show that L11 has high efficiency in converting organic nitrogen to ammonia nitrogen in an organic nitrogen-rich environment, and has the potential to be applied to the treatment of high-nitrogen organic waste.

[0049] Example 3: Denitrification ability characterization of Alcaligenes

[0050] 1. Direct ammonia oxidation ability determination of strain M8

[0051] Strain M8 was pre-cultured in LB medium to complete strain activation. Then, 1.5 mL of strain seed liquid was inoculated into a 250 mL closed bottle containing 50 mL of HNM medium (C / N=10), and the HNM medium contained 200 mg / L of (NH4)2SO4. The closed bottle was incubated at 35°C and 160 rpm on a rotary shaker. The bacterial growth (OD 600 ), NH4 + -N, NH2OH-N, NO2 - -N, NO3 - -N content was measured every 12 h. The amount of nitrogen generated was measured at 36 h and 72 h.

[0052] The above NH4 + -N was determined by Nessler's reagent spectrophotometry, total nitrogen was determined by alkaline potassium persulfate method, nitrite was determined by N-(1-naphthyl)-diaminoethane photometry, nitrate was determined by phenol disulfonic acid photometry, hydroxylamine was determined by 8-hydroxyquinoline photometry, N2 was determined by GC-TCL, and the growth of bacteria was determined by spectrophotometer at 600 nm.

[0053] The direct ammonia oxidation ability and denitrification ability of the strain were tested in HNM medium, and it was found that: Figure 6As shown, in the ammonia conversion process, ammonia consumption is accompanied by cell growth, and strain M8 can ultimately consume 86% of NH4 + -N. A large amount of hydroxylamine accumulates in the process, which is ultimately completely consumed, accompanied by the production of a small amount of nitrite and nitrate. M8 produces nitrogen gas during the aerobic conversion of ammonia. In addition, most of the consumed ammonia is converted into cellular nitrogen and organic nitrogen.

[0054] 2. Direct ammonia oxidation capacity of strain M8 under different NH4 + -N concentrations

[0055] Prepare high-ammonia nitrogen nitrification medium with different (NH4)2SO4 contents of 500 mg / L, 1000 mg / L, 1500 mg / L, 2000 mg / L, and 2500 mg / L, respectively. The strain is cultured according to the above method, then inoculated into 100 mL of high-ammonia nitrogen nitrification medium with different ammonium contents at an inoculation amount of 3%, and set up three repeats, and placed in a constant temperature shaker at 30°C and 160 r / min. The bacterial growth (OD 600 ), NH4 + -N content is measured every 24 h to analyze the direct ammonia oxidation capacity of the strain under high-ammonia nitrogen conditions.

[0056] The results are shown in Table 1. Strain M8 grows well and has good direct ammonia oxidation capacity in the range of 500-2500 mg / L of ammonia nitrogen concentration. When the initial ammonia concentration is 500, 1000, and 1500 mg / L of NH4 + -N, the substrate can be almost completely removed in 24, 120, and 120 h, respectively, with removal rates of 500, 198.14, and 300 mg / L / d, respectively. When the initial ammonia concentration reaches 2500 mg / L of NH4 + -N, about 83% of ammonia is removed within 7 days.

[0057] Table 1 Denitrification capacity of Alcaligenes sp. M8 under different NH4 + -N concentrations

[0058]

[0059] The strain M8 of the present application exhibits excellent ammonia nitrogen tolerance characteristics, which can maintain normal metabolic activity in an environment with an ammonia nitrogen concentration as high as 2500 mg / L. At the same time, the strain has a high removal capacity for high-concentration ammonia nitrogen, and when the initial ammonia nitrogen concentration is ≤1500 mg / L, the removal efficiency of the strain for NH4 + -N reaches 100%. The above characteristics indicate that the strain has significant engineering application value in the fields of biological strengthening treatment of high-concentration ammonia nitrogen wastewater and denitrification of landfill leachate.

[0060] Example 4: Two bacterial co-cultivation strategies promote the removal of organic nitrogen

[0061] An equal amount of strain M8 and strain L11 bacterial liquid was inoculated into 100 mL of synchronous ammonification nitrification medium with a total inoculation amount of 3%. The inoculated flask was cultured at 30°C with a rotation speed of 160 rpm. All experiments were repeated three times. The concentration of NH4 + -N, NO3 - -N, NO2 - -N, TN, NH2OH, OD 600 was detected periodically during the culture process.

[0062] The above-mentioned synchronous ammonification nitrification medium includes: tryptone 0.83 g, (NH4)2SO4 2.36 g, sodium succinate 16.88 g, K2HPO4·3H2O 6.5 g, KH2PO4 1.5 g, trace elements 1 mL, EDTA-2Na 0.1 g, KCl 0.3 g, NaCl 0.25 g, MgSO4·7H2O 2 g, H2O 1 L.

[0063] The results are shown in Figure 7 , 96 h ammonia nitrogen decreased from 503.1 mg / L to 1.31 mg / L, ammonia nitrogen removal rate was 99.73%, almost completely removed. The total nitrogen concentration decreased from 605.1 mg / L to 216.5 mg / L, the total nitrogen removal rate was 64.25%, indicating that the co-cultivation system had a good nitrogen removal rate.

[0064] Example 5: Effect of different inoculation times on co-cultivation

[0065] Two strains were inoculated into the same medium at different times, strain L11 was inoculated into the synchronous ammonification nitrification medium at the initial culture stage (0 h), and strain M8 was inoculated in batches, inoculated at 0, 24, 48, and 72 h, respectively. The inoculated flask was cultured at 30°C with a rotation speed of 160 rpm. All experiments were repeated three times. The concentration of NH4 + -N, NO3 - -N, NO2 - -N, NH2OH, TN, OD 600 was detected periodically during the culture process.

[0066] Different inoculation times have a significant impact on the synchronous growth and functional expression of microorganisms in the co-cultivation system, and optimization of time can promote the rapid establishment of microbial community and efficient synergistic effect, thereby improving the nitrogen removal efficiency of the whole system.

[0067] The results are shown in Figure 8As shown, with the increase of inoculation time of strain M8 from 0 to 72 h, the total nitrogen removal efficiency of the co-culture increased. When strain M8 was inoculated at 48 h, the 96 h TN removal efficiency of the co-culture reached 75.95%, which was significantly improved compared with the culture inoculated with strain L11 at 0 h. Considering the time cost and total nitrogen removal efficiency, the effect of inoculating M8 at 48 h after inoculating L11 was the best.

[0068] Example 6: Construction of a composite microbial denitrification system

[0069] The screened ammoniating bacteria L11, direct ammonia-oxidizing bacteria M8, and the existing high-efficiency denitrification composite microbial inoculant in patent CN116179423A including Bacillus halodurans S1, Bacillus subtilis L2, and Rhizomicrobium B5 were inoculated and compounded in equal proportions to construct a more efficient denitrification composite microbial inoculant to improve its environmental tolerance and stability of the microbial community structure. The obtained optimal denitrification combination was used as the strain combination for constructing an "ammoniation-direct ammonia oxidation" coupling system. The combination arrangement is shown in Table 2.

[0070] Table 2: Table of compound arrangement combination

[0071]

[0072] Bacillus halodurans (Bacillus halodurans) strain ZJB21169, preservation number CCTCC NO: M20221659; Bacillus subtilis (Bacillus subtilis) strain ZJB21170, preservation number CCTCC NO: M20221658; Rhizomicrobium (Rhizomicrobium) strain ZJB21172, preservation number CCTCC NO: M20221657. Bacillus haynesii Bacillus subtilis stain Stenotrophomonas riizophila stain

[0073] Figure 9 The five strains were combined to find the optimal denitrification combination. Compared with the two-two combination, in the three-three combination, all experimental groups showed excellent ammonia nitrogen removal capacity, and the ammonia nitrogen removal rate was more than 95%. Among them, the strain combination #11+8+1 reached the peak value of ammonia nitrogen removal efficiency, and the ammonia nitrogen degradation rate was 99.83%, and the nitrate nitrogen degradation rate was 73.32%. In the four-four combination and the five-five combination, the ammonia nitrogen degradation rate was slightly higher than that of the two-two combination and the three-three combination, and the ammonia nitrogen degradation rate was more than 98%, and the highest nitrate nitrogen degradation rate was 50.98%. Therefore, the combination #11+8+1 was selected as the strain combination for constructing an "ammoniation-direct ammonia oxidation" coupling system.

[0074] Example 6: Application and effect comparison of coupled nitrogen catabolism composite microbial inoculant in biological denitrification treatment of landfill leachate ​​​

[0075] The constructed ammoniation-direct amination oxidation coupling system is made into a coupling nitrogen catabolism complex microbial inoculant. The preparation method of the coupling nitrogen catabolism complex microbial inoculant is referred to the Chinese patent CN116179423A disclosed by the laboratory, and specifically includes adding 3 g of L11, M8 and S1 wet microbial bodies into 1000 mL of water respectively, adding 0.5% of sodium benzoate as a preservative, and adding 0.1% of magnesium chloride as a protective agent, so as to prepare the inoculant. The prepared inoculant is diluted by 1:3000, and then is added to an aeration tank in an A area of a sewage treatment plant, and no inoculant is added in a B area as a control group. The TN, COD and ammonia nitrogen concentration changes in each section are monitored every day.

[0076] The coupling nitrogen catabolism complex microbial inoculant further includes a nutritional enhancer. The nutritional enhancer includes, in terms of mass fraction, 0.245-0.45 g / L of amino acids, 0.09-0.223 g / L of metal salts, 0-0.05 g / L of choline, and 0-0.05 g / L of B vitamins. The amino acids include at least one of alanine, glutamic acid, valine, proline and leucine. The metal salts include calcium salt, magnesium salt, ferrous salt and copper salt. The B vitamins include at least one of pantothenic acid and pyridoxine. The amino acids include, in terms of mass fraction, 0.2-0.5 g / L of alanine, 0-0.04 g / L of glutamic acid, 0-0.04 g / L of valine, 0-0.04 g / L of proline, and 0-0.04 g / L of leucine. The metal salts include 0.02-0.05 g / L of calcium salt, 0.02-0.03 g / L of magnesium salt, 0.01-0.025 g / L of ferrous salt, and 0.01-0.02 g / L of copper salt. The calcium salt is CaCl2, the magnesium salt is MgSO4, the ferrous salt is FeSO4, and the copper salt is CuSO4. The B vitamins include, in terms of mass fraction, 0-0.03 g / L of pantothenic acid and 0-0.02 g / L of pyridoxine. The mass of at least one of the substances is not 0.

[0077] Table 3 Nutritional enhancer configuration component table of coupling nitrogen catabolism complex microbial inoculant

[0078]

[0079] The operation data of the sewage treatment system show that, in terms of ammonia nitrogen treatment efficiency, the coupling nitrogen catabolism complex microbial inoculant has a treatment efficiency of 90% or above, and the treatment efficiency of the control group is 70% or below. Figure 10As shown, the influent concentration in Zone A (microbial agent addition group) fluctuated significantly (1000-2500 mg / L), with a mean of 1507.77 mg / L, which was basically the same as the average influent concentration of 1497.77 mg / L in Zone B (control group). After bio-enhanced treatment, the ammonia nitrogen in the effluent from the sedimentation tank in Zone A was stably controlled at around 9.44 mg / L (σ²=46.36), with a removal rate of 99.37%, which was significantly better than the 0-60 mg / L (σ²=151.51) and 98.71% removal rate in Zone B. The results indicate that after adding the compound microbial agent to Zone A, the variance of the ammonia nitrogen effluent concentration fluctuation decreased by 69.4% compared with the control group Zone B, and the removal rate increased by 0.59%, with a significant difference (p<0.05), highlighting the synergistic effect of the microbial agent on the stable removal of ammonia nitrogen.

[0080] like Figure 11 As shown, during the 92-day operating cycle, the influent COD concentration in both zones was around 15000 mg / L. After three-stage treatment—mixing tank-biological rotating disc-aeration tank—the effluent COD in zone A remained stable at around 455 mg / L, with a removal rate of 96.45%; while the effluent in zone B fluctuated significantly, with an effluent concentration around 600 mg / L, and the removal rate decreased to 95.46%. The results indicate that the COD variance in zone A was reduced by 82.2% compared to zone B, and the removal rate was increased by 0.99%, confirming the optimizing effect of the bacterial agent on the degradation stability of complex organic matter.

[0081] like Figure 12 As shown, the total nitrogen (TN) in the influent of both zones was characterized by a high concentration of approximately 2500 mg / L. After system treatment, the TN effluent concentration in zone A was approximately 73 mg / L, with a removal rate of 96.84%, significantly better than the 125 mg / L and 94.60% removal rate in zone B. The results indicate that the TN removal rate in zone A was 2.24% higher than that in zone B, and the variance of the effluent concentration decreased by 78.7%, with a significant difference (p<0.05). This confirms that the addition of the bacterial agent improved the system's resistance to high nitrogen loads.

[0082] In summary, the coupled nitrogen-decomposing metabolic composite microbial agent and its application method in landfill leachate denitrification treatment provided by this invention stabilized ammonia nitrogen in the effluent of the wastewater treatment system at 9.44 mg / L and reduced COD to 455.98 mg / L, significantly superior to traditional processes. This indicates that this technology has broad application prospects in biological denitrification treatment, especially in landfill leachate denitrification treatment.

Claims

1. An ammoniating-direct ammoximation coupling system characterized by, The system is composed of Bacillus lysine ( Lysinibacillus boronitolerans ZJB24343 and Alcaligenes faecalis ( Alcaligenes faecalis Composed of ZJB24344, the lysine-containing Bacillus ( Lysinibacillus boronitolerans ZJB24343 has the accession number CCTCC NO: M20251029, and the described Alcaligenes faecalis ( Alcaligenes faecalis The accession number of ZJB24344 is CCTCC NO: M20251030.

2. The application of the coupling system of ammoniation and direct ammonia oxidation in the denitrification treatment of landfill leachate sewage according to claim 1.

3. Use according to claim 2, wherein the compound is ###0002### Lysine Bacillus ( Lysinibacillus boronitolerans ZJB24343 and Alcaligenes faecalis ( Alcaligenes faecalis ZJB24344 was successively inoculated into nitrogen-containing wastewater, including Bacillus lysine (…). Lysinibacillus boronitolerans ZJB24343 was inoculated at 0h, and Alcaligenes feces ( Alcaligenes faecalis ZJB24344 was administered 48 hours later.

4. The use according to claim 2, wherein the compound is ###0002### The Bacillus licheniformis (Bacillus licheniformis) Lysinibacillus boronitolerans The inoculation amount of Bacillus licheniformis (Bacillus licheniformis) Alcaligenes faecalis ) ZJB24344 is 1:

1.

5. A coupled nitrogen catabolism composite microbial inoculant, characterized in that, The ammonia-direct amination oxidation coupling system of claim 1, and any one or more of Paenibacillus huaihensis ( Bacillus haynesii ) ZJB21169, with the preservation number of CCTCC NO: M20221659, Bacillus subtilis ( Bacillus subtilis stain ) ZJB21170, with the preservation number of CCTCC NO: M20221658, or Stenophomonas rhizophila ( Stenotrophomonas riizophila stain ) ZJB21172, with the preservation number of CCTCC NO: M20221657.​​​ 6. The coupled nitrogen catabolism composite microbial inoculum according to claim 5, wherein, It also includes preservatives and protective agents, the preservative is sodium benzoate, and the protective agent is magnesium chloride.

7. The coupled nitrogen catabolism composite microbial inoculant of claim 6, wherein, The wet bacteria in the bacterial agent are mixed with 0.5% preservatives and 0.1% protective agents at a ratio of 3g / 1000 mL.

8. The application of the coupling nitrogen catabolism complex microbial agent in the denitrification treatment of sewage according to any one of claims 5-7.

9. The application of the coupling nitrogen catabolism complex microbial agent in the denitrification treatment of landfill leachate according to any one of claims 5-7.

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