Coupling nitrogen catabolism compound microbial agent and application thereof

Abstract

The invention discloses a coupled nitrogen catabolism compound microbial agent and application thereof, and belongs to the technical field of biology. According to the method, an efficient ammonification strain (lysinibacillus L11) and a direct ammoxidation strain (alcaligenes M8) are screened, an ammonification-Dirammox coupling system is constructed, the strain co-culture time sequence (L11 is inoculated 48 hours earlier) is optimized, and organic nitrogen is directly converted into N2. According to the method, the total nitrogen removal rate in the synchronous ammoniation and nitrification culture medium reaches 96.84%, and the ammonia nitrogen removal rate reaches 99.37%. In practical application, the ammonia nitrogen in effluent of a sewage treatment system is stabilized at 9.44 mg / L and COD (Chemical Oxygen Demand) is reduced to 455.98 mg / L by adding the microbial agent, which is obviously superior to that of the traditional process. The method provided by the invention solves the problems of low treatment efficiency of high-ammonia-nitrogen wastewater and serious secondary pollution, and is suitable for co-treatment of aging leachate and perishable landfill 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. To achieve the synergistic and efficient removal of organic nitrogen-ammonia-nitrogen, it is of great 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: The present application provides a lysine bacillus ( Lysinibacillus boronitolerans ) ZJB24343 and alkaligenes coprophilus ( Alcaligenes faecalis ) ZJB24344, the preservation number of the lysine bacillus ( Lysinibacillus boronitolerans ) ZJB24343 is CCTCC NO: M20251029, and the preservation number of the alkaligenes coprophilus ( Alcaligenes faecalis ) ZJB24344 is CCTCC NO: M20251030.

[0008] The present application provides the application of the above-mentioned ammonia and direct ammonia oxidation coupled system in wastewater denitrification treatment.

[0009] Further, in the application, the lysine bacillus ( Lysinibacillus boronitolerans ) ZJB24343 and the alkaligenes coprophilus ( Alcaligenes faecalis ) ZJB24344 are inoculated into nitrogen-containing wastewater in turn, wherein the lysine bacillus ( Lysinibacillus boronitolerans ) ZJB24343 is inoculated at 0h, and the alkaligenes coprophilus ( Alcaligenes faecalis ) ZJB24344 is inoculated with a delay of 48h.

[0010] Further, in the application, the inoculation amount of the lysine bacillus ( Lysinibacillus boronitolerans ) ZJB24343 and the alkaligenes coprophilus ( Alcaligenes faecalis ) ZJB24344 is 1:1.

[0011] The present application provides a coupled nitrogen catabolism composite microbial inoculant containing the above-mentioned ammonia and direct ammonia oxidation coupled system.

[0012] Furthermore, this coupled nitrogen-degrading metabolic complex microbial agent also includes Bacillus hainanensis (…). Bacillus haynesii Bacillus subtilis ( Bacillus subtilis ) and root-loving oligotrophosomes ( Stenotrophomonas riizophila ) .

[0013] Furthermore, the coupled nitrogen-degrading metabolic complex microbial agent also includes a preservative and a protectant, wherein the preservative is sodium benzoate and the protectant is magnesium chloride.

[0014] Furthermore, in this coupled nitrogen-degrading metabolic composite microbial agent, wet bacterial cells are mixed with 0.5% preservative and 0.1% protectant at a ratio of 3g / 1000 mL.

[0015] The fourth aspect of this invention provides the application of the above-mentioned coupled nitrogen decomposition and metabolism composite microbial agent in wastewater denitrification treatment.

[0016] The fifth aspect of this invention provides the application of the above-mentioned coupled nitrogen decomposition and metabolism composite microbial agent in the denitrification treatment of landfill leachate.

[0017] This invention constructs an "ammoniation-Dirammox" coupling system by screening highly efficient ammonifying strains (Lysine Bacillus L11) and direct ammonia-oxidizing strains (Alcaligenes M8), and optimizes the co-culture sequence of the strains (L11 inoculated 48 hours in advance) to achieve the direct conversion of organic nitrogen into N2. This method achieves a total nitrogen removal rate of 96.84% and an ammonia nitrogen removal rate of 99.37% in a simultaneous ammoniation and nitrification medium. In practical applications, the addition of the bacterial agent stabilized the ammonia nitrogen in the wastewater treatment system at 9.44 mg / L and reduced COD to 455.98 mg / L, significantly superior to traditional processes. This invention solves the problems of low treatment efficiency and severe secondary pollution in high-ammonia nitrogen wastewater and is suitable for the co-treatment of leachate from aging plants and perishable landfills. Attached Figure Description

[0018] Figure 1 This is a graph demonstrating the nitrogen-denitrifying ability of the ammonifying bacteria in this invention. Figure 2 This is a diagram verifying the nitrogen removal capacity of the nitrifying bacteria of this invention; Figure 3 This is a phylogenetic tree of the 16S rDNA of Bacillus lysine in this invention; Figure 4 This is a phylogenetic tree of the 16S rDNA of Alcaligenes bacillus of the present invention; Figure 5 This is a graph showing the ammonification ability determination of Bacillus lysine in this invention; Figure 6 This is a graph showing the determination of the direct ammonia oxidation capacity of Alcaligenes in this invention; Figure 7 Denitrification ability of the co-culture system of the present application; Figure 8 Denitrification ability of the co-culture system of the present application under different inoculation times; Figure 9 Combination of the coupled nitrogen catabolism complex microbial inoculant of the present application; Figure 10 NH4 + -N concentration change of the coupled nitrogen catabolism complex microbial inoculant of the present application applied in the water body after the aerobic section of the actual sewage treatment; Figure 11 COD concentration change of the coupled nitrogen catabolism complex microbial inoculant of the present application applied in the water body after the aerobic section of the actual sewage treatment; Figure 12 TN concentration change of the coupled nitrogen catabolism complex microbial inoculant of the present application applied in the water body after the aerobic section of the actual sewage treatment. DETAILED DESCRIPTION

[0019] The present application will be further described in conjunction with specific examples, but the protection scope of the present application is not limited to this.

[0020] Example 1: Screening and identification of ammonifying bacteria and nitrifying bacteria The ammonifying bacteria screening method is as follows: 3ml sample is taken from sewage and directly inoculated in a 250ml flask, 100ml ammonifying culture medium is added in the flask, 160rpm, 30℃, and cultured for 72h, then 10ml of the suspension is added into 100ml fresh ammonifying culture medium, and cultured for 72h under the same conditions, and repeated for three times, and the dominant bacteria will be enriched. The final culture is diluted 10 -7 times on LB solid culture medium, and incubated at 30℃ for 2-3 days, and different strains are separated according to the size and color of the morphology to obtain pure strain.

[0021] Further, the nitrifying bacteria screening method is the same as above, only the ammonifying culture medium is replaced by nitrifying culture medium.

[0022] 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 0.2 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.

[0023] A plurality of ammonification bacteria and nitrification bacteria are screened from the LB solid medium. The ammonification bacteria screened are verified using the ammonification medium. Specifically, 3 mL of pre-cultured bacteria liquid is inoculated into 100 mL of culture medium, and the culture is carried out at a rotation speed of 160 rpm at 30°C. Ammonium nitrogen (NH4 + -N) and total nitrogen (TN) are detected periodically during the culture. The nitrification bacteria screened are verified using the nitrification medium, and the method is the same as above. Ammonium nitrogen (NH4 + -N) is detected periodically during the culture. All experiments are repeated three times.

[0024] , wherein A is the concentration of nitrogen elements in the initial culture medium (mg / L), and B is the concentration of nitrogen elements in the culture medium at the end (mg / L). The results are shown in Table 2. Figure 1 The results are shown in Table 2. The ammonia nitrogen generation amount of each ammonification strain is 72 h. The higher the ammonia nitrogen generation amount, the higher the efficiency of converting organic nitrogen to ammonia nitrogen, and the higher the efficiency of decomposing organic nitrogen. Among them, the ammonia nitrogen generation amount of L11 is the highest, which is 234.12 mg / L, and the ammonia nitrogen generation rate of 72 h is 3.25 mg / (L·h).

[0025] The nitrification bacteria are verified using the nitrification medium, Figure 2 and the ammonia nitrogen removal efficiency of each nitrification strain is 72 h. Among them, the ammonia nitrogen removal rate of M8 is the highest, which is 100% (500 mg / L).

[0026] 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.

[0027] 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.

[0028] 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.

[0029] The above-mentioned strains were preserved.

[0030] 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.

[0031] 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.

[0032] Example 2: Analysis of the ammonification ability of Bacillus lysine-containing bacteria Strain L11 was inoculated in LB medium for activation culture. When the bacteria entered the logarithmic growth phase, 3% of the seed liquid was inoculated into a 100 mL shake flask containing ammoniation medium with 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. Bacterial growth (OD 600 ), NH4 + -N content was measured every 24 h.

[0033] 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.

[0034] Example 3: Denitrification ability characterization of Alcaligenes 1. Direct ammonia oxidation ability determination of strain M8 Strain M8 was pre-cultured in LB medium to activate the strain. 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. Bacterial growth (OD 600 ), NH4 + -N, NH2OH-N, NO2 - -N, NO3 - -N content was measured every 12 h. Nitrogen production was measured at 36 h and 72 h.

[0035] The above NH4 + -N was determined by the Nash reagent spectrophotometric method, total nitrogen was determined by the alkaline potassium persulfate method, nitrite was determined by the N-(1-naphthyl)-diaminoethane spectrophotometric method, nitrate was determined by the phenol disulfonic acid spectrophotometric method, hydroxylamine was determined by the 8-hydroxyquinoline spectrophotometric method, N2 was determined by GC-TCL, and bacterial growth was determined by a spectrophotometer at 600 nm.

[0036] The direct ammonia oxidation ability and denitrification ability of the strain were tested in HNM medium, and it was found that, as shown in Figure 6 , during the ammonia conversion process, ammonia consumption was accompanied by cell growth, and strain M8 could ultimately consume 86% of the NH4 +-N. Hydroxylamine accumulated massively in this process, and was eventually completely consumed, accompanied by the production of small amounts of nitrite and nitrate. M8 produced nitrogen gas during aerobic conversion of ammonia. In addition, most of the consumed ammonia was converted into cellular nitrogen and organic nitrogen.

[0037] 2. Direct ammonia oxidation capacity of strain M8 under different NH4 + -N concentrations 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.

[0038] 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, and the removal rates are 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.

[0039] Table 1 Denitrification capacity of Alcaligenes sp. M8 under different NH4 + -N concentrations

[0040] The strain M8 of the present application exhibits excellent ammonia nitrogen tolerance characteristics, and 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 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.

[0041] Example 4: Two bacteria co-culture strategies promote removal of organic nitrogen Equal amounts of strain M8 and strain L11 bacterial liquid were inoculated into 100 mL of synchronous ammonification and nitrification medium with a total inoculation amount of 3%. The inoculated flasks were cultured at 30°C with a rotation speed of 160 rpm. All experiments were repeated three times. The concentrations of NH4 + -N, NO3 - -N, NO2 - -N, TN, NH2OH, OD 600 were detected periodically during the culture process.

[0042] The above-mentioned synchronous ammonification and nitrification medium comprises: 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.

[0043] The results are shown in Figure 7 As shown in the table, the ammonia nitrogen was reduced from 503.1 mg / L to 1.31 mg / L after 96 h, and the ammonia nitrogen removal rate was 99.73%, which was almost completely removed. The total nitrogen concentration was reduced from 605.1 mg / L to 216.5 mg / L, and the total nitrogen removal rate was 64.25%, indicating that the co-culture system had a good nitrogen removal rate.

[0044] Example 5: Effect of different inoculation times on co-culture Two strains were inoculated into the same medium at different times. Strain L11 was inoculated into the synchronous ammonification and nitrification medium at the initial culture stage (0 h), and strain M8 was inoculated in batches at 0, 24, 48, and 72 h. The inoculated flasks were cultured at 30°C with a rotation speed of 160 rpm. All experiments were repeated three times. The concentrations of NH4 + -N, NO3 - -N, NO2 - -N, NH2OH, TN, OD 600 were detected periodically during the culture process.

[0045] Different inoculation times have a significant effect on the synchronous growth and functional expression of microorganisms in the co-culture system. Optimization of the time can promote the rapid establishment of microbial communities and efficient synergistic action, thereby improving the nitrogen removal efficiency of the entire system.

[0046] 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.

[0047] Example 6: Construction of a composite microbial denitrification system 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.

[0048] Table 2: Table of compound arrangement combination

[0049] Among them, Bacillus halodurans (Bacillus halodurans) strain No. ZJB21169, preservation number CCTCC NO: M20221659; Bacillus subtilis (Bacillus subtilis) strain No. ZJB21170, preservation number CCTCC NO: M20221658; Rhizomicrobium (Rhizomicrobium) strain No. ZJB21172, preservation number CCTCC NO: M20221657. Bacillus haynesii Bacillus subtilis stain Stenotrophomonas riizophila stain

[0050] Figure 9 The degradation of ammonia nitrogen and nitrate nitrogen of different compound results is shown in the schematic diagram. 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.

[0051] Example 6: Application and effect comparison of coupled nitrogen catabolism composite microbial inoculant in biological denitrification treatment of landfill leachate ​​​The constructed ammoniation-direct ammonia 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 laboratory published Chinese patent CN116179423A, and specifically includes adding 3 g of L11, M8 and S1 wet microbial bodies into 1000 mL of water respectively, then adding 0.5% of sodium benzoate as a preservative and 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 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.

[0052] The coupling nitrogen catabolism complex microbial inoculant further comprises a nutritional enhancer. The nutritional enhancer comprises, by 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 comprise at least one of alanine, glutamic acid, valine, proline and leucine. The metal salts comprise at least one of calcium salts, magnesium salts, ferrous salts and copper salts. The B vitamins comprise at least one of pantothenic acid and pyridoxine. The amino acids comprise, by 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 comprise 0.02-0.05 g / L of calcium salts, 0.02-0.03 g / L of magnesium salts, 0.01-0.025 g / L of ferrous salts and 0.01-0.02 g / L of copper salts. The calcium salts are CaCl2, the magnesium salts are MgSO4, the ferrous salts are FeSO4 and the copper salts are CuSO4. The B vitamins comprise, by 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.

[0053] Table 3 Nutritional enhancer configuration component table of coupling nitrogen catabolism complex microbial inoculant

[0054] 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 20% increase in ammonia nitrogen removal rate, a 20% increase in ammonia nitrogen removal efficiency and a 20% increase in ammonia nitrogen removal capacity. 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.

[0055] 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.

[0056] 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.

[0057] 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 includes Bacillus lysine ( Lysinibacillus boronitolerans ZJB24343 and Alcaligenes faecalis ( Alcaligenes faecalis 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 ammonia-direct ammonia oxidation coupling system of claim 1 in the denitrification treatment of landfill leachate sewage.

3. The use according to claim 1, wherein Lysine Bacillus ( Lysinibacillus boronitolerans ZJB24343 and Alcaligenes faecalis ( Alcaligenes faecalis ZJB24344 was successively inoculated into nitrogen-containing wastewater, including Bacillus lysine-containing bacteria (…). Lysinibacillus boronitolerans ZJB24343 was inoculated at 0h, and Alcaligenes feces ( Alcaligenes faecalis ZJB24344 was administered 48 hours later.

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

1.

5. The coupling nitrogen catabolism complex microbial agent containing the ammonia-direct ammonia oxidation coupling system of claim 1.

6. The coupled nitrogen catabolism composite microbial inoculum according to claim 5, wherein, Also includes Bacillus hainanensis ( Bacillus haynesii ), Bacillus subtilis ( Bacillus subtilis ) and root-loving oligotrophosomes ( Stenotrophomonas riizophila ) .

7. The coupled nitrogen catabolism composite microbial inoculum according to claim 5, wherein, Preservatives and protectants are also included, the preservative being sodium benzoate and the protectant being magnesium chloride.

8. The coupled nitrogen catabolism composite microbial inoculum according to claim 7, wherein, The wet bacteria in the microbial agent are mixed with 0.5% preservatives and 0.1% protectants at a ratio of 3g / 1000 mL.

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

10. The application of the coupling nitrogen catabolism complex microbial agent of any one of claims 5-8 in the denitrification treatment of landfill leachate.

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