Pseudomonas, fungicide and iron composite material combined preparation for denitrification nitrogen removal as well as method and application of pseudomonas, fungicide and iron composite material combined preparation

Abstract

The invention provides a pseudomonas for denitrification nitrogen removal, a microbial inoculum, an iron composite material combined preparation as well as a method and application thereof, and relates to a biological denitrification technology. The invention provides pseudomonas sp. AH40, the preservation number is CCTCC (China Center for Type Culture Collection) NO: M20221280, and the pseudomonas is preserved in the China Center for Type Culture Collection on August 15, 2022 and has the aerobic denitrification capability; the invention also provides an iron composite material, the iron composite material can be combined with pseudomonas AH40 to prepare a combined preparation, and the combined preparation can further improve the nitrogen removal rate in the culture medium.

Description

Technical Field

[0001] This invention pertains to biological denitrification technology, specifically to Pseudomonas bacteria, and more specifically to Pseudomonas bacteria, bacterial agents, iron composite material combination preparations, methods, and applications used for denitrification. Background Technology

[0002] Nitrate is the main form of nitrogen in wastewater treatment plant effluent. A total nitrogen content of 15 mg / L meets the Class A (GB18918-2002) standard for urban wastewater treatment plant effluent, but it still far exceeds the Class V (GB3838-2002) water quality standard for surface water (2 mg / L). Deep denitrification of wastewater treatment plant effluent is a prerequisite for resource utilization and an inevitable choice for reducing the nitrogen pollution load on receiving water bodies. Therefore, the advanced treatment of low-concentration nitrate water bodies is receiving increasing attention.

[0003] Novel biological ammonia removal technologies, particularly those based on aerobic denitrifying strains, have garnered widespread attention. Heterotrophic nitrifying aerobic denitrifying bacteria can unify nitrification and denitrification processes in both time and space, enabling biological nitrogen removal to be completed within a single reactor. This significantly reduces nitrogen removal costs in wastewater treatment and substantially improves nitrogen removal efficiency, achieving highly efficient and environmentally friendly wastewater treatment. Furthermore, due to the wide range of aerobic denitrifying bacteria sources, rapid cell growth, high nitrogen removal efficiency, and strong adaptability to external environments, they maintain high nitrogen removal efficiency even in extreme environments such as those with high ammonia nitrogen levels or heavy metal ions. Therefore, they show great promise for applications in various scenarios, including domestic sewage, wastewater treatment plant effluent, and aquaculture wastewater.

[0004] Electron transfer reactions are central to many biological processes, especially respiration, where microorganisms can transfer intracellularly generated electrons to intracellular electron acceptors such as nitrates, oxygen, and sulfates through a series of redox reactions. Urban wastewater treatment plant effluents generally have low carbon-to-nitrogen ratios, and limited carbon sources reduce electron transfer efficiency, thus limiting the sustainability of deep denitrification. Therefore, seeking feasible strategies to enhance the denitrification performance of biological treatment processes is urgent. When microorganisms live in micron-thick biofilms, access to certain chemicals is limited, or when electron donors and acceptors are spatially separated, rapid electron transfer faces challenges. However, some microorganisms can utilize conductive or semi-conductive materials (such as magnetite, hematite, and graphite) as functional "tools" to aid electron transfer (Dong H. Mineral-microbe interactions: a review. Front Earth Sci China, 2010). Nanoparticles have demonstrated unprecedented advantages in enhancing electron transport and metabolic activity in microorganisms, while atomic materials, with their unique active site structure and uniform dispersion of loaded atoms, possess characteristics such as superior catalytic activity, high selectivity, and sustained stability (Liu G, Robertson AW, Li MMJ, Kuo WC, Darby MT, Muhieddine MH, Lin YC, Suenaga K, Stamatakis M, Warner JH. MoS2 monolayer catalyst doped with isolated Co atoms for the hydrodeoxygenation reaction. Nat Chem, 2017).

[0005] Existing technology CN115786181A provides a wastewater treatment agent that treats low C / N wastewater through the coupling of a bacterial strain with pyrite. The strain provided by this technology exhibits stronger denitrification efficiency and better carbon source utilization under the synergistic effect of pyrite. The strain is a *Pseudomonas aeruginosa* strain from Hunan. Pseudomonas hunanensis The strain PAD-1 has the accession number CCTCC NO: M2022138. The disadvantages of this technology include low nitrogen removal rate and the potential for water pollution from impurities in the pyrite.

[0006] Li Qiulin et al. found that iron-carbon coupled denitrification (MEBD) was more effective in treating low C / N wastewater, achieving a nitrogen removal efficiency of 63.89%, which was higher than that of iron-carbon microelectrolysis (ICME) and denitrifying bacteria (DB) (46.59% and 37.09%, respectively). In addition, they investigated the reduction of nitrates (NO3) by zero-valent iron and iron-carbon microelectrolysis within a pH range of 4-7.- The performance of NO3- was investigated, and the effects of pH and the ratio of iron to activated carbon on the removal of nitrates in an iron-carbon microelectrolysis system were explored. It was found that under acidic conditions (pH=4), the efficiency of the microelectrolysis system in reducing nitrates was significantly better than that of the zero-valent iron system. The activated carbon showed a weaker adsorption effect on nitrates, and the microelectrolysis system exhibited better adsorption of NO3-. - The removal rate of NO3- can reach 88.86%. Under neutral conditions, zero-valent iron (NO3) can be reduced. - The efficiency of (NO3-N) in the iron-carbon micro-electrolysis system is only 6.84%, while the efficiency of (NO3-N) in the iron-carbon micro-electrolysis system is much lower. - The removal rate of nitrogen (N-N) remained around 50%. The micro-electrolysis system showed the best reduction effect on nitrates when the ratio of iron to activated carbon was 3:1. The disadvantages of this technology are low nitrogen removal rate and long overall reaction time. Summary of the Invention

[0007] This invention addresses the problems of low nitrogen removal rate and long reaction time in existing technologies by providing a combined formulation of *Pseudomonas* bacteria, bacterial agent, and iron composite material for denitrification, along with its method and application. This invention provides a *Pseudomonas* (… Pseudomonas The 16S rDNA sequence of Pseudomonas aeruginosa (S.) AH40, as shown in SEQ ID NO.1, was deposited at the China Center for Type Culture Collection on August 15, 2022, with accession number CCTCC NO: M20221280, and possesses aerobic denitrification capabilities. This invention also provides an iron composite material that can be used in combination with Pseudomonas aeruginosa AH40 to prepare a combined formulation, which can further improve the nitrogen removal rate and nitrogen removal efficiency in the culture medium.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: On the one hand, the present invention provides a Pseudomonas aeruginosa ( Pseudomonas The genera *AH40*, with accession number CCTCCNO: M20221280, was deposited at the China Center for Type Culture Collection on August 15, 2022, and its 16S rDNA sequence is the sequence shown in SEQ ID NO.1.

[0009] On the other hand, the present invention provides a microbial agent, wherein the microbial agent is the above-mentioned Pseudomonas AH40 or a preparation of Pseudomonas AH40.

[0010] Preferably, the preparation of Pseudomonas AH40 includes a culture of Pseudomonas AH40, a culture extract, a lyophilized powder, a fermentation broth, a fermentation broth precipitate, a fermentation broth supernatant, or a fermentation broth extract.

[0011] On the other hand, the present invention provides the application of the above-mentioned Pseudomonas AH40 or the above-mentioned bacterial agent in denitrification and nitrogen removal in water bodies.

[0012] On the other hand, the present invention provides a method for denitrification in water, wherein the method involves adding the above-mentioned Pseudomonas AH40 or the above-mentioned bacterial agent to the water to carry out denitrification.

[0013] Preferably, Pseudomonas aeruginosa AH40 or bacterial agents are added to artificial culture media or wastewater containing nitrates, nitrites or ammonium salts, and denitrification can be carried out under suitable conditions.

[0014] On the other hand, the present invention provides a combination formulation comprising the above-mentioned Pseudomonas AH40 or the above-mentioned bacterial agent.

[0015] Preferably, the combined formulation further comprises an iron composite material.

[0016] Preferably, the preparation method of the iron composite material includes the following steps: (1) ZnCl2 was dissolved in a solvent mixture consisting of N,N-dimethylformamide, anhydrous ethanol, deionized water and ammonium hydroxide, and sonicated to form a homogeneous solution. Then, triacetylacetone iron was added and mixed well. Then, 1H-1,2,3-triazole was added and a precipitate was formed. The precipitate was collected, washed and dried to obtain Fe-MET-6. (2) Fe-MET-6 was heated at a nitrogen atmosphere at a temperature greater than 900°C for 1-5 hours to obtain Fe-ISAs; (3) Under a nitrogen atmosphere, FeSO4·7H2O was dissolved in an ethanol solution, and sodium borohydride solution was added dropwise until a black substance was produced. After stirring, the precipitate was collected and washed. All the precipitates were dispersed in anhydrous ethanol to obtain Fe-NPs / ethanol solution. (4) Take Fe-ISAs and disperse them in anhydrous ethanol, sonicate, then add Fe-NPs / ethanol solution, stir and dry to obtain iron composite material.

[0017] Preferably, the preparation method of the combined formulation includes the following steps: S1. Add the bacterial culture of Pseudomonas aeruginosa AH40 to a nitrate-containing medium for culture, and then dry to obtain bacterial powder; S2. Add the iron composite material to the bacterial powder, with a mass ratio of bacterial powder to iron composite material of 1:1, and mix well to obtain the combined preparation.

[0018] On the other hand, the present invention provides the application of the above-mentioned combined formulation in denitrification of water bodies.

[0019] Preferably, the above-mentioned combined preparation is added to an artificial culture medium or wastewater containing nitrates, nitrites or ammonium salts, and denitrification can be carried out under suitable conditions.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1. The Pseudomonas aeruginosa AH40 provided by this invention can grow in a culture medium with NH4Cl, KNO3, and NaNO2 as the sole nitrogen source, and within 14 hours, NH4Cl... + -N removal rate is 100%, aerobic denitrification NO3 - The removal rates of -N were 99.46%, and NO2... - -N removal rates were 99.96%.

[0021] 2. The combined use of iron composite materials and Pseudomonas aeruginosa AH40 further improved the denitrification rate; at the same time, the promoting effect of iron composite materials on the denitrification process of strain AH40 increased with the increase of dosage, which not only significantly improved the activity of the electron transport system of strain AH40, but also significantly improved the activity of nitrite reductase of strain AH40.

[0022] Preservation Instructions Bacterial species name: Pseudomonas aeruginosa AH40; Category Naming: Pseudomonas sp. AH40; Preservation institution: China Center for Type Culture Collection; Abbreviation for depository institution: CCTCC; Address: No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province (within the campus of Wuhan University); Date of deposit: August 15, 2022; Accession number: CCTCC NO: M20221280. Attached Figure Description

[0023] Figure 1 Characterization of strain AH40: Colony morphology (a), Gram staining micrograph (b), and scanning electron microscope image (c) of strain AH40.

[0024] Figure 2 A phylogenetic tree was constructed for the 16S rDNA gene of strain AH40.

[0025] Figure 3 For NO3 - Changes in the composition of the culture medium with -N as the sole nitrogen source.

[0026] Figure 4 For NO2 - Changes in the composition of the culture medium with -N as the sole nitrogen source.

[0027] Figure 5 For NH4 + Changes in the composition of the culture medium with -N as the sole nitrogen source.

[0028] Figure 6 This is a scanning electron microscope image of an iron composite material.

[0029] Figure 7 This is a transmission electron microscope image of an iron composite material.

[0030] Figure 8 High-angle annular dark-field scanning transmission electron microscope image of iron composite material Figure 9 The effect of combined formulations on denitrification performance.

[0031] Figure 10 The effect of the combined formulation on the activity of the electron transport system. (Different letters represent significant differences between data points, P < 0.05; the same letter represents no significant differences between data points, P > 0.05.) Figure 11 The effect of the combined formulation on the activity of denitrification-related enzymes (different letters represent significant differences between data, P<0.05; the same letter represents no significant differences between data, P>0.05). Detailed Implementation It is worth noting that the raw materials used in this invention are all commercially available products, and their sources are not specifically limited.

[0032] Preparation of culture medium: Enrichment medium (DBM): KNO3 5.0 g / L, sodium succinate hexahydrate 11.1 g / L, KH2PO4 1.0 g / L, Na2HPO4·12H2O 7.03 g / L, MgSO4·7H2O 0.13 g / L, trace elements 2 mL / L, pH 7.0.

[0033] Bromothymol blue solid medium (BTB, denitrification identification medium): KNO3 5.0 g / L, sodium succinate hexahydrate 11.1 g / L, KH2PO4 1.0 g / L, Na2HPO4·12H2O 7.03 g / L, MgSO4·7H2O 0.13 g / L, trace elements 2 mL / L, 1% bromothymol blue, 2% agar powder, pH 7.0~7.3.

[0034] Nitrogen-nitrogen medium: KNO3 0.108 g / L, C6H 12 O6 (glucose) 0.5624 g / L, KH2PO4 0.226 g / L, Na2HPO4·12H2O 0.792 g / L, MgSO4·7H2O 0.01 g / L, trace elements 2 mL / L; of which NO3 - -N content is 15 mg / L.

[0035] Nitrite medium: NaNO2 0.0738 g / L, C6H12 O6 (glucose) 0.5624 g / L, KH2PO4 0.226 g / L, Na2HPO4·12H2O 0.792 g / L, MgSO4·7H2O 0.01 g / L, trace elements 2 mL / L; of which NO2 - -N content is 15 mg / L.

[0036] Ammonia nitrogen medium: NH4Cl 0.058 g / L, C6H 12 O6 (glucose) 0.5624 g / L, KH2PO4 0.226 g / L, Na2HPO4·12H2O 0.792 g / L, MgSO4·7H2O 0.01 g / L, trace elements 2 mL / L; including NH4 + -N content is 15 mg / L.

[0037] Trace elements: FeCl2·4H2O 1.8g / L, CoCl2·6H2O 0.25g / L, NiCl2·6H2O 0.01g / L, CuCl2·2H2O 0.01g / L, MnCl2·4H2O 0.70g / L, ZnCl20.1g / L, H3BO30.5g / L, Na2MoO4·2H2O, NaSeO3·5H2O 0.01g / L.

[0038] Sterilization procedure: Pour 150 ml of culture medium into a 250 ml Erlenmeyer flask, and sterilize in an autoclave at 120°C for 20 minutes. The culture medium contains C6H. 12 The sterilizer temperature for O6 culture medium was set to 115℃ to prevent glucose carbonization.

[0039] Culture conditions for the strain: After inoculation into the culture medium, the culture medium was placed in a constant temperature incubator at 150 rpm and 30°C.

[0040] Example 1: Screening, isolation and identification of strains Inoculate 5 mL of activated sludge into denitrification enrichment medium (DBM), and after three days of culture, inoculate 5 mL of the culture into fresh denitrification enrichment medium. Repeat this process five times. Finally, serially dilute the culture solution from the last round and spread it onto denitrification identification medium (BTB). The target colonies were selected, purified by streak filtration, and verified using denitrification test medium (nitrate medium). Finally, the Pseudomonas strain AH40 was obtained.

[0041] Strain AH40 is a Gram-negative bacterium with round, milky-white, moist colonies that have a smooth surface and short, rod-shaped cells. Figure 1The image shows the colony morphology of strain AH40 (a), a Gram-stained micrograph (b), and a scanning electron microscope image (c).

[0042] Molecular biological identification: Using universal primers for 16S rDNA gene amplification and AH40 bacterial culture as a template, PCR amplification was performed, yielding a PCR product of 1396 bp, the sequence of which is shown in SEQ ID NO.1 below: Figure 2 As shown, it was found to be related to Pseudomonas ( Pseudomonas The 16S rDNA gene sequence similarity of the strain was 100%. Based on the morphological characteristics, physiological and biochemical properties, and molecular biological identification characteristics of the strain, it was identified as belonging to Pseudomonas and named Pseudomonas AH40. Pseudomonas sp. AH40 was deposited at the China Center for Type Culture Collection on August 15, 2022, with accession number CCTCC NO: M20221280.

[0043] Example 2: Aerobic denitrification performance of strain AH40 Strain strain AH40 was first inoculated into enrichment medium and incubated in a constant temperature incubator at 150 rpm and 30℃. When the OD600 reached 0.25, 2% v / v of the culture was inoculated into nitrate nitrogen medium, nitrite nitrogen medium, and ammonia nitrogen medium, respectively, to adapt strain AH40 to the three nitrogen sources at 15 mg / L, thus obtaining three activated bacterial suspensions of AH40 with different nitrogen sources. The activated bacterial suspension of AH40 in nitrate nitrogen medium with 2% v / v OD600 = 0.25 was added to fresh nitrate nitrogen medium and incubated in a constant temperature incubator at 150 rpm and 30℃. Samples were taken periodically to determine OD600 (representing biomass), TN (representing total nitrogen), and TRN (representing soluble total nitrogen). After filtration through a 0.22 μm filter membrane, the NO3 content in the culture medium was measured. - -N, NO2 - -N and NH4 + The content of -N. The experimental procedure using sodium nitrite and ammonium chloride as nitrogen sources is exactly the same as that of the nitrate nitrogen medium experiment, with initial NO2. - -N and NH4 + The concentration of -N was set at 15 mg / L, and all tests were performed in triplicate.

[0044] Regarding detection methods, OD 600 TN, NO3 - -N, NO2 - -N and NH4 + -N content was measured using a spectrophotometer, OD 600 The values ​​were directly measured at a wavelength of 600 nm. TN and TRN were determined using an alkaline potassium persulfate oxidation-UV spectrophotometric method. NO3 - -N was determined using ultraviolet spectrophotometry, NO2 - -N is produced using the sulfonamide-naphthylethylenediamine hydrochloride method, NH4 + -N was determined using Nessler's reagent method.

[0045] When NO3 - The detection results when -N is used as the sole nitrogen source are as follows: Figure 3As shown, the abundance of strain AH40 increased continuously from 0 to 14 hours; simultaneously, after 14 hours of culture, NO3... - The concentration of nitrogen-1,N decreased from the initial 14.74 mg / L to 0.08 mg / L, achieving a removal rate of 99.46%. The removal rate of dissolved nitrogen in the water sample reached 95.71%, and the accumulation of ammonia nitrogen and nitrite nitrogen was low, almost zero.

[0046] When NO2 - When -N is the only nitrogen source, the detection results are as follows: Figure 4 As shown, after strain AH40 was cultured for 14 hours, NO2... - The NO2- concentration decreased from the initial 15.31 mg / L to 0.0055 mg / L, achieving a removal rate of 99.96%. - -N was almost completely removed. There was no accumulation of ammonia nitrogen, and very little accumulation of nitrate nitrogen.

[0047] When NH4 + When -N is the only nitrogen source, the detection results are as follows: Figure 5 As shown, NH4 + The removal rate of -N was 100%, and the accumulation of nitrite and nitrate was minimal.

[0048] Example 3: Preparation of Fe / CN composite material Using metal-organic frameworks (MOFs) as precursors, single-atom iron Fe-ISAs immobilized on nitrogen-doped porous carbon materials were synthesized; molecular iron Fe-NPs were obtained by reducing ferric sulfate with sodium borohydride; then, molecular iron was adsorbed onto the single-atom iron materials through physical stirring to obtain a dual-scale iron composite material Fe / CN. The specific preparation method is as follows: (1) Synthesis of 0.5% Fe-MET-6: 2.00 g ZnCl2 was dissolved in a solvent mixture consisting of 20 mL N,N-dimethylformamide (DMF), 20 mL anhydrous ethanol, 30 mL deionized water, and 8 mL 30% ammonium hydroxide under stirring. The mixture was sonicated for 5 min to form a homogeneous solution. Then, 26 mg of iron triacetylacetone was added, and the mixture was stirred for 30 min. 2.5 mL of 1H-1,2,3-triazole was added dropwise to the mixture to form a precipitate. The mixture was allowed to stand at room temperature for 24 h. The solid was collected by centrifugation, washed several times with ethanol, and dried overnight in a constant temperature oven at 70 °C to obtain 0.5% Fe-MET-6.

[0049] (2) 0.5% Fe-MET-6 was placed in a quartz boat and then heated in a tube furnace at 920°C for 3 hours at a heating rate of 5°C / min under a flowing nitrogen atmosphere to obtain 0.5% Fe-ISAs.

[0050] (3) Synthesis of Fe-NPs: Under vigorous stirring and a nitrogen atmosphere, 3g of FeSO4·7H2O was dissolved in 150ml of 30% ethanol / water solution (V / V), and 0.3M sodium borohydride solution was added dropwise to reduce Fe. 2+ Stir until a black substance is produced, then wash three times with ethanol. Finally, disperse all the precipitate in 40 ml of anhydrous ethanol to obtain a Fe-NPs / ethanol solution for later use.

[0051] (4) Synthesis of Fe / CN: Take 60 mg of 0.5% Fe-ISAs and disperse it in 49 ml of anhydrous ethanol. Sonicate for 5 min, then add 1 ml of the above Fe-NPs / ethanol solution, stir for 8 h, and dry to obtain powdered Fe / CN.

[0052] Example 4: Relevant Characterization of Iron Composite Materials Figure 6 This is a scanning electron microscope image of the iron composite material. The image shows that the material has a porous structure, which increases the contact area with bacteria. Figure 7 , Figure 8 The images show transmission electron microscopy (TEM) images and high-angle annular dark-field scanning TEM images. It can be seen from the images that the iron composite material contains single-atom iron, and that the nano-iron is loaded on its carbon-based support.

[0053] Example 5: Preparation of the combined formulation A 2% v / v AH40 activated bacterial solution (prepared according to the method in Example 2, OD600=0.25) was added to nitrate nitrogen medium and cultured until OD600=0.25. The bacterial solution was then freeze-dried under vacuum to obtain bacterial powder. This bacterial powder was mixed with the Fe / CN prepared in Example 3 at a mass ratio of 1:1 to obtain the combined formulation.

[0054] Example 6: Effect of combined formulation on denitrification This example further investigates the effect of the combined formulation compared to the single strain AH40 on the denitrification process. 18 mg of the combined formulation and 9 mg of AH40 bacterial powder (both prepared in Example 5) were added to 150 ml of nitrification medium, respectively. Simultaneously, only 9 mg of iron composite material (prepared in Example 3) was added to 150 ml of nitrification medium as a control group to exclude the potential influence of the iron composite material on denitrification. All three groups were incubated in a constant temperature incubator at 150 rpm and 30°C, with three parallel experiments for each group. Samples were taken periodically, filtered through a 0.22 μm filter membrane, and the NO3 content in the culture medium was measured. - -N.

[0055] from Figure 9It can be seen that the single iron composite material did not affect the nitrate content in the culture medium, but the reduction rate of nitrate nitrogen increased with the addition of the combined preparation. By 10h, the nitrate content in the experimental group with only strain AH40 powder added was still 5.19 mg / L, with a removal rate of only 64.5%; while the nitrate in the experimental group with the combined preparation was almost completely removed, with a removal rate of 96.1%, indicating that the application of the combined preparation can effectively increase the removal efficiency of nitrate in water.

[0056] Example 7: Effect of combined formulation on the activity of electron transport system This example further investigates the effect of the combined formulation on the activity of the electron transport system. 18 mg of the combined formulation and 9 mg of AH40 bacterial powder (both prepared in Example 5) were added to 150 ml of nitrate-nitrogen medium, respectively. Both groups were incubated in a constant temperature incubator at 150 rpm and 30°C, with three parallel experiments for each group. Samples were taken at 8 h. 10 ml of the sample was placed in a 10 ml centrifuge tube and centrifuged at 8000 rpm for 10 minutes to collect the bacterial precipitate. The bacterial precipitate, 0.2 mg NADH (reducing coenzyme I), and 200 μL of 0.5% 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetraazole chloride (INT) were mixed and reacted at 30°C in the dark for 30 minutes. Then, 100 μL of formaldehyde was added to terminate the reaction, and the cell slurry was harvested by centrifugation at 10,000 rpm for 3 min. Formazan orange (INF) was extracted from the cell slurry using methanol as solvent (500 μL, 96%). Using the solvent as a blank, the OD490 was detected at a wavelength of 490 nm using a spectrophotometer to calculate the activity of the electron transport system.

[0057] from Figure 10 It can be seen that the activity of the electron transport system in the combined formulation experimental group (1.0975 ± 0.1511) was significantly higher than that in the experimental group with only strain AH40 added (2.3012 ± 0.2129), which was 2.09 times that of the AH40 experimental group.

[0058] Example 8: Effect of combined formulation on denitrification-related enzyme activity This embodiment further investigated the effect of the combined formulation on the activity of denitrification-related enzymes. 18 mg of the combined formulation and 9 mg of AH40 bacterial powder (both prepared in Example 5) were added to 150 ml of nitrate-containing medium, respectively. Both groups were incubated in a constant temperature incubator at 150 rpm and 30°C, with three parallel experiments for each group. Samples were taken at 8 hours. 10 ml of the sample was placed in a 10 ml centrifuge tube and centrifuged at 8000 rpm for 10 minutes to collect the bacterial pellet. The pellet was then washed three times with PBS (50 mmol / L, pH=7.2) and resuspended. The bacterial resuspension was subjected to intermittent ultrasonic disruption at 2-second intervals (4°C, 200W, 3 min) in an ice bath using an ultrasonic cell disruptor. The suspension was then centrifuged at 4°C and 12000 rpm for 10 min. The collected supernatant was the cell extract, used for subsequent enzyme activity assays. The total protein concentration in the cell extract was determined using the Coomassie Brilliant Blue assay.

[0059] Periplasmic nitrate reductase (NAP) activity assay: 100 μL of cell extract was added to a 2 mL reaction flask of Mixture I. The reaction was started, and NO2 was measured using a spectrophotometer after 30 min of reaction. - The amount of -N produced. Mixture I consisted of 10 mmol / L PBS (pH=7.4), 5 mmol / L Na₂S₂O₄, 10 mmol / L methyl viologen, and 1 mmol / L KNO₃. Nitrate reductase activity is the amount of NO₂ produced per unit time per unit protein action. - The amount of -N was measured by ultraviolet spectrophotometry.

[0060] Nitrite reductase (NIR) activity assay: Add 100 μL of cell extract to a 2 mL reaction flask containing mixture II and start the reaction. After reacting for 30 min, measure NO2. - The amount of -N consumed. Mixture II consists of 10 mmol / L PBS (pH=7.4), 5 mmol / L Na2S2O4, 10 mmol / L methyl amethyst, and 1 mmol / L NaNO2. Nitrite reductase activity is the amount of NO2 reduced per unit time per unit protein. - The amount.

[0061] Figure 11 It can be seen that the combined preparation has a weak effect on nitrate reductase activity, but significantly increases nitrite reductase activity. The combined preparation experimental group (0.1442 ± 0.0193) is 3.17 times that of the strain AH40 experimental group (0.4578 ± 0.0286).

[0062] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A type of Pseudomonas AH40 ( Pseudomonas sp. AH40), characterized in that, Its accession number is CCTCC NO: M20221280, and it was deposited at the China Center for Type Culture Collection on August 15, 2022.

2. A microbial agent, characterized in that, The bacterial agent comprises Pseudomonas AH40 as described in claim 1 or a preparation of Pseudomonas AH40.

3. The microbial agent according to claim 2, characterized in that, The preparations of Pseudomonas AH40 include cultures of Pseudomonas AH40, culture extracts, lyophilized powders, fermentation broths, fermentation broth precipitates, fermentation broth supernatants, or fermentation broth extracts.

4. The application of the Pseudomonas AH40 of claim 1 or the bacterial agent of any one of claims 2-3 in denitrification of water bodies.

5. A method for nitrification denitrification in water, characterized in that, The method involves adding the Pseudomonas aeruginosa AH40 of claim 1 or the bacterial agent of any one of claims 2-3 into the water body to carry out denitrification.

6. A combination formulation, characterized in that, The combined formulation comprises Pseudomonas AH40 as described in claim 1 or the bacterial agent as described in any one of claims 2-3.

7. The combination formulation according to claim 6, characterized in that, The combined formulation also includes an iron composite material.

8. The combination formulation according to claim 7, characterized in that, The preparation method of the iron composite material includes the following steps: (1) ZnCl2 was dissolved in a solvent mixture consisting of N,N-dimethylformamide, anhydrous ethanol, deionized water and ammonium hydroxide, and sonicated to form a homogeneous solution. Then, triacetylacetone iron was added and mixed well. Then, 1H-1,2,3-triazole was added and a precipitate was formed. The precipitate was collected, washed and dried to obtain Fe-MET-6. (2) Fe-MET-6 was heated at a nitrogen atmosphere at a temperature greater than 900°C for 1-5 hours to obtain Fe-ISAs; (3) Under a nitrogen atmosphere, FeSO4·7H2O was dissolved in an ethanol solution, and sodium borohydride solution was added dropwise until a black substance was produced. After stirring, the precipitate was collected and washed. All the precipitates were dispersed in anhydrous ethanol to obtain Fe-NPs / ethanol solution. (4) Take Fe-ISAs and disperse them in anhydrous ethanol, sonicate, then add Fe-NPs / ethanol solution, stir and dry to obtain iron composite material.

9. The combination formulation according to claim 7 or 8, characterized in that, The preparation method of the combined formulation includes the following steps: S1. Add the bacterial culture of Pseudomonas aeruginosa AH40 to a nitrate-containing medium for culture, and then dry to obtain bacterial powder; S2. Add the iron composite material to the bacterial powder, with a mass ratio of bacterial powder to iron composite material of 1:1, and mix well to obtain the combined preparation.

10. The application of the combined formulation according to any one of claims 6-9 in denitrification of water bodies.

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