Stress-resistant heterotrophic nitrification-aerobic denitrification nitrogen removal bacterium and application thereof

Abstract

The invention relates to the field of microorganisms, and discloses a stress-resistant heterotrophic nitrification-aerobic denitrification nitrogen removal bacterium and application thereof. The pseudomonas is named as H1, the microorganism classification name of the pseudomonas is Pseudomonas sihuiensis, the preservation number of the pseudomonas is CGMCC (China General Microbiological Culture Collection Center) NO: 33481, the preservation date of the pseudomonas is January 20, 2025, and the preservation place of the pseudomonas is China General Microbiological Culture Collection Center. The pseudomonas H1 disclosed by the invention can still realize efficient denitrification in a heterotrophic nitrification-aerobic denitrification way in a high-salt and high-nitrogen water body environment containing toxic pollutants with benzene ring functional groups, so that the pseudomonas H1 has great application potential in biological treatment of nitrogen-containing wastewater.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, in particular to a stress-resistant heterotrophic nitrification-aerobic denitrification bacterium and application thereof. Background Art

[0002] With the rapid development of industries such as chemical, pharmaceutical, printing and dyeing, and food processing, the discharge of high-salinity chemical wastewater has increased annually, and its pollution problem has become increasingly prominent. High-salinity chemical wastewater is characterized by high salinity (>10g / L), high ammonia nitrogen (>250mg / L), complex organic pollutant composition, poor biodegradability, and high toxicity. Efficient nitrogen removal in this high-salinity and toxic environment is a key and difficult issue in wastewater treatment. If treatment does not meet standards, the eutrophication of water bodies caused by nitrogen pollution will have many negative impacts on the environment and will also hinder the sustainable development of the chemical industry. Therefore, the development of green and efficient denitrification technologies is of great significance.

[0003] Microbial denitrification technology has gradually become the mainstream technology for treating nitrogen-containing wastewater because of its cleanliness and high efficiency. Traditional biological denitrification refers to the process in which organic nitrogen undergoes nitrification and denitrification reactions under the combined action of aerobic autotrophic nitrifying bacteria and anaerobic heterotrophic denitrifying bacteria, and is eventually converted into gaseous nitrogen. At present, studies have discovered a new type of denitrifying bacteria, in which nitrification and denitrification can occur simultaneously under heterotrophic and aerobic conditions, and organic pollutants can be removed while denitrifying. These bacteria are synchronous heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria (e.g., CN114480159A). This type of bacteria has the advantages of rapid growth, strong environmental tolerance, and high denitrification efficiency. It fundamentally breaks through the technical bottleneck of the traditional nitrification-denitrification process, which requires zoned operation and strict oxygen control, and provides a more efficient and economical solution for the biological denitrification of high-salt chemical wastewater.

[0004] However, the HN-AD bacteria that have been isolated so far have a good denitrification effect under laboratory simulated wastewater conditions, but the denitrification efficiency is significantly reduced and the effect is extremely unstable in the actual high-salt chemical wastewater environment. This is because: (1) The high salt content in high-salt chemical wastewater will destroy the microbial cell structure, thereby inhibiting microbial growth and reproduction, resulting in a significant reduction in denitrification efficiency; (2) Chemical wastewater usually contains toxic pollutants with benzene ring functional groups, such as sulfonamide antibiotics, aniline and other pollutants. The toxicity of these pollutants will reduce microbial activity, slow down the reaction rate, and worsen the treatment effect. Therefore, screening heterotrophic nitrification-aerobic denitrification strains that can stably perform denitrification performance in high-salt, high-nitrogen, and toxic pollutant-containing environments is the basis for building efficient microbial denitrification technology, and is of great significance for promoting the synergistic increase in pollution reduction and carbon reduction in high-salt chemical wastewater treatment. Summary of the Invention

[0005] To address the above technical problems, the present invention provides a stress-resistant heterotrophic nitrification-aerobic denitrification bacterium and its applications. The present invention provides a novel Pseudomonas sp. H1 strain that can achieve efficient denitrification through a heterotrophic nitrification-aerobic denitrification pathway even in high-salt, high-nitrogen environments containing toxic pollutants with benzene rings. Therefore, it has great potential for application in the biological treatment of nitrogenous wastewater.

[0006] The specific technical solutions of the present invention are: In the first aspect, the present invention provides a stress-resistant heterotrophic nitrification-aerobic denitrification bacterium, which is named H1, and its microbial classification is named Pseudomonas sihuiensis, the preservation number is CGMCC NO: 33481, the preservation date is January 20, 2025, and the preservation location is China General Microbiological Strain Collection Administration Center; the 16SrRNA sequence of the above-mentioned Pseudomonas H1 is shown in SEQ ID NO: 1.

[0007] The morphological and physiological and biochemical characteristics of the Pseudomonas H1 strain of the present invention are as follows: it is a Gram-negative bacterium that can grow under aerobic facultative conditions. After culturing on a solid plate of enriched acclimation medium for 7 days, the strain colonies are round, with regular edges, a smooth and moist surface, a slightly raised center, and an opaque yellow color. Figure 1 The temperature growth range is 15-45℃ (optimum 35℃), and the pH growth range is 5.0-9.0 (optimum 7.5). PCR amplification and sequencing were performed using bacterial 16S rRNA universal primers. The sequencing results were compared with the 16S rRNA gene sequence homology of other strains in the GenBank gene database using BLAST software. Strains with higher sequence similarity were selected, and a phylogenetic tree was constructed using the neighbor-joining method (NJ) using MEGA 11.0 software ( Figure 2 ), the results showed that the gene sequence similarity between strain H1 and Pseudomonas sihuiensis was over 98%. In summary, combined with the morphological characteristics and 16S rRNA gene sequence analysis results of strain H1, the strain was preliminarily identified as Pseudomonas sihuiensis and named Pseudomonas sihuiensis H1.

[0008] The Pseudomonas H1 obtained by screening in the present invention has the ability to remove nitrogen from wastewater through the heterotrophic nitrification-aerobic denitrification pathway. Specifically, the strain can convert organic nitrogen in wastewater into inorganic nitrogen. Furthermore, the present invention also found that during the process of converting ammonia nitrogen, nitrite nitrogen or nitrate nitrogen respectively, there will be no accumulation of nitrite nitrogen and nitrate nitrogen. This simultaneous nitrification and denitrification treatment can significantly improve the efficiency of microbial denitrification, reduce the residence time of nitrogen in the water body, thereby reducing the risk of eutrophication of the water body, and at the same time reduce the risk of secondary pollution that may be caused by these intermediate products, which is of great significance for protecting the ecological environment of the water body and human health.

[0009] In addition, compared with the characteristics of most strains that have poor survival ability or low denitrification activity in water environments with high salt, high nitrogen, and toxic pollutants containing benzene ring functional groups, the Pseudomonas H1 of the present invention has a very strong resistance to stress of high salt, high nitrogen, and high concentrations of toxic pollutants containing benzene ring functional groups in water bodies, and still has efficient denitrification ability in the corresponding water environment.

[0010] In a second aspect, the present invention provides a microbial denitrification preparation containing Pseudomonas and / or its bacterial culture; wherein the Pseudomonas is named H1, its microbial classification is named Pseudomonas sihuiensis, the preservation number is CGMCC NO: 33481, the preservation date is January 20, 2025, and the preservation location is China General Microbiological Strain Collection Center; the 16S rRNA sequence of the Pseudomonas H1 is shown in SEQ ID NO: 1.

[0011] In a third aspect, the present invention provides the use of the aforementioned stress-resistant heterotrophic nitrification-aerobic denitrification bacteria or the aforementioned microbial denitrification preparation in wastewater denitrification. The Pseudomonas and / or its bacterial culture denitrifies wastewater via a heterotrophic nitrification-aerobic denitrification pathway.

[0012] Furthermore, the salinity of the wastewater is ≤80 g / L (the optimal salinity range is 20-60 g / L).

[0013] Furthermore, the nitrogen-containing substances in the wastewater are one or more of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and organic nitrogen.

[0014] Furthermore, the nitrogen concentration in the wastewater is ≤1500 mg / L (the optimal nitrogen concentration range is 200-500 mg / L).

[0015] Furthermore, the wastewater contains toxic pollutants with benzene ring functional groups.

[0016] Still more preferably, the toxic pollutant containing a benzene ring functional group is one or more of sulfamethoxazole, aniline, catechol, protocatechuic acid, benzoic acid and butyl benzoate.

[0017] The present invention further discovered that when the toxic pollutants containing benzene ring functional groups are aniline, catechol, protocatechuic acid, benzoic acid, and butyl benzoate, Pseudomonas H1 can not only tolerate and degrade these compounds, but also use these compounds as substrates to enhance its growth activity. Among them, Pseudomonas H1 grows fastest in an environment containing catechol and protocatechuic acid. However, when the benzene ring side chain in the compound is more complex (such as aniline, benzoic acid, and butyl benzoate), it is relatively difficult for the strain to utilize it, and the growth activity promotion effect is relatively weak.

[0018] Furthermore, the concentration of toxic pollutants in the wastewater is ≤1000 mg / L (the optimal concentration of toxic pollutants is 20-200 mg / L).

[0019] In a fourth aspect, the present invention provides a method for denitrification of wastewater based on microorganisms: Pseudomonas and / or its bacterial culture is added to wastewater to achieve denitrification through a heterotrophic nitrification-aerobic denitrification pathway.

[0020] The Pseudomonas is named H1, its microbial classification is named Pseudomonas sihuiensis, its preservation number is CGMCC NO: 33481, the preservation date is January 20, 2025, and the preservation location is China General Microbiological Strain Collection Center; the 16S rRNA sequence of the Pseudomonas H1 is shown in SEQ ID NO: 1.

[0021] Preferably, the inoculation amount of Pseudomonas and / or its bacterial culture in the wastewater is 1%-50% (v / v).

[0022] Preferably, the denitrification temperature is 15-45°C and the denitrification time is 6-24h.

[0023] Furthermore, the salinity of the wastewater is ≤80 g / L (the optimal salinity range is 20-60 g / L); and / or Furthermore, the nitrogen-containing substances in the wastewater are one or more of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and organic nitrogen.

[0024] Furthermore, the nitrogen concentration in the wastewater is ≤1500 mg / L (the optimal nitrogen concentration range is 200-500 mg / L).

[0025] Still more preferably, the toxic pollutant containing a benzene ring functional group is one or more of sulfamethoxazole, aniline, catechol, protocatechuic acid, benzoic acid and butyl benzoate.

[0026] Furthermore, the concentration of toxic pollutants with benzene ring functional groups in the wastewater is ≤1000 mg / L (the optimal concentration of toxic pollutants with benzene ring functional groups is 20-200 mg / L).

[0027] Compared with the prior art, the present invention has the following beneficial effects: (1) The Pseudomonas H1 strain of the present invention exhibits excellent growth adaptability and denitrification capabilities in high-salt, high-nitrogen, and water environments containing toxic pollutants with benzene rings. Therefore, it has significant application potential and advantages in the treatment of wastewater containing high-salt, high-ammonia nitrogen, and toxic pollutants with benzene rings.

[0028] (2) The Pseudomonas sp. H1 of the present invention has the ability to rapidly remove nitrogen from wastewater through a heterotrophic nitrification-aerobic denitrification pathway. Furthermore, the strain does not accumulate nitrite nitrogen and nitrate nitrogen during the process of converting ammonia nitrogen, nitrite nitrogen, or nitrate nitrogen, respectively. This simultaneous nitrification and denitrification process can significantly improve the efficiency of microbial denitrification, reduce the residence time of nitrogen in the water body, thereby reducing the risk of eutrophication of the water body, and at the same time reduce the risk of secondary pollution caused by these intermediate products, which is of great significance for protecting the water ecological environment and human health.

[0029] (3) The Pseudomonas sp. H1 strain of the present invention not only tolerates and degrades toxic pollutants containing benzene ring functional groups, but also utilizes these compounds as substrates to enhance its growth activity. The growth enhancement effect is best in an environment containing catechol and protocatechuic acid, and the degradation rate is also the best. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a photo of the colony morphology of the H1 strain.

[0031] Figure 2 This is the phylogenetic tree of strain H1 constructed based on 16S rRNA gene sequence homology.

[0032] Figure 3 The growth and denitrification characteristics of H1 strain when using ammonia nitrogen and nitrate nitrogen as mixed nitrogen sources.

[0033] Figure 4 The growth and denitrification characteristics of strain H1 when using ammonia nitrogen and nitrite nitrogen as mixed nitrogen sources.

[0034] Figure 5 The growth and denitrification characteristics of H1 strain under different salinities when ammonia nitrogen was the only nitrogen source ((a) OD 600 , (b) ammonia nitrogen, (c) nitrite nitrogen and (d) nitrate nitrogen).

[0035] Figure 6The growth and denitrification characteristics of H1 strain under different nitrogen concentrations when ammonia nitrogen was the only nitrogen source ((a) OD 600 , (b) ammonia nitrogen, (c) nitrite nitrogen and (d) nitrate nitrogen).

[0036] Figure 7 This is the degradation of aniline by strain H1 under high salt conditions.

[0037] Figure 8 This is the denitrification performance of H1 strain under mixed organic pollutant conditions.

[0038] Figure 9 The degradation of aniline by strain H1 at different concentrations is shown.

[0039] Figure 10 The growth and denitrification characteristics of H1 strain under different concentrations of aniline ((a) OD 600 , (b) ammonia nitrogen, (c) nitrite nitrogen and (d) nitrate nitrogen).

[0040] Figure 11 This is the utilization of aniline, catechol and protocatechuic acid as substrates by H1 strain. DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to the embodiments.

[0042] Overall embodiment A stress-resistant heterotrophic nitrification-aerobic denitrifying Pseudomonas strain was named H1, its microbial classification was named Pseudomonas sihuiensis, the preservation number was CGMCC NO: 33481, the preservation date was January 20, 2025, and the preservation location was China General Microbial Strain Collection Management Center.

[0043] The 16S rRNA sequence of the above-mentioned Pseudomonas sp. H1 is shown in SEQ ID NO: 1.

[0044] A microbial denitrification preparation, comprising Pseudomonas and / or a bacterial culture thereof; wherein the Pseudomonas is named H1, its microbial classification is named Pseudomonas sihuiensis, its preservation number is CGMCC NO: 33481, its preservation date is January 20, 2025, and its preservation location is China General Microbiological Strain Collection Administration Center; the 16S rRNA sequence of the Pseudomonas H1 is shown in SEQ ID NO: 1.

[0045] Application of the stress-resistant heterotrophic nitrification-aerobic denitrification Pseudomonas or the microbial denitrification preparation in wastewater denitrification: the Pseudomonas and / or its bacterial culture denitrifies wastewater through heterotrophic nitrification-aerobic denitrification.

[0046] In some specific implementation cases, the salinity of the wastewater is ≤80 g / L; in some more preferred implementation cases, the salinity of the wastewater is in the range of 20-60 g / L.

[0047] Furthermore, the nitrogen-containing substances in the wastewater are one or more of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and organic nitrogen; in some specific implementation cases, the nitrogen concentration in the wastewater is ≤1500 mg / L, and in some more preferred implementation cases, the nitrogen concentration in the wastewater is in the range of 200-500 mg / L.

[0048] Furthermore, the wastewater contains toxic pollutants with benzene ring functional groups; in some preferred implementation cases, the toxic pollutants containing benzene ring functional groups are one or more of sulfamethoxazole, aniline, catechol, protocatechuic acid, benzoic acid and butyl benzoate; in some preferred implementation cases, the concentration of toxic pollutants with benzene ring functional groups in the wastewater is ≤1000 mg / L; in some more preferred implementation cases, the concentration range of toxic pollutants with benzene ring functional groups in the wastewater is 20-200 mg / L.

[0049] A microbial-based wastewater denitrification method involves adding Pseudomonas and / or its bacterial culture to wastewater to achieve denitrification via a heterotrophic nitrification-aerobic denitrification pathway. The Pseudomonas is designated H1, and its microbial classification is Pseudomonas sihuiensis. Its deposit number is CGMCC NO: 33481, and its deposit date is January 20, 2025, at the China General Microbiological Collection Center. The 16S rRNA sequence of Pseudomonas H1 is shown in SEQ ID NO: 1.

[0050] In some preferred embodiments, the inoculation amount of Pseudomonas and / or its bacterial culture in the wastewater is 1%-50% (v / v).

[0051] In some preferred implementation cases, the denitrification temperature is 15-45°C and the denitrification time is 6-24 hours.

[0052] In some preferred implementation cases, the salinity of the wastewater is ≤80 g / L; in some more preferred implementation cases, the salinity of the wastewater is in the range of 20-60 g / L.

[0053] In some preferred implementation cases, the nitrogen concentration in the wastewater is ≤1500 mg / L; in some more preferred implementation cases, the nitrogen concentration in the wastewater is in the range of 200-500 mg / L.

[0054] In some specific implementation cases, the nitrogen-containing substances in the wastewater are one or more of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and organic nitrogen.

[0055] In some preferred implementation cases, the toxic pollutant containing a benzene ring functional group is one or more of sulfamethoxazole, aniline, catechol, protocatechuic acid, benzoic acid and butyl benzoate.

[0056] In some preferred implementation cases, the concentration of toxic pollutants with benzene ring functional groups in the wastewater is ≤1000 mg / L, and the concentration of toxic pollutants with benzene ring functional groups in the wastewater ranges from 20 to 200 mg / L. Specific embodiments Example 1: Domestication, isolation and identification of strains (1) Culture medium Enriched acclimation medium: K2HPO4·3H2O 1.50 g / L, NaNO3 1.1 g / L, NH4Cl 0.7 g / L, KH2PO4 0.5 g / L, NaCl 20 g / L, MgSO4·7H2O 0.2 g / L, sulfamethoxazole (SMX) 20.0 mg / L.

[0058] High-salt LB liquid medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 20.0 g / L, sulfamethoxazole (SMX) 20.0 mg / L.

[0059] The solid culture medium is prepared by adding 1.5% (w / v) agar to the corresponding liquid culture medium formula. The sterilization method is high-temperature and high-pressure steam sterilization at 121° C. for 30 minutes.

[0060] (2) Domestication The sludge from the sewage treatment plant of Yangzhou Tianhe Pharmaceutical Co., Ltd. was added to the enrichment and acclimation culture medium, and cultured in a constant temperature incubator at 35°C and 150 r / min for one week to obtain the first-generation bacterial community. Then, part of the first-generation bacterial community was taken to fresh enrichment and acclimation culture medium and cultured for one week. The above subculture process was repeated multiple times in the same proportion to obtain a stable primary bacterial community.

[0061] (3) Purification and separation Part of the primary bacterial culture solution was mixed with a concentration gradient of 10 -3 , 10 -4 , 10 -5 and 10 -6The strain was diluted 100 μL at each concentration and evenly spread on an enriched acclimation solid medium. The culture was then inverted and cultured in a 35°C constant temperature biochemical incubator for 7 days. Independent colonies with obvious morphological differences were picked and isolated by streaking on the plate. After repeated purification, strain H1 was obtained.

[0062] (4) Identification The morphological and physiological and biochemical characteristics of the strain H1 were identified as follows: it is a Gram-negative bacterium that can grow under aerobic conditions. After 7 days of culture on a solid plate of enriched acclimation medium, the colonies of the strain were round, with regular edges, a smooth and moist surface, a slightly raised center, and an opaque yellow color. Figure 1 The temperature growth range is 15-45℃ (optimum 35℃), and the pH growth range is 5.0-9.0 (optimum 7.5). PCR amplification and sequencing were performed using universal primers for bacterial 16S rRNA. The sequencing results were compared with the 16S rRNA gene sequence homology of other strains in the GenBank gene database using BLAST software. Strains with higher sequence similarity were selected, and a phylogenetic tree was constructed using the neighbor-joining method (NJ) using MEGA 11.0 software ( Figure 2 ), the results showed that the gene sequence similarity between strain H1 and Pseudomonas sihuiensis was over 98%. In summary, combined with the morphological characteristics and 16S rRNA gene sequence analysis results of strain H1, the strain was preliminarily identified as Pseudomonas sihuiensis and named Pseudomonas sihuiensis H1.

[0063] The 16S rRNA sequence of Pseudomonas sp. H1 is shown in SEQ ID NO: 1:

[0064] Example 2: Heterotrophic nitrification-aerobic denitrification capability of Pseudomonas sp. H1 in a high-salt environment (1) Preparation of culture medium Heterotrophic nitrification-aerobic denitrification medium 1: C6H5Na3O74.74g / L, NH4Cl 0.25g / L, NaNO3 0.406g / L, NaCl 20g / L, MgSo4·7H2O 0.1g / L, KH2PO4 1.5g / L, K2HPO4·3H2O 7.9g / L, pH 7.0.

[0065] Heterotrophic nitrification-aerobic denitrification medium 2: C6H5Na3O7 4.74 g / L, NH4Cl 0.25 g / L, NaNO2 0.25 g / L, NaCl 20 g / L, MgSO4·7H2O 0.1 g / L, KH2PO4 1.5 g / L, K2HPO4·3H2O7.9 g / L, pH 7.0.

[0066] Simulated wastewater medium 1 with ammonium chloride as the sole nitrogen source: C6H5Na3O7 4.74 g / L, NH4Cl 0.25 g / L, MgSO4·7H2O 0.1 g / L, KH2PO4 1.5 g / L, K2HPO4·3H2O 7.9 g / L, pH 7.0.

[0067] Simulated wastewater medium 2 with ammonium chloride as the sole nitrogen source: C6H5Na3O7 4.74 g / L, NaCl 20 g / L, MgSO4·7H2O 0.1 g / L, KH2PO4 1.5 g / L, K2HPO4·3H2O 7.9 g / L, pH 7.0.

[0068] Simulated wastewater culture medium 3: KH2PO4 1 g / L, K2HPO4 1 g / L, NaCl 20 g / L, salt solution 10 mL, salt solution: MgSO4 0.5 g / L, MnSO4 0.17 g / L, H38O3 0.116 g / L, ZnSO4·7H2O 0.115 g / L, FeSO4·7H2O 0.3 g / L, pH 7.0, aniline 200 mg / L.

[0069] Simulated wastewater culture medium 4: C6H5Na3O74.74g / L, NH4Cl 0.5g / L, NaCl 20g / L, K2HPO4·3H2O1.5g / L, KH2PO40.5g / L, MgSO4·7H2O0.1g / L, SMX 20mg / L, aniline 200mg / L.

[0070] Simulated wastewater culture medium 5: C6H5Na3O74.74g / L, NH4C10.5g / L, NaCl 20g / L, K2HPO4·3H2O1.5g / L, KH2PO40.5g / L, MgSO4·7H2O0.1g / L, aniline concentration gradient 200mg / L, 400mg / L, 600mg / L, 800mg / L, 1000mg / L, volume 100mL.

[0071] Simulated wastewater culture medium 6: NH4C10.5 g / L, NaCl 20 g / L, K2HPO4·3H2O1.5 g / L, KH2PO40.5 g / L, MgSO4·7H2O0.1 g / L, volume 100 mL.

[0072] The sterilization method is high temperature and high pressure steam sterilization at 121℃ for 30 minutes.

[0073] (2) Preparation of seed solution Pseudomonas H1 was cultured in a solid enrichment acclimation medium (same as in Example 1) at 35°C for 7 days, and a single colony was picked. The single colony was inoculated into 10 mL of high-salt LB liquid medium (same as in Example 1) and cultured at 150 r / min and 35°C until the logarithmic phase (cultured for 12 hours). The above bacterial liquid was transferred to 60 mL of high-salt LB liquid medium (same as in Example 1) and cultured at 35°C and 150 r / min until the OD 600 =1.0, the cells were collected by centrifugation, washed twice with physiological saline, and resuspended to prepare a cell suspension as the seed solution.

[0074] (3) Denitrification characteristics of Pseudomonas H1 on mixed nitrogen sources (ammonia nitrogen + nitrate nitrogen) The seed solution was transferred to heterotrophic nitrification-aerobic denitrification medium 1 at a 20% (v / v) inoculation rate, cultured at 35°C and 150 r / min, and samples were taken every 12 hours to determine NH4 + -N, NO3 - -N, NO2 - -N and OD 600 The results are as follows Figure 3 shown.

[0075] according to Figure 3 The data show that at a salinity of 20 g / L and a pH of 7.0, Pseudomonas H1 had no obvious growth lag period in medium 1. After 12 h of culture, the OD 600 The concentrations of ammonia nitrogen and nitrate nitrogen reached a peak, and a simultaneous downward trend was observed for both. Compared to nitrate nitrogen, Pseudomonas H1 had a faster removal rate for ammonia nitrogen, rapidly degrading an initial concentration of 65 mg / L of ammonia nitrogen within 12 hours, achieving a removal rate of nearly 100%. Simultaneously, the nitrate nitrogen removal rate was 68.19%, and no nitrite nitrogen accumulated during the experiment. The total nitrogen removal rate in heterotrophic nitrification-aerobic denitrification medium 1 reached 7.79 mg / L / h. This demonstrates that Pseudomonas H1 possesses both nitrification and denitrification capabilities under high-salt, SMX-containing stress conditions.

[0076] (4) Denitrification characteristics of Pseudomonas sp. H1 on mixed nitrogen sources (ammonia nitrogen + nitrite nitrogen) The seed solution was transferred to heterotrophic nitrification-aerobic denitrification medium 2 at a 20% (v / v) inoculation rate, cultured at 35°C and 150 r / min, and samples were taken every 12 h to determine NH4 + -N, NO3 - -N, NO2 - -N and OD 600 The results are as follows Figure 4 shown.

[0077] according to Figure 4 The data show that Pseudomonas H1 exhibited no significant lag phase in the mixed nitrogen source (ammonia nitrogen + nitrite nitrogen) medium 2, rapidly removing 62 mg / L of ammonia nitrogen within 12 hours, with a removal rate approaching 100%. However, Pseudomonas H1 was less effective at removing nitrite nitrogen than ammonia nitrogen, with a removal rate of only 75.42% in 12 hours and a maximum removal rate of 93.01% at 36 hours. Nitrate nitrogen accumulation was virtually nonexistent during the experiment. Pseudomonas H1 achieved a mixed nitrogen removal rate of 8.55 mg / L / h in medium 2.

[0078] (5) Denitrification characteristics of H1 strain at different salinities when ammonia nitrogen was the only nitrogen source The Pseudomonas H1 cell suspension was transferred into the simulated wastewater medium 1 with ammonium chloride as the only nitrogen source at a 20% (v / v) inoculation rate. The salinity gradient was set to 20, 40, 60, 80, and 100 g / L. The culture was carried out at 35°C and 150 rpm. The NH4 + -N, NO3 - -N, NO2 - -N and OD 600 The results are as follows Figure 5 shown.

[0079] The ammonia nitrogen removal performance of Pseudomonas H1 under different salinity conditions is shown in the figure. Figure 5 As shown, in a 20 g / L salinity medium, the strain rapidly removed 128 mg / L of ammonia nitrogen within 12 hours, achieving a 100% removal rate. In salinity media of 40 and 60 g / L, ammonia nitrogen removal rates reached their maximum values ​​of 97.11% and 89.58% within 36 and 48 hours, respectively. However, at higher salinity levels (80 and 100 g / L), the strain lacked denitrification capabilities, as high salinity inhibits microbial activity, reduces the strain's growth and metabolic capacity, and can even lead to its death. Furthermore, no nitrite nitrogen accumulated in the medium, and the minimal nitrate nitrogen accumulation (only 0.5-1 mg / L) was rapidly removed during subsequent experiments, demonstrating that Pseudomonas H1 possesses strong nitrification-denitrification capabilities and can tolerate high salt loads to achieve rapid denitrification.

[0080] (6) Denitrification characteristics of H1 strain under different nitrogen concentrations when ammonia nitrogen is the only nitrogen source. The Pseudomonas H1 cell suspension was transferred into the simulated wastewater medium 2 with ammonium chloride as the only nitrogen source at a 20% (v / v) inoculum volume. The ammonia nitrogen concentration gradient was set to 100, 200, 300, 400, and 500 mg / L. The culture was carried out at 35 °C and 150 rpm. Samples were taken every 12 h to determine the NH4 + -N, NO3 - -N, NO2 - -N and OD 600 The results are as follows Figure 6 shown.

[0081] Study on the denitrification characteristics of Pseudomonas H1 under different ammonia nitrogen concentrations Figure 6 As shown, at low ammonia nitrogen concentrations (100 mg / L), Pseudomonas H1 achieved a 100% ammonia nitrogen removal rate within 12 hours. However, at high ammonia nitrogen concentrations (200-500 mg / L), the removal rate of Pseudomonas H1 gradually decreased with increasing ammonia nitrogen concentrations, reaching 71.22%, 67.95%, 61.75%, and 60.75% at 60 hours in cultures containing 200, 300, 400, and 500 mg / L ammonia nitrogen, respectively. Pseudomonas H1 maintained a high ammonia nitrogen removal rate at concentrations of 100-500 mg / L, reaching a rate of 11-14 mg / L / h within 12 hours. Furthermore, there was no significant accumulation of nitrite and nitrate nitrogen in the culture medium, demonstrating that Pseudomonas H1 possesses strong nitrification-denitrification capabilities, can tolerate high ammonia nitrogen loads, and achieve rapid nitrogen removal. Under the stress conditions of high salt, high ammonia nitrogen and the presence of the toxic pollutant SMX, Pseudomonas H1 removed nitrogen in a shorter time (12h) and at a faster rate than other strains, and the ammonia nitrogen removal rate remained stable at a higher level.

[0082] (7) Removal of toxic pollutants with benzene ring functional groups by H1 strain under high salt conditions. Pseudomonas H1 cell suspension was transferred into simulated wastewater medium 3 at a 20% (v / v) inoculation rate and cultured at 35°C and 150 r / min. Samples were taken every 24 h to determine the concentrations of aniline and NH4 + -N. The result is as follows Figure 7 shown.

[0083] Removal of toxic pollutants with benzene ring functional groups by Pseudomonas H1 under high salt conditions Figure 7 As shown in the figure, the aniline removal rate at 84 hours was 43.77%, and the ammonia nitrogen accumulation was only 6.95 mg / L. This shows that under high salt conditions, the H1 strain can effectively degrade the toxic pollutant aniline with a benzene ring functional group, and while degrading aniline, it can also undergo simultaneous nitrification and denitrification to remove NH4 +-N. This fully demonstrates that the H1 strain can tolerate environmental pressures such as high salt and toxic pollutants containing benzene ring functional groups, and exert stable denitrification performance.

[0084] (8) Denitrification performance of H1 strain under mixed organic pollutant conditions The Pseudomonas H1 cell suspension was transferred into the simulated wastewater medium 4 at a 20% (v / v) inoculation rate and cultured at 35°C and 150 r / min. NH4 was sampled and measured every 12 h. + -N, NO3 - -N, NO2 - -N、OD 600 , aniline and SMX residual concentrations. Figure 8 shown.

[0085] Denitrification performance of Pseudomonas sp. H1 under mixed organic pollutants Figure 8 As shown, the ammonia nitrogen removal rate was 93.10% within 12 hours, and the aniline removal rate was 46.41% within 48 hours. There was no accumulation of nitrate and nitrite nitrogen during the cultivation process. These results demonstrate that the H1 strain maintained stable and efficient denitrification performance even under conditions of mixed organic pollutants, effectively degrading aniline while removing ammonia nitrogen from the culture medium. This demonstrates that the H1 strain not only denitrifies under the stress of toxic pollutants containing benzene ring functional groups, but also effectively degrades toxic pollutants.

[0086] (9) Degradation ability and denitrification performance of H1 strain at different aniline concentrations The Pseudomonas H1 cell suspension was transferred into the simulated wastewater medium 5 at a 20% (v / v) inoculation rate and cultured at 35°C and 150 r / min. NH4 + -N, NO3 - -N, NO2 - -N、OD 600 and the residual concentration of aniline. Figure 9-10 shown.

[0087] Under high concentrations of aniline (200-1000 mg / L), the strain maintained a strong growth trend, growing rapidly within 12 hours with almost no lag phase. It also demonstrated efficient and stable denitrification capabilities within 12 hours. However, as aniline was degraded and removed, ammonia nitrogen accumulated to varying degrees in the later stages of the culture, reaching approximately 30-35 mg / L at all concentrations at 48 hours. Furthermore, no nitrite nitrogen was detected during the culture process, and nitrate nitrogen accumulation in the culture medium was minimal (<1 mg / L).

[0088] Furthermore, the H1 strain demonstrated excellent aniline degradation under high-concentration conditions, achieving removal rates of 35.11%, 42.27%, 45.21%, 35.21%, and 40.36% at concentrations of 200-1000 mg / L, respectively. Furthermore, the H1 strain exhibited even higher aniline degradation capacity at moderate aniline concentrations of 400 and 600 mg / L compared to other concentrations. This demonstrates that the H1 strain can consistently exert its denitrification and aniline degradation capabilities under high-concentration aniline stress conditions.

[0089] (10) Determination of the broad spectrum of substrates of H1 strain Five common aromatic compounds—aniline, catechol, protocatechuic acid, benzoic acid, and butyl benzoate—were selected as test substrates and added to simulated wastewater medium 6 to a final concentration of 200 mg / L. A 20% (v / v) inoculum of Pseudomonas H1 cell suspension was inoculated into the medium and incubated at 35°C and 150 rpm to determine whether strain H1 could utilize these substrates for growth. Samples were taken every 24 hours to determine the residual concentrations of aniline, catechol, and protocatechuic acid.

[0090] Table 1 shows the substrate utilization of strain H1. While all substrates were successfully utilized by H1, the utilization varied significantly. H1 grew rapidly in media containing catechol and protocatechuic acid as sole carbon sources. However, complex benzene side chains were less readily utilized, leading to slower growth.

[0091] Table 1: Substrate utilization of H1 strain Test substrate Strain growth activity aniline + Catechol ++ Protocatechuic acid ++ benzoic acid + Butyl benzoate + Note: (++) indicates vigorous growth; (+) indicates possible growth.

[0092] The utilization of aniline, catechol and protocatechuic acid by H1 strain is as follows Figure 11 As shown, strain H1 significantly outperformed aniline in degradation of catechol and protocatechuic acid. At 4 days, strain H1 achieved degradation rates of 10.91%, 27.16%, and 24.61% for aniline, catechol, and protocatechuic acid, respectively. This finding, combined with the strain's growth in various media, supports the conclusion that vigorous growth and high activity are associated with superior degradation of BTEX. Furthermore, catechol and protocatechuic acid contain phenolic hydroxyl groups, which have simpler side chain structures than other BTEX compounds, such as aniline, making them more readily bioavailable for microbial utilization.

[0093] Unless otherwise specified, the raw materials and equipment used in the present invention are commonly used in the art; the methods used in the present invention are conventional methods in the art unless otherwise specified.

[0094] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A stress-resistant heterotrophic nitrification-aerobic denitrification Pseudomonas, characterized by: The strain was named H1, its microbial classification was named Pseudomonas sihuiensis, its deposit number was CGMCC NO: 33481, its deposit date was January 20, 2025, and its deposit location was China General Microbiological Strain Collection Center; The 16S rRNA sequence of Pseudomonas sp. H1 is shown in SEQ ID NO:

1.

2. A microbial denitrification preparation, characterized in that: Containing Pseudomonas and / or its bacterial cultures; The Pseudomonas is named H1, its microbial classification is named Pseudomonas sihuiensis, the preservation number is CGMCCNO: 33481, the preservation date is January 20, 2025, and the preservation location is China General Microbiological Strain Collection Center; the 16S rRNA sequence of Pseudomonas H1 is shown in SEQ ID NO:

1.

3. Use of the stress-resistant heterotrophic nitrification-aerobic denitrifying Pseudomonas denitrificans according to claim 1 or the microbial denitrification preparation according to claim 2 in wastewater denitrification, characterized in that: The Pseudomonas and / or bacterial culture thereof denitrifies wastewater through a heterotrophic nitrification-aerobic denitrification pathway.

4. The use according to claim 3, characterized in that: The wastewater contains toxic pollutants and / or salts with benzene ring functional groups.

5. The use according to claim 4, characterized in that: The toxic pollutant containing a benzene ring functional group is one or more of sulfamethoxazole, aniline, catechol, protocatechuic acid, benzoic acid and butyl benzoate; and / or The concentration of toxic pollutants containing benzene ring functional groups in the wastewater is ≤ 1000 mg / L; and / or The salinity of the wastewater is ≤80g / L.

6. The use according to claim 3, characterized in that: The nitrogen-containing substances in the wastewater are one or more of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and organic nitrogen; and / or The nitrogen concentration in the wastewater is ≤1500 mg / L.

7. A method for denitrification of wastewater based on microorganisms, characterized in that: Adding Pseudomonas and / or its bacterial culture into wastewater to achieve denitrification through heterotrophic nitrification-aerobic denitrification pathway; The Pseudomonas is named H1, its microbial classification is named Pseudomonas sihuiensis, the preservation number is CGMCCNO: 33481, the preservation date is January 20, 2025, and the preservation location is China General Microbiological Strain Collection Center; the 16S rRNA sequence of Pseudomonas H1 is shown in SEQ ID NO:

1.

8. The wastewater denitrification method according to claim 7, characterized in that: The inoculation amount of Pseudomonas and / or its bacterial culture in the wastewater is 1-50% (v / v).

9. The wastewater denitrification method according to claim 7, characterized in that: The denitrification temperature is 15-45°C and the denitrification time is 6-24h.

10. The biological denitrification method for nitrogen-containing wastewater according to claim 7, characterized in that: The salinity of the wastewater is ≤80g / L; and / or The nitrogen concentration in the wastewater is ≤1500 mg / L; and / or The toxic pollutant containing a benzene ring functional group is one or more of sulfamethoxazole, aniline, catechol, protocatechuic acid, benzoic acid and butyl benzoate; and / or The concentration of toxic pollutants containing benzene ring functional groups in the wastewater is ≤1000 mg / L.

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