A salt-tolerant nitrate-reducing bacterium capable of degrading aniline and removing nitrogen and phosphorus and application thereof

Abstract

The application discloses a salt-tolerant aniline high-efficiency degradation and denitrification and phosphorus removal nitrate-reducing bacterium and application thereof, and belongs to the wastewater treatment and microbial technology field. Nitratireductor The strain is sp. AS9, is preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC No. 36404 and a preservation date of October 30, 2025. The strain has high-salt high-nitrogen and phosphorus high-concentration aniline tolerance, can take aniline as the only carbon and nitrogen source, can efficiently degrade aniline under low, medium and high-concentration aniline conditions, and can simultaneously realize denitrification and phosphorus removal. In particular, the aniline degradation efficiency and denitrification capacity of the strain are more prominent in a high-concentration aniline environment. In addition, the AS9 strain can simultaneously perform nitrification-denitrification denitrification and phosphorus removal reaction, and has great application potential and technical advantages in the treatment of wastewater containing high-salt high-nitrogen and phosphorus aniline compounds.

Description

Technical Field

[0001] This invention relates to the fields of wastewater treatment and microbial technology, specifically to a salt-tolerant nitrate-reducing bacterium that efficiently degrades aniline and synergistically removes nitrogen and phosphorus, and its applications. Background Technology

[0002] Aniline, an important chemical raw material and highly biotoxic pollutant, is widely found in industrial wastewater from dyeing, pharmaceuticals, pesticides, and rubber industries. Its stable nature and difficulty in natural degradation pose a serious threat to the ecological environment and human health. Biological methods are considered ideal for treating aniline-containing wastewater due to their low cost and environmental friendliness. Although there has been extensive research on aniline-degrading bacteria, such as *Pseudomonas*, *Rhodococcus*, *Daucus*, *Achromobacter*, and *Delft*, currently reported aniline-degrading bacteria still face significant technical bottlenecks when dealing with complex industrial wastewater, particularly in simultaneously meeting the demands of treating complex pollution such as high salt, high nitrogen and phosphorus, and high concentrations of aniline.

[0003] (1) Existing strains have single functions and cannot achieve synergistic removal of pollutants. Wastewater treatment often requires the removal of multiple pollutants, but existing technologies mostly use strains with single functions and do not have the ability to synergistically remove nitrogen and phosphorus by salt-tolerant aniline degradation. For example, the aniline-degrading bacteria Aristolochicus intermedius H52 and Tauella bacillus BAA19 disclosed in patents CN116355793B and CN116042450B, although they have aniline degradation ability and a certain denitrification ability, have not been reported to have phosphorus removal performance. And patent CN107723264B discloses a salt-tolerant nitrate-reducing bacterium XHNA1, whose function is limited to removing inorganic nitrogen from aquaculture water and does not involve aniline degradation and phosphorus removal.

[0004] (2) It is difficult to achieve both high-efficiency degradation and denitrification and phosphorus removal functions under high-salt conditions. High-salt conditions generally inhibit microbial activity. Although the above-mentioned aniline-degrading bacteria (such as H52 and BAA19) have a certain salt tolerance, the upper limit of salinity is 5%, and the aniline degradation capacity and denitrification efficiency decrease significantly when the salinity increases.

[0005] (3) Under the inhibition of high concentrations of aniline, the metabolic diversity of the strains is limited. Currently reported aniline-degrading bacteria require an external carbon source for co-metabolism when treating aniline wastewater, making it difficult for them to exert their degradation effect using aniline as the sole carbon source. Moreover, the addition of an external carbon source leads to a delay in aniline degradation, and excessive or incompletely utilized external carbon sources will remain in the treatment system. This not only increases treatment costs but may also cause the chemical oxygen demand (COD) of the effluent to increase instead of decrease, resulting in secondary pollution. In addition, the addition of carbon sources must be maintained at an appropriate concentration; otherwise, the biological system will be difficult to stabilize, which increases the operational difficulty of the system. For example, the Delftibacterium AD1 disclosed in patent CN110373352B can better exert its aniline degradation ability under the condition of an external carbon source.

[0006] In summary, current microbial treatment technologies face three core challenges when dealing with complex industrial wastewater containing high salt, high nitrogen and phosphorus, and high concentrations of aniline: single-function strains, low synergistic removal efficiency of pollutants under the inhibition of high salt and high toxicity, and complex process flows. Therefore, developing multifunctional strains capable of tolerating extreme high-salt environments, achieving efficient degradation using high-concentration aniline as the sole carbon source, and simultaneously completing deep denitrification and phosphorus removal is urgently needed and has significant technological value for revolutionizing biological treatment processes for such wastewater and achieving efficient, low-consumption, one-step purification. This can provide microbial agent resources for the green and efficient treatment of wastewater containing high salt, high concentrations of aniline compounds, and nitrogen and phosphorus pollutants, and has broad application value in water pollution control. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a salt-tolerant nitrate-reducing bacterium that efficiently degrades aniline and synergistically removes nitrogen and phosphorus, and its application.

[0008] The technical solution adopted in this invention is: According to a first aspect of the present invention, the present invention provides a salt-tolerant, highly efficient aniline-degrading, synergistic denitrification and phosphorus-removing strain, said strain being a nitrate-reducing bacterium. Nitratireductor sp.AS9 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36404 and deposit date of October 30, 2025.

[0009] Furthermore, the 16S rRNA sequence of the AS9 strain is shown in SEQ ID No. 1.

[0010] According to a second aspect of the present invention, the present invention provides a microbial agent, immobilized microbial preparation, or activated sludge enhancement agent comprising the aforementioned salt-tolerant aniline-efficient degradation and synergistic denitrification and phosphorus removal strains. These agents or preparations may be in the form of freeze-dried powder, bacterial liquid, immobilized granules, etc., thereby facilitating large-scale preparation, long-term storage, transportation, and addition and engineering application in actual aniline-containing wastewater treatment systems.

[0011] According to a third aspect of the present invention, the present invention also provides the application of the salt-tolerant aniline-efficient degradation and synergistic denitrification and phosphorus removal strains in the degradation of aniline in aniline-containing wastewater.

[0012] Furthermore, the strain of this invention can efficiently degrade aniline using aniline as the sole carbon and nitrogen source, thus enabling it to treat wastewater where aniline is the sole carbon and nitrogen source. Similarly, the aforementioned microbial agents, immobilized microbial preparations, or activated sludge enhancing agents are also suitable for wastewater where aniline is the sole carbon and nitrogen source. According to a specific embodiment of the present invention, the concentration of aniline in the aniline-containing wastewater is 0.2-20 g / L; the strain of the present invention still has aniline degradation activity at high aniline concentrations, therefore, it is suitable for the degradation of aniline in wastewater with an aniline concentration of 1-10 g / L, more preferably 6-10 g / L.

[0013] Furthermore, the strain of the present invention has salt tolerance characteristics, and the concentration of inorganic salts in the aniline-containing wastewater it can treat can be 5-80 g / L; preferably 10-80 g / L, more preferably 10-50 g / L, more preferably 15-25 g / L, for example 20 g / L.

[0014] More preferably, the salt-tolerant aniline-resistant, highly efficient degradative, synergistic denitrification and phosphorus removal strain provided by the present invention is suitable for degrading aniline in wastewater and simultaneously removing nitrogen and phosphorus from the wastewater. Preferably, the denitrification in the present invention refers to the removal of nitrogen-containing compounds after aniline degradation, as well as ammonia nitrogen, nitrate nitrogen, or nitrite nitrogen compounds originally present in the aniline wastewater. Preferably, the total concentration of ammonia nitrogen, nitrate nitrogen, or nitrite nitrogen compounds in the wastewater is ≤1000 mg / L. The phosphorus removal refers to the removal of phosphate, monohydrogen phosphate, or dihydrogen phosphate from the aniline-containing wastewater. Preferably, the phosphate concentration in the wastewater is ≤2000 mg / L, more preferably 200-1500 mg / L, and even more preferably 500-1000 mg / L.

[0015] Compared with the prior art, the beneficial effects of the present invention include: 1) Most existing aniline-degrading bacteria are only suitable for treating low-concentration aniline wastewater. When the aniline concentration is ≥4 g / L, the degradation performance of the strains is inhibited or basically lost, and they are difficult to survive in high-salt environments, lacking the ability to simultaneously remove nitrogen and phosphorus. The nitrate-reducing bacterium AS9 strain screened in this invention has tolerance and degradation ability for high-salt (20 g / L), high-nitrogen, high-phosphorus, and high-concentration aniline (it still has good aniline degradation ability at a aniline concentration of 10 g / L), and possesses excellent performance in simultaneous denitrification and phosphorus removal of high-concentration aniline. The nitrate-reducing bacterium AS9 of this invention can remove nitrogen-containing compounds after aniline degradation, as well as ammonia nitrogen, nitrate nitrogen, or nitrite nitrogen originally present in aniline wastewater, achieving wastewater denitrification. In addition, it can also remove phosphorus simultaneously, making it very suitable for treating high-salt, high-nitrogen, and high-phosphorus aniline wastewater currently present in industry.

[0016] 2) The nitrate-reducing bacterium AS9 of this invention can degrade aniline in wastewater using aniline as the sole carbon and nitrogen source, without relying on an external carbon source, thus avoiding secondary pollution caused by increased COD in the effluent due to carbon source residue. Simultaneously, this strain possesses salt tolerance and simultaneous nitrogen and phosphorus removal capabilities, making it highly suitable for the treatment of high-concentration aniline wastewater. Furthermore, the nitrate-reducing bacterium AS9 exhibits extremely strong degradation ability under aniline-only carbon and nitrogen source conditions, significantly outperforming other bacteria under the same conditions and also surpassing the degradation ability of previously reported aniline-degrading strains under external carbon source conditions.

[0017] 3) Existing nitrate-reducing bacteria are mainly used to convert nitrates into nitrogen-containing gases, but they cannot effectively degrade aniline or remove phosphorus in high-salt environments, thus limiting their application. The AS9 strain of nitrate-reducing bacteria discovered in this invention can use aniline as the sole carbon and nitrogen source to degrade aniline while simultaneously removing nitrogen and phosphorus from water. Furthermore, it can tolerate high salt and high concentrations of aniline, demonstrating significant application potential and advantages in the treatment of wastewater containing aniline compounds, especially in high-salt, high-nitrogen, and high-phosphorus environments. Attached Figure Description

[0018] Figure 1 The image shows the growth status of AS9 strain on solid culture medium plates.

[0019] Figure 2 A phylogenetic tree of AS9 strains constructed based on the 16S rRNA sequence.

[0020] Figure 3 Degradation performance of AS9 strain under different high-salt, low-concentration aniline conditions (A: aniline, B: NH4) + -N、C:NO3 - -N、D: NO2 - -N).

[0021] Figure 4 Degradation performance of AS9 strain under different high-salt concentrations of aniline (A: aniline, B: NH4) + -N、C:NO3 - -N、D: NO2 - -N).

[0022] Figure 5 Degradation performance of AS9 strain under different high-salt, high-concentration aniline conditions (A: aniline, B: NH4) + -N、C:NO3 - -N、D: NO2 - -N).

[0023] Figure 6The denitrification performance of AS9 strain under different nitrogen source conditions (A: ammonia nitrogen as the sole nitrogen source, B: nitrate nitrogen as the sole nitrogen source, C: nitrite nitrogen as the sole nitrogen source, D: ammonia nitrogen and nitrate nitrogen as a mixed nitrogen source).

[0024] Figure 7 The nitrogen and phosphorus removal performance of strain AS9 under ammonia nitrogen as the sole nitrogen source was investigated.

[0025] Figure 8 Comparison of aniline degradation performance between AS9 strain and aniline-degrading bacterium Pseudomonas H1 (A: Comparison of degradation capacity of AS9 strain and Pseudomonas H1 under the condition of aniline as the sole carbon and nitrogen source; B: Comparison of aniline degradation capacity of AS9 strain under the condition of aniline as the sole carbon and nitrogen source and the aniline degradation capacity of Pseudomonas H1 under the condition of additional carbon source).

[0026] Figure 9 The study investigated the aniline degradation capacity and denitrification and phosphorus removal performance of strain AS9 under aniline as the sole carbon and nitrogen source (A: aniline degradation and denitrification and phosphorus removal under 5 g / L aniline, B: aniline degradation and denitrification and phosphorus removal under 10 g / L aniline). Detailed Implementation

[0027] The following examples provide those skilled in the art with guidance on how to manufacture and evaluate the invention. These examples are merely illustrative of the present disclosure and do not limit its scope. While every effort has been made to ensure accuracy regarding numerical values ​​(e.g., quantities, temperatures, etc.), some errors and deviations should be considered. Unless otherwise stated, temperatures are in °C or at ambient temperature, and pressures are at or near atmospheric pressure.

[0028] I. Domestication, isolation, purification and identification of bacterial strains 1. Culture medium Enrichment and acclimatization medium: Aniline 200 mg / L, NaCl 20 g / L, KH₂PO₄ 1 g / L, K₂HPO₄ 1 g / L, salt solution 10 mL / L, pH adjusted to 7.0. Salt solution composition: MgSO₄ 0.5 g / L, MnSO₄ 0.17 g / L, H₃BO₃ 0.116 g / L, ZnSO₄·7H₂O 0.115 g / L, FeSO₄·7H₂O 0.3 g / L.

[0029] High-salt LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 20 g / L.

[0030] 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 autoclaving at 121 ℃ for 20 min.

[0031] 2. Domestication Activated sludge from the biochemical treatment system of the wastewater treatment plant of Zhejiang Haisheng Pharmaceutical Co., Ltd. was added to an enrichment and acclimatization medium and cultured with shaking in a constant temperature incubator at 35 ℃ and 150 r / min for one week to obtain a first-generation bacterial population. Then, it was added to fresh enrichment and acclimatization medium at a 20% (v / v) inoculation ratio and cultured for another week. This passage process was repeated multiple times at the same ratio to obtain a stable primary bacterial population.

[0032] 3. Purification and separation Part of the primary bacterial culture medium was prepared at a concentration gradient of 10. -3 10 -4 10 -5 and 10 -6 The culture was diluted 1-2 times, and 100 μL of each dilution was evenly spread onto enrichment and acclimatization solid medium and incubated upside down at 35 ℃ for 5 days. Independent colonies with obvious morphological differences were picked and streaked onto plates for isolation and repeated purification to obtain strain AS9.

[0033] 4. Identification The morphological and physiological-biochemical characteristics of the strain are as follows: Gram-negative bacteria, growing under facultative aerobic conditions. After culturing for 5 days on enriched and acclimatized solid medium, the colonies are round, with regular edges, a smooth, moist, and sticky surface, a slightly convex center, and a yellow, opaque color. Figure 1 The optimal temperature range for growth is 25-45 ℃ (optimal 35 ℃), and the optimal pH range is 5.0-9.0 (optimal 7.0). 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 high sequence similarity were selected, and a phylogenetic tree was constructed using MEGA 11.0 software via neighbor-joining (NJ) method. Figure 2 The results showed that strain AS9 was related to... Nitratireductor kimnyeongensis The gene sequence similarity of strain B390.1 is over 98%, hence the name. Nitratireductor sp. AS9 strain (nitrate-reducing bacteria). For ease of description, the following examples will refer to it as nitrate-reducing bacteria AS9 strain or AS9 strain. The 16S rRNA sequence of AS9 strain is shown in SEQ ID No. 1. Figure 2A phylogenetic tree of the AS9 strain, constructed based on its 16S rRNA sequence, is presented. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36404, and the deposit date is October 30, 2025.

[0034] II. The ability of AS9 strain to synergistically denitrify and remove phosphorus in aniline degradation under high-salt conditions 1. Preparation of culture medium High-salt, low-concentration aniline (sole carbon and nitrogen source) simulated wastewater culture medium 1: Aniline (200-1000 mg / L), NaCl 20 g / L, KH₂PO₄ 1 g / L, K₂HPO₄ 1 g / L, salt solution 10 mL / L, pH adjusted to 7.0. Salt solution composition: MgSO₄ 0.5 g / L, MnSO₄ 0.17 g / L, H₃BO₃ 0.116 g / L, ZnSO₄·7H₂O 0.115 g / L, FeSO₄·7H₂O 0.3 g / L.

[0035] High-salt, medium-concentration aniline (sole carbon and nitrogen source) simulated wastewater culture medium 2: aniline (1-5 g / L), NaCl 20 g / L, KH₂PO₄ 1 g / L, K₂HPO₄ 1 g / L, salt solution 10 mL / L, pH adjusted to 7.0. Salt solution composition: MgSO₄ 0.5 g / L, MnSO₄ 0.17 g / L, H₃BO₃ 0.116 g / L, ZnSO₄·7H₂O 0.115 g / L, FeSO₄·7H₂O 0.3 g / L.

[0036] High-salt, high-concentration aniline (sole carbon and nitrogen source) simulated wastewater culture medium 3: aniline (6-10 g / L), NaCl 20 g / L, KH₂PO₄ 1 g / L, K₂HPO₄ 1 g / L, salt solution 10 mL / L, pH adjusted to 7.0. Salt solution composition: MgSO₄ 0.5 g / L, MnSO₄ 0.17 g / L, H₃BO₃ 0.116 g / L, ZnSO₄·7H₂O 0.115 g / L, FeSO₄·7H₂O 0.3 g / L.

[0037] High-salt simulated wastewater culture medium 4: aniline 200 mg / L, C6H5Na3O7 4.74 g / L, NH4Cl 0.5 g / L, NaCl 20 g / L, K2HPO4·3H2O 1.5 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, pH of the culture medium adjusted to 7.0.

[0038] High-salt heterotrophic nitrification-aerobic denitrification medium 1: C6H5Na3O7 4.74 g / L, NH4Cl 0.5 g / L, NaCl 20 g / L, MgSO4·7H2O 0.1 g / L, KH2PO4 1.5 g / L, K2HPO4·3H2O 7.9 g / L, pH of the medium adjusted to 7.0.

[0039] High-salt heterotrophic nitrification-aerobic denitrification medium 2: C6H5Na3O7 4.74 g / L, NaNO3 0.5 g / L, NaCl 20 g / L, MgSO4·7H2O 0.1 g / L, KH2PO4 1.5 g / L, K2HPO4·3H2O 7.9 g / L, pH of the medium adjusted to 7.0.

[0040] High-salt heterotrophic nitrification-aerobic denitrification medium 3: C6H5Na3O7 4.74 g / L, NH4Cl 0.5 g / L, NaNO3 0.5 g / L, NaCl 20 g / L, MgSO4·7H2O 0.1 g / L, KH2PO4 1.5 g / L, K2HPO4·3H2O 7.9 g / L, pH of the medium adjusted to 7.0.

[0041] High-salt heterotrophic nitrification-aerobic denitrification medium 4: C6H5Na3O7 4.74 g / L, NaNO2 0.5 g / L, NaCl 20 g / L, MgSO4·7H2O 0.1 g / L, KH2PO4 1.5 g / L, K2HPO4·3H2O 7.9 g / L, pH of the medium adjusted to 7.0.

[0042] The sterilization method is high-temperature and high-pressure steam sterilization at 121 ℃ for 30 min.

[0043] 2. Preparation of Seed Liquid Strains AS9 were cultured at 35 °C for 5 days in enrichment and acclimatization solid medium (same as in Example 1), and single colonies were picked. Each single colony was inoculated into 10 mL of high-salt LB liquid medium and cultured at 150 r / min and 35 °C until the logarithmic growth phase (12 h). The bacterial culture was then transferred to 60 mL of high-salt LB liquid medium and cultured at 35 °C and 150 r / min until the logarithmic growth phase. The cells were collected by centrifugation, washed twice with physiological saline, and resuspended to prepare a cell suspension. The OD of the cell suspension was adjusted. 600 1.0 was used as seed solution.

[0044] 3. Investigation on the degradation ability of nitrate-reducing bacteria strain AS9 under high-salt, low-concentration aniline conditions. The seed culture was transferred to a high-salt, low-concentration aniline (sole carbon and nitrogen source) simulated wastewater culture medium 1 at an inoculation rate of 20% (v / v). The medium was incubated at 35 °C and 150 r / min, and samples were taken every 12 h to determine the levels of aniline and NH4+. + -N, NO3 - -N and NO2 - -N concentration, results as follows Figure 3 As shown, strain AS9 completely degraded 200 mg / L aniline within 36 h under high-salt conditions, achieving a degradation rate of 100%. Under other concentrations, at 72 h, the aniline degradation rates at 400, 600, 800, and 1000 mg / L were 93.91%, 55.09%, 37.11%, and 32.82%, respectively. Furthermore, strain AS9 did not accumulate nitrite or nitrate nitrogen during aniline degradation, and the residual ammonia nitrogen (<30 mg / L) was far lower than the theoretically expected ammonia nitrogen production after aniline degradation (approximately 15 mg / L of ammonia nitrogen is produced for every 100 mg / L of aniline degraded). This indicates that strain AS9 possesses aniline tolerance and degradation capabilities, enabling simultaneous aniline degradation and denitrification.

[0045] 4. Investigation on the degradation ability of nitrate-reducing bacteria strain AS9 under high salt concentration aniline conditions. The seed culture was transferred to high-salt, medium-concentration aniline (sole carbon and nitrogen source) simulated wastewater medium 2 at an inoculation rate of 20% (v / v), and cultured at 35 ℃ and 150 r / min. Samples were taken every 24 h to determine aniline and NH4+. + -N, NO3 - -N and NO2 - -N concentration, results as follows Figure 4 As shown in the figure, the activity of strain AS9 was not inhibited in simulated wastewater with medium concentrations of aniline. Within 5 days, the aniline degradation rates for simulated wastewater with initial aniline concentrations of 1, 2, 3, 4, and 5 g / L were 51.54%, 34.99%, 28.49%, 27.29%, and 26.96%, respectively. With increasing aniline concentration, the ammonia nitrogen accumulation decreased, indicating that strain AS9 stably exerted its synergistic denitrification ability for aniline degradation. Furthermore, strain AS9 did not accumulate nitrite or nitrate nitrogen during the degradation of medium-concentration aniline, indicating that strain AS9 possesses tolerance and degradation capabilities for medium-concentration aniline, and exhibits superior denitrification ability under medium-concentration aniline conditions.

[0046] 5. Investigation on the degradation ability of nitrate-reducing bacteria strain AS9 under high-salt and high-concentration aniline conditions. The seed culture was transferred to high-salt, high-concentration aniline (sole carbon and nitrogen source) simulated wastewater medium 3 at an inoculation rate of 20% (v / v), and cultured at 35 ℃ and 150 r / min. Samples were taken every 24 h to determine aniline and NH4+.+ -N, NO3 - -N and NO2 - -N concentration, results as follows Figure 5 As shown, the AS9 strain's activity was not inhibited under high-salt, high-concentration aniline conditions, exhibiting stable aniline degradation performance. Within 132 h, the aniline degradation rates of simulated wastewater with initial aniline concentrations of 6, 7, 8, 9, and 10 g / L were 33.58%, 34.26%, 35.31%, 32.89%, and 29.18%, respectively. The corresponding aniline removal amounts were 2-2.5 g / L, with an average degradation rate of 15-19 mg / L / h, indicating that the AS9 strain possesses tolerance and degradation capabilities for high-concentration aniline. Furthermore, no nitrite or nitrate nitrogen accumulated during the degradation of high-concentration aniline by the AS9 strain, and NH4+ was not generated under high-concentration aniline cultivation conditions. + The accumulation of -N was relatively low, and NH4+ was present at 132 h. + The -N accumulation was only 2-3 mg / L, far lower than the theoretical amount of ammonia nitrogen produced after aniline degradation, indicating that the AS9 strain has excellent performance in simultaneous denitrification of high-concentration aniline.

[0047] 6. Investigation on the denitrification characteristics of nitrate-reducing bacterium AS9 strain to single and mixed nitrogen sources (ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, ammonia nitrogen + nitrate nitrogen). The seed culture was transferred to high-salt heterotrophic nitrification-aerobic denitrification media 1, 2, 3, and 4 at an inoculation rate of 20% (v / v), and cultured at 35 ℃ and 150 r / min. NH4 was measured every 12 h. + -N, NO3 - -N, NO2 - -N concentration and OD 600 The result is as follows Figure 6As shown, strain AS9 grew well under conditions with ammonia nitrogen, nitrate nitrogen, nitrite nitrogen as the sole nitrogen source, and under mixed nitrogen source conditions. Specifically, when ammonia nitrogen (initial concentration 134.93 mg / L) was used as the sole nitrogen source, strain AS9 achieved a 98.04% removal rate after 12 h of cultivation, with no accumulation of nitrate nitrogen or nitrite nitrogen throughout the entire cultivation process. When nitrate nitrogen (initial concentration 85.05 mg / L) was used as the sole nitrogen source, strain AS9 achieved a 97.86% removal rate after 36 h of cultivation, with no accumulation of nitrite nitrogen throughout the entire cultivation process. When nitrite nitrogen (initial concentration 111.15 mg / L) was used as the sole nitrogen source, strain AS9 achieved a 98.29% removal rate after 48 h of cultivation, with no accumulation of ammonia nitrogen or nitrate nitrogen throughout the entire process. When ammonia nitrogen (initial concentration 128.58 mg / L) and nitrate nitrogen (initial concentration 83.76 mg / L) were used as mixed nitrogen sources, the AS9 strain achieved removal rates of 90.40% and 100% for ammonia nitrogen and nitrate nitrogen respectively after 12 h of culture, indicating that the AS9 strain has excellent simultaneous nitrification-denitrification nitrogen removal performance under high salt and high nitrogen stress conditions.

[0048] 7. Investigation on the nitrogen and phosphorus removal performance of nitrate-reducing bacteria strain AS9 under conditions where ammonia nitrogen is the sole nitrogen source. The seed culture was transferred to high-salt heterotrophic nitrification-aerobic denitrification medium 1 at an inoculum of 20% (v / v) and cultured at 35℃ and 150 r / min. PO4 was measured every 12 h. 3- -P, NH4 + -N, NO3 - -N, NO2 - -N concentration and OD 600 The result is as follows Figure 7 As shown. AS9 strain's response to NH4+ within 12 hours. + -N and PO4 3 The average removal rates of -P were 9.92 mg / L / h and 84.13 mg / L / h, respectively. At 24 h, both nitrogen and phosphorus removal efficiencies were close to 100%, and NH4+ removal efficiency was also low. + -N and PO4 3 The average removal rates of -P were 6.11 mg / L / h and 52.94 mg / L / h, respectively. Furthermore, strain AS9 did not accumulate nitrite or nitrate nitrogen when using ammonia nitrogen as the sole nitrogen source for denitrification and phosphorus removal, indicating that strain AS9 possesses excellent salt tolerance for simultaneous denitrification and phosphorus removal.

[0049] 8. Comparison of the aniline degradation capacity of strain AS9 under aniline as the sole carbon and nitrogen source with that of aniline-degrading bacterium Pseudomonas H1 (CGMCC No. 33481) under both aniline as the sole carbon and nitrogen source and with an added carbon source. The aniline-degrading bacterium Pseudomonas H1 (CGMCC No. 33481) has been proven to have salt-tolerant aniline degradation ability. Therefore, in Example 8, the aniline degradation performance of strain AS9 and strain H1 was compared to verify the aniline degradation advantage of strain AS9 in this technology.

[0050] The AS9 strain seed culture was transferred at an inoculum rate of 20% (v / v) to a high-salt, low-concentration aniline (200 mg / L) simulated wastewater medium 1 (aniline as the sole carbon and nitrogen source). The H1 strain seed culture was transferred to both high-salt, low-concentration aniline (200 mg / L) simulated wastewater medium 1 (aniline as the sole carbon and nitrogen source) and high-salt simulated wastewater medium 4 (external carbon source), respectively. The cultures were incubated at 35 ℃ and 150 r / min, and aniline concentration was measured every 12 h. Figure 8 As shown in Figure A, under the condition that aniline is the only carbon and nitrogen source, the aniline degradation rate of strain AS9 is close to 100% after 36 h, while the aniline degradation rate of strain Pseudomonas H1 is only 7.99% after 36 h. This indicates that strain AS9 has a better salt-tolerant aniline degradation ability than the aniline-degrading bacterium Pseudomonas H1.

[0051] A comparison of the aniline degradation capacity of strain AS9 with aniline as the sole carbon and nitrogen source and that of aniline-degrading bacteria Pseudomonas H1 with an additional carbon source is provided. Figure 8 As shown in B, under the condition of an external carbon source, the aniline degradation rate of Pseudomonas H1 strain at 36 h was only 26.59%, which was significantly lower than that of AS9 strain. This indicates that AS9 strain has better aniline degradation performance with aniline as the sole carbon and nitrogen source compared to aniline-degrading Pseudomonas H1 strain.

[0052] 9. Investigation on the aniline degradation capacity and denitrification and phosphorus removal performance of strain AS9 under conditions where aniline is the sole carbon and nitrogen source. The AS9 strain seed culture was transferred at an inoculum rate of 20% (v / v) to simulated wastewater media with high salt and high concentrations of aniline (aniline was the sole carbon and nitrogen source, with aniline concentrations of 5 g / L and 10 g / L, respectively). The media were incubated at 35 ℃ and 150 r / min, and samples were taken every 12 h to measure aniline and PO4. 3- -P and NH4 + -N concentration.

[0053] like Figure 9 As shown in Figure A, under a high concentration of aniline of 5 g / L, the average degradation rate of aniline by strain AS9 was 11.93 mg / L / h. A small amount of ammonia nitrogen accumulated during the aniline degradation process (around 1 mg / L), but this was far lower than the theoretically expected amount of ammonia nitrogen produced by aniline degradation. However, under a high concentration of aniline of 10 g / L (…), Figure 9In the study (B), strain AS9 exhibited an average aniline degradation rate of 21.83 mg / L / h, with no ammonia nitrogen accumulation during the degradation process, indicating that strain AS9 demonstrated superior aniline degradation and denitrification capabilities under high aniline concentrations. Furthermore, under high aniline concentrations, strain AS9 could remove approximately 420 mg / L of PO4 within 12 hours. 3- -P removal rate close to 100% indicates that AS9 strain has excellent tolerance and degradation ability to high concentrations of aniline, as well as excellent simultaneous nitrogen and phosphorus removal performance.

[0054] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A salt-tolerant strain of aniline that efficiently degrades and synergistically removes nitrogen and phosphorus, characterized in that, The strain is a nitrate-reducing bacterium. Nitratireductor sp. AS9 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36404 and deposit date of October 30, 2025.

2. The salt-tolerant aniline-resistant, highly efficient degradative, synergistic denitrification and phosphorus removal strain according to claim 1, characterized in that, The nitrate-reducing bacteria Nitratireductor The 16S rRNA sequence of sp. AS9 is shown in SEQ ID No.

1.

3. A microbial agent, immobilized microbial preparation, or activated sludge enhancement agent comprising the salt-tolerant aniline-efficient degradation and synergistic denitrification and phosphorus removal strains as described in claim 1.

4. The application of the salt-tolerant aniline-efficient degradation and synergistic denitrification and phosphorus removal strain according to claim 1, characterized in that, Used to degrade aniline in aniline-containing wastewater.

5. The application according to claim 4, characterized in that, The application is as follows: Salt-tolerant aniline-efficient degradation and synergistic denitrification and phosphorus removal strains can use aniline as the sole carbon and nitrogen source in wastewater for aniline degradation.

6. The application according to claim 4 or 5, characterized in that, The salt-tolerant aniline-efficient degradation and synergistic denitrification and phosphorus removal strains degrade aniline in wastewater while simultaneously removing nitrogen and phosphorus from the wastewater.

7. The application according to claim 4 or 5, characterized in that, The concentration of aniline in the aniline-containing wastewater is 0.2-20 g / L; preferably 1-10 g / L, more preferably 6-10 g / L.

8. The application according to claim 4 or 5, characterized in that, The concentration of inorganic salts in the aniline-containing wastewater is 5-80 g / L; preferably 10-50 g / L, and more preferably 15-25 g / L.

9. The application according to claim 6, characterized in that, The denitrification includes removing nitrogen-containing compounds produced by the degradation of aniline, as well as ammonia nitrogen, nitrate nitrogen, or nitrite nitrogen originally present in the wastewater.

10. The application according to claim 6, characterized in that, The phosphorus removal process involves removing phosphate, monohydrogen phosphate, or dihydrogen phosphate from aniline-containing wastewater. The phosphate concentration in the wastewater is 100-2000 mg / L, preferably 200-1500 mg / L, and more preferably 500-1000 mg / L.

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