A sulfonamide antibiotic-resistant denitrifying phosphate-accumulating bacteria and its application in denitrification and phosphorus removal

Abstract

This invention discloses a denitrifying polyphosphate-accumulating bacterium resistant to sulfonamide antibiotics and its application in nitrogen and phosphorus removal. The denitrifying polyphosphate-accumulating bacterium is *Acinetobacter junii* BWFJF1, with accession number GDMCC NO: 63199. This strain exhibits excellent resistance to sulfonamide antibiotics, enabling stable growth in wastewater containing high concentrations of sulfonamides while maintaining efficient nitrogen and phosphorus removal. This strain effectively overcomes the problem of low nitrogen and phosphorus removal efficiency of traditional microorganisms under sulfonamide antibiotic stress, providing a new microbial option for treating sulfonamide-contaminated wastewater.

Description

Technical Field

[0001] This invention relates to the technical field of nitrogen and phosphorus removal, specifically to a denitrifying polyphosphate-accumulating bacterium resistant to sulfonamide antibiotics and its application in nitrogen and phosphorus removal. Background Technology

[0002] Sulfonamide antibiotics are widely used in the medical, agricultural, and livestock industries due to their broad spectrum, stability, and low cost. However, most sulfonamide antibiotics are not effectively absorbed and utilized, ultimately entering water bodies through discharge, leading to serious antibiotic pollution problems.

[0003] In wastewater treatment, denitrifying polyphosphate-accumulating bacteria (DPCA) are functional microorganisms capable of denitrifying using nitrate nitrogen as an electron acceptor and over-absorbing phosphate under anoxic conditions. These microorganisms have significant application value in the removal of pollutants from water bodies. However, traditional DPCA bacteria are often inhibited by sulfonamide antibiotics when treating wastewater, leading to a significant decrease in their nitrogen and phosphorus removal efficiency. Especially in water environments with high concentrations of sulfamethoxazole contamination, the growth and metabolic activities of traditional microbial communities are significantly inhibited, making it difficult to maintain normal nitrogen and phosphorus removal functions, resulting in a marked decrease in phosphorus removal efficiency.

[0004] Therefore, finding a denitrifying polyphosphate-accumulating bacterium that can maintain high denitrification efficiency and significantly improve the removal of total phosphorus in wastewater contaminated with high concentrations of sulfamethoxazole is of great technical value for the treatment of antibiotic-contaminated wastewater, in order to overcome the problem of poor denitrification and phosphorus removal efficiency of microorganisms in high-concentration antibiotic wastewater in existing technologies. Summary of the Invention

[0005] To overcome the aforementioned defects and deficiencies in the prior art, the present invention provides a denitrifying polyphosphate-accumulating bacterium resistant to sulfonamide antibiotics and its application in denitrification and phosphorus removal.

[0006] The first objective of this invention is to provide the application of Acinetobacter junii BWFJF1 in the removal of pollutants from water bodies.

[0007] A second objective of this invention is to provide the use of Acinetobacter junii BWFJF1 in the preparation of formulations for the removal of pollutants from water bodies.

[0008] A third objective of this invention is to provide a method for removing pollutants from water.

[0009] This invention claims protection for the following:

[0010] Application of Acinetobacter junii BWFJF1 in the removal of pollutants from water bodies. Acinetobacter junii BWFJF1 was deposited at Guangdong Provincial Microbial Culture Collection Center on February 25, 2023, with accession number GDMCC NO: 63199.

[0011] The water body contains sulfonamide antibiotics;

[0012] The pollutants include nitrogen pollutants and / or phosphorus pollutants.

[0013] Preferably, the nitrogen pollutant is a nitrate nitrogen pollutant.

[0014] Preferably, the phosphorus pollutant is a total phosphorus pollutant.

[0015] Preferably, the sulfonamide antibiotic is sulfamethoxazole.

[0016] Preferably, the removal of nitrogen and phosphorus pollutants from the water is carried out simultaneously.

[0017] The application of Acinetobacter junii BWFJF1 in the preparation of agents for removing pollutants from water bodies, wherein Acinetobacter junii BWFJF1 was deposited at Guangdong Provincial Microbial Culture Collection Center on February 25, 2023, with accession number GDMCC NO: 63199;

[0018] The water body contains sulfonamide antibiotics;

[0019] The pollutants include nitrogen pollutants and / or phosphorus pollutants.

[0020] Preferably, the nitrogen pollutant is a nitrate nitrogen pollutant.

[0021] Preferably, the phosphorus pollutant is a total phosphorus pollutant.

[0022] Preferably, the sulfonamide antibiotic is sulfamethoxazole.

[0023] Preferably, the removal of nitrogen and phosphorus pollutants from the water body is carried out simultaneously.

[0024] A method for removing pollutants from water bodies involves adding Acinetobacter junii BWFJF1 to the water body. Acinetobacter junii BWFJF1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on February 25, 2023, with accession number GDMCC NO: 63199.

[0025] The water body contains sulfonamide antibiotics;

[0026] The pollutants include nitrogen pollutants and / or phosphorus pollutants.

[0027] Preferably, the nitrogen pollutant is a nitrate nitrogen pollutant.

[0028] Preferably, the phosphorus pollutant is a total phosphorus pollutant.

[0029] Preferably, the sulfonamide antibiotic is sulfamethoxazole.

[0030] Preferably, the inoculum size of Acinetobacter junii BWFJF1 is 1-3%.

[0031] Preferably, the removal of nitrogen and phosphorus pollutants from the water body is carried out simultaneously.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] This invention discloses a denitrifying polyphosphate-accumulating bacterium resistant to sulfonamide antibiotics and its application in simultaneous nitrogen and phosphorus removal. The denitrifying polyphosphate-accumulating bacterium is *Acinetobacter junii* BWFJF1, with accession number GDMCC NO: 63199, located at 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou. This strain exhibits excellent resistance to sulfonamide antibiotics, able to grow stably in wastewater containing high concentrations of sulfonamides while maintaining efficient nitrogen and phosphorus removal. This strain effectively overcomes the problem of low nitrogen and phosphorus removal efficiency of traditional microorganisms under sulfonamide antibiotic stress, providing a new microbial option for treating sulfonamide-contaminated wastewater. Attached Figure Description

[0034] Figure 1 The growth curves of Acinetobacter junii BWFJF1 under different concentrations of SMX are shown.

[0035] Figure 2 The denitrification and phosphorus removal efficiency of Acinetobacter junii BWFJF1 under 0 mg / L SMX conditions was evaluated.

[0036] Figure 3 The denitrification and phosphorus removal efficiency of Acinetobacter junii BWFJF1 under 0.5 mg / L SMX conditions was evaluated.

[0037] Figure 4The denitrification and phosphorus removal efficiency of Acinetobacter junii BWFJF1 under 1 mg / L SMX conditions was evaluated.

[0038] Figure 5 The denitrification and phosphorus removal efficiency of Acinetobacter junii BWFJF1 under 5 mg / L SMX conditions was evaluated.

[0039] Figure 6 The denitrification and phosphorus removal efficiency of Acinetobacter junii BWFJF1 under 10 mg / L SMX conditions was evaluated.

[0040] Figure 7 This is a comparison of the denitrification effects of Acinetobacter junii BWFJF1 under different concentrations of SMX.

[0041] Figure 8 This is a comparison of the phosphorus removal efficiency of Acinetobacter junii BWFJF1 under different concentrations of SMX. Detailed Implementation

[0042] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0043] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0044] The Acinetobacter junii BWFJF1 used in the examples was deposited at the Guangdong Provincial Center for Microbial Culture Collection on February 25, 2023, with accession number GDMCC NO: 63199, located at 5th Floor, Building 59, No. 100 Courtyard, Xianlie Middle Road, Guangzhou. It is the Acinetobacter junii BWFJF1 in Chinese Invention Patent CN116555117A.

[0045] Example 1: Growth tolerance of Acinetobacter junii BWFJF1 under different concentrations of sulfamethoxazole (SMX)

[0046] I. Experimental Methods

[0047] 1. Preparation of culture medium

[0048] The culture medium formula is: CH3COONa 2g / L, KNO3 0.35g / L, K2HPO4 0.1g / L, MgSO4·7H2O 0.04g / L, KCl 0.018g / L, and trace elements 2mL / L;

[0049] The trace element formula is as follows: Fe2(SO4)3 1.67g / L, CuSO4·5H2O 0.027g / L, KI 0.162g / L, ZnSO4 0.108g / L, CoCl2·6H2O 0.15g / L, and disodium EDTA 10g / L.

[0050] SMX was added after the culture medium was sterilized, so that the concentrations of SMX in the culture medium were 0, 0.5, 1, 5 and 10 mg / L, respectively.

[0051] 2. Inoculation and Culture

[0052] Activated Acinetobacter junii BWFJF1 (accession number GDMCC NO: 63199, OD) was used. 600 =1.5) Inoculate at a rate of 1% (v / v) into culture media containing different concentrations of SMX, with three replicates for each concentration. Incubate in a constant temperature shaker at 30°C and 110 rpm for 48 h.

[0053] The absorbance (OD) was measured at a wavelength of 600 nm using a UV spectrophotometer. 600 The growth of the strain was monitored every 6 hours, and growth curves of Acinetobacter junii BWFJF1 at different concentrations of SMX were plotted.

[0054] II. Experimental Results

[0055] The results are as follows Figure 1 As shown, all concentrations of the strains exhibited an upward trend in the early stages of growth. However, with increasing culture time, the nutrients in the culture medium were gradually consumed, leading to limited bacterial growth. Under the control group (0 mg / L SMX), the strain showed the most vigorous growth in the early stages, with OD values ​​reaching [value missing] after 24 hours of culture. 600 It reaches its peak, then declines slightly, but maintains a high level of biomass overall.

[0056] As the SMX concentration increased, at concentrations of 0.5 mg / L and 1 mg / L, the growth status of the strains was similar to or even slightly better than that of the control group at certain stages, indicating that low concentrations of SMX did not inhibit the growth of the strains, but may have had a slight promoting effect.

[0057] Under high concentrations of SMX (5 mg / L and 10 mg / L), the growth of the strain was slightly inhibited but remained stable overall. This indicates that Acinetobacter junii BWFJF1 has significant SMX tolerance and can still grow stably even under high SMX concentrations.

[0058] Example 2: Denitrification and phosphorus removal characteristics of Acinetobacter jumbo at 0 mg / L SMX conditions

[0059] I. Experimental Methods

[0060] The culture medium was prepared according to Example 1. After sterilization, SMX was not added to the culture medium, and three parallel samples were set up.

[0061] Acinetobacter junii BWFJF1 (GDMCC NO: 63199) was inoculated into the culture medium at a rate of 1% (v / v) and cultured at 30℃ and 110 rpm for 48 h. Samples were taken every 6 h, and the bacterial suspension was centrifuged at 10000 rpm for 10 min at 4℃ to test the concentrations of nitrate nitrogen, nitrite nitrogen and total phosphorus in the supernatant.

[0062] II. Experimental Results

[0063] The results are as follows Figure 2 As shown, under SMX-free conditions, the nitrate nitrogen concentration of Acinetobacter junii BWFJF1 decreased from an initial concentration of approximately 48.14 mg / L to approximately 3.78 mg / L in 24 h and further to approximately 1.87 mg / L in 48 h, achieving a removal rate of approximately 96.12%. This indicates that Acinetobacter junii BWFJF1 has a highly efficient nitrate nitrogen removal capacity under SMX-free conditions. Nitrite nitrogen accumulated to some extent but remained at a low level.

[0064] The total phosphorus concentration decreased from an initial concentration of approximately 25.64 mg / L to approximately 10.77 mg / L over 24 hours, with a removal rate of approximately 58.00%, indicating that Acinetobacter junii BWFJF1 has a good ability to simultaneously remove nitrogen and phosphorus.

[0065] Example 3: Denitrification and phosphorus removal characteristics of Acinetobacter jumbo under 0.5 mg / L SMX stress.

[0066] I. Experimental Methods

[0067] The culture medium was prepared according to Example 1. After sterilization, SMX was added to make the concentration of SMX in the culture medium 0.5 mg / L, and three parallel samples were set up.

[0068] Acinetobacter junii BWFJF1 (GDMCC NO: 63199) was inoculated into the culture medium at a rate of 1% (v / v) and cultured at 30℃ and 110 rpm for 48 h. Samples were taken every 6 h, and the bacterial suspension was centrifuged at 10000 rpm for 10 min at 4℃ to test the concentrations of nitrate nitrogen, nitrite nitrogen and total phosphorus in the supernatant.

[0069] II. Experimental Results

[0070] The results are as follows Figure 3 As shown, under 0.5 mg / L SMX conditions, the nitrate nitrogen concentration of Acinetobacter junii BWFJF1 decreased from an initial concentration of approximately 48.73 mg / L to approximately 4.72 mg / L after 24 hours and further decreased to approximately 2.64 mg / L after 48 hours, achieving a removal rate of approximately 94.58%, which was only about 1.54 percentage points different from the blank control group. Nitrite nitrogen accumulated to some extent but remained at a low level.

[0071] The total phosphorus concentration decreased from an initial concentration of approximately 26.67 mg / L to approximately 7.31 mg / L within 24 hours, with a significant increase in removal rate to approximately 72.59%. This indicates that under the condition of 0.5 mg / L SMX, the strain can effectively perform simultaneous nitrogen and phosphorus removal, and significantly improve phosphorus removal capacity while maintaining a basically unchanged denitrification capacity.

[0072] Example 4: Denitrification and phosphorus removal characteristics of Acinetobacter jumbo under 1 mg / L SMX stress.

[0073] I. Experimental Methods

[0074] The culture medium was prepared according to Example 1. After sterilization, SMX was added to make the concentration of SMX in the culture medium 1 mg / L, and three parallel samples were set up.

[0075] Acinetobacter junii BWFJF1 (GDMCC NO: 63199) was inoculated into the culture medium at a rate of 1% (v / v) and cultured at 30℃ and 110 rpm for 48 h. Samples were taken every 6 h, and the bacterial suspension was centrifuged at 10000 rpm for 10 min at 4℃ to test the concentrations of nitrate nitrogen, nitrite nitrogen and total phosphorus in the supernatant.

[0076] II. Experimental Results

[0077] The results are as follows Figure 4As shown, under 1 mg / L SMX conditions, the nitrate nitrogen concentration of Acinetobacter junii BWFJF1 decreased from approximately 49.51 mg / L initially to approximately 4.96 mg / L after 24 hours, and further decreased to approximately 2.33 mg / L after 48 hours, achieving a removal rate of approximately 95.29%. This indicates that Acinetobacter junii BWFJF1 can still efficiently remove nitrate nitrogen under relatively high SMX concentrations. Nitrite nitrogen accumulated to some extent but remained at a low level.

[0078] The total phosphorus concentration decreased from approximately 25.68 mg / L to approximately 4.97 mg / L in 24 hours, with a removal rate of approximately 80.65%, which was significantly higher than that of the blank control group.

[0079] Example 5: Denitrification and phosphorus removal characteristics of Acinetobacter jumbo under 5 mg / L SMX stress.

[0080] I. Experimental Methods

[0081] The culture medium was prepared according to Example 1. After sterilization, SMX was added to make the concentration of SMX in the culture medium 5 mg / L, and three parallel samples were set up.

[0082] Acinetobacter junii BWFJF1 (GDMCC NO: 63199) was inoculated into the culture medium at a rate of 1% (v / v) and cultured at 30℃ and 110 rpm for 48 h. Samples were taken every 6 h, and the bacterial suspension was centrifuged at 10000 rpm for 10 min at 4℃ to test the concentrations of nitrate nitrogen, nitrite nitrogen and total phosphorus in the supernatant.

[0083] II. Experimental Results

[0084] The results are as follows Figure 5 As shown, under 5 mg / L SMX conditions, the nitrate nitrogen concentration of Acinetobacter junii BWFJF1 decreased from an initial concentration of approximately 48.93 mg / L to approximately 5.53 mg / L after 24 hours and further to approximately 5.03 mg / L after 48 hours, with a removal rate of approximately 89.72%, slightly lower than the blank control group. Nitrite nitrogen accumulated to some extent and remained at a low level.

[0085] The total phosphorus concentration decreased from an initial concentration of approximately 26.06 mg / L to approximately 4.10 mg / L within 24 hours, with a removal rate of approximately 84.27%, demonstrating good phosphorus removal efficiency. Even under high SMX concentrations, the strain can still effectively perform simultaneous nitrogen and phosphorus removal, maintaining highly efficient denitrification and nitrogen removal functions, while significantly improving phosphorus removal efficiency.

[0086] Example 6: Denitrification and phosphorus removal characteristics of Acinetobacter jumbo under 10 mg / L SMX stress.

[0087] I. Experimental Methods

[0088] The culture medium was prepared according to Example 1. After sterilization, SMX was added to make the concentration of SMX in the culture medium 10 mg / L, and three parallel samples were set up.

[0089] Acinetobacter junii BWFJF1 (GDMCC NO: 63199) was inoculated into the culture medium at a rate of 1% (v / v) and cultured at 30℃ and 110 rpm for 48 h. Samples were taken every 6 h, and the bacterial suspension was centrifuged at 10000 rpm for 10 min at 4℃ to test the concentrations of nitrate nitrogen, nitrite nitrogen and total phosphorus in the supernatant.

[0090] II. Experimental Results

[0091] The results are as follows Figure 6 As shown, under 10 mg / L SMX conditions, the removal of nitrate nitrogen by Acinetobacter junii BWFJF1 was slightly inhibited, with the concentration decreasing from an initial concentration of approximately 48.34 mg / L to approximately 9.81 mg / L after 30 h, representing a removal rate of approximately 79.71%. Nitrite nitrogen accumulated to some extent and remained at a low level.

[0092] The total phosphorus concentration decreased from approximately 26.97 mg / L to approximately 3.41 mg / L within 24 hours, with a removal rate of approximately 87.36%. The removal rate reached approximately 88.32% within 30 hours, representing an increase of approximately 29.36 percentage points compared to the control group. This indicates that *Acinetobacter junii* BWFJF1 maintains highly efficient nitrogen and phosphorus removal performance even under high SMX concentration conditions, while significantly promoting phosphorus removal.

[0093] The comparison of nitrogen and phosphorus removal efficiency of Acinetobacter junii BWFJF1 under different concentrations of SMX is shown in the figure below. Figure 7 and Figure 8As shown in the figure, Acinetobacter junii BWFJF1 exhibits significant resistance to sulfonamide antibiotics. Even under high concentrations of SMX (10 mg / L), it maintains good growth, metabolism, and denitrification and phosphorus removal functions. Furthermore, under high concentrations, Acinetobacter junii BWFJF1 significantly promotes phosphorus removal efficiency (total phosphorus removal rate increases by approximately 30% within 24 hours), indicating that this strain possesses strong functional stability and environmental adaptability under sulfonamide antibiotic stress.

[0094] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of Acinetobacter junii BWFJF1 for removing pollutants from water bodies, characterized in that, The Acinetobacter junii BWFJF1 strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on February 25, 2023, with accession number GDMCC NO: 63199. The water body contains sulfonamide antibiotics, specifically sulfamethoxazole. The pollutants include nitrogen pollutants and / or phosphorus pollutants.

2. Use according to claim 1, characterized in that, The nitrogen pollutant is a nitrate nitrogen pollutant.

3. Use of Acinetobacter junii BWFJF1 for the preparation of a preparation for the removal of pollutants from water bodies, characterized in that, The Acinetobacter junii BWFJF1 strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on February 25, 2023, with accession number GDMCC NO: 63199. The water body contains sulfonamide antibiotics, specifically sulfamethoxazole. The pollutants include nitrogen pollutants and / or phosphorus pollutants.

4. Use according to claim 3, characterized in that, The nitrogen pollutant is a nitrate nitrogen pollutant.

5. A method of removing a contaminant from a body of water, characterized by, Acinetobacter junii BWFJF1 was added to the water. Acinetobacter junii BWFJF1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on February 25, 2023, with accession number GDMCC NO: 63199. The water body contains sulfonamide antibiotics, specifically sulfamethoxazole. The pollutants include nitrogen pollutants and / or phosphorus pollutants.

6. The method of claim 5, wherein, The nitrogen pollutant is a nitrate nitrogen pollutant.

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