Sulfamethoxazole-nitrate dual-effect degrading bacterial strain and application thereof

Abstract

The present application discloses a sulfamethoxazole-nitrate nitrogen double-effect degradation strain, which is Burkholderia Stardust46, preserved in China General Microbiological Culture Collection Center on July 22, 2025, named as Burkholderia sp. Stardust46, and the preservation number is CGMCC No: 35337. The present application also discloses the application of the sulfamethoxazole-nitrate nitrogen double-effect degradation strain in degrading sulfamethoxazole and nitrate nitrogen in wastewater. The strain of the present application has strong tolerance to SMX and nitrate nitrogen, can grow efficiently under the stress conditions of SMX with a concentration of 40 mg / L and nitrate nitrogen with a concentration of 200 mg / L, and maintain high efficient aerobic denitrification, and has good tolerance to high-concentration antibiotic impact.

Description

Technical Field

[0001] This invention belongs to the field of microbial strain technology, specifically relating to sulfamethoxazole-nitrate nitrogen dual-effect degrading strains, as well as the domestication and isolation methods and applications of sulfamethoxazole-nitrate nitrogen dual-effect degrading strains. Background Technology

[0002] Sulfamethoxazole (SMX), a commonly used sulfonamide antibiotic, is widely used in medical, agricultural, and food production fields. However, only a small portion of antibiotics can be absorbed by organisms; the majority is excreted in feces or urine as the original drug and metabolites, inevitably being released into the natural environment. This continuous influx not only induces the spread of antibiotic resistance genes (ARGs) but also adversely affects the stability of wastewater treatment systems.

[0003] Livestock and poultry breeding wastewater not only contains high concentrations of antibiotics, but also high concentrations of ammonia nitrogen. The ammonia nitrogen in the wastewater will be converted into nitrate nitrogen and nitrite nitrogen by microorganisms. If it is directly discharged into the environment, it will cause problems such as eutrophication of water bodies, decline in aquatic biodiversity and nitrite poisoning.

[0004] While existing biological treatment technologies are highly effective at removing conventional pollutants, the simultaneous removal of antibiotics and nitrates remains a challenge in wastewater treatment. On the one hand, the presence of high concentrations of antibiotics significantly inhibits the activity of denitrifying bacteria, leading to decreased nitrogen removal efficiency. On the other hand, single-function strains struggle to simultaneously address the needs of antibiotic degradation and nitrogen conversion. Traditional denitrification is anaerobic, which suffers from drawbacks such as the need for additional carbon sources, complex processes, and high operating costs. Aerobic denitrification, however, can simultaneously perform nitrification and denitrification, simplifying the process and saving costs. Therefore, the domestication and purification of multifunctional strains possessing good cultivation characteristics, high-efficiency denitrification performance, and SMX degradation capabilities is not only a cutting-edge scientific issue in environmental microbiology but also a pressing practical need for upgrading wastewater treatment technologies. Summary of the Invention

[0005] The primary objective of this invention is to provide a sulfamethoxazole-nitrate nitrogen dual-effect degrading strain that can degrade SMX and has aerobic denitrification denitrification capability.

[0006] A second objective of this invention is to provide a method for the domestication and isolation of sulfamethoxazole-nitrate nitrogen-degrading strains.

[0007] A third objective of this invention is to provide the application of sulfamethoxazole-nitrate nitrogen dual-effect degrading strains in wastewater treatment.

[0008] The first technical solution adopted in this invention is a sulfamethoxazole-nitrate nitrogen dual-effect degrading strain, namely Burkholderia sp. Stardust46, which was deposited at the China General Microbiological Culture Collection Center on July 22, 2025, with the microbial name Burkholderia sp. Stardust46 and the accession number CGMCC No: 35337.

[0009] The second technical solution adopted in this invention is a method for the domestication and isolation of sulfamethoxazole-nitrate nitrogen dual-effect degrading strains, specifically as follows: Activated sludge was centrifuged at high speed, and the lower layer of bacteria was inoculated into denitrification liquid medium. The SMX concentration in the denitrification liquid medium was gradually increased for acclimatization. After acclimatization, the bacterial solution was serially diluted and evenly spread on BTB denitrification solid medium. It was incubated at 30℃ for 3-5 days. Single colonies with a blue discoloration zone of ≥1.5cm in diameter around the colony were picked and purified by streak plating three times. The purified strain was inoculated into denitrification liquid medium, and its SMX concentration was adjusted to 40mg / L. The residual concentrations of nitrate nitrogen and SMX were measured every 24 hours. Bifunctional strains with aerobic denitrification and SMX degradation capabilities were selected. The target strain was amplified by 16S rRNA gene PCR, and the strain classification was confirmed by NCBI BLAST comparison. The strain was identified as Burkholderia Stardust46.

[0010] The specific denitrification liquid culture medium is as follows: KNO3, carbon source, KH2PO4, MgSO4·7H2O, pH 7.0±0.2, trace element solution, pH adjusted to 7.0±0.2 with KOH solution, and sterilized in an autoclave. The trace element solution components include: EDTA, FeSO4, ZnSO4, MnCl2, H3BO3, CoCl2, CuCl2, NiCl2, NaMoO4; The carbon source is any one or more of glucose, sodium succinate, and sodium acetate.

[0011] The BTB denitrification solid medium is prepared by adding bromothymol blue aqueous solution, SMX and agar powder to the denitrification liquid medium, autoclaving in an autoclave, and then cooling.

[0012] The third technical solution adopted in this invention is the application of sulfamethoxazole-nitrate nitrogen dual-effect degrading strains in the degradation of sulfamethoxazole and nitrate nitrogen in wastewater.

[0013] The invention is further characterized in that, The degradation temperature of the sulfamethoxazole-nitrate nitrogen dual-effect degrading strain is 25-37℃.

[0014] The degradation pH of the sulfamethoxazole-nitrate nitrogen dual-effect degrading strain was 7.0.

[0015] The sulfamethoxazole-nitrate nitrogen dual-effect degrading strain has a carbon-to-nitrogen ratio of C / N ≥ 10.

[0016] The beneficial effects of this invention are: (1) The sulfamethoxazole-nitrate nitrogen dual-effect degrading strain of the present invention has the denitrification ability of simultaneous SMX degradation and aerobic denitrification, realizing the isolation and identification of dual-function strains.

[0017] (2) The sulfamethoxazole-nitrate nitrogen dual-effect degrading strain of the present invention has strong tolerance to SMX and nitrate nitrogen. It can grow efficiently and maintain efficient aerobic denitrification under stress conditions of 40 mg / L SMX and 200 mg / L nitrate nitrogen, and has good tolerance to high concentration antibiotic shock.

[0018] (3) When the sulfamethoxazole-nitrate nitrogen dual-effect degrading strain of the present invention was inoculated into organic carbon wastewater with a concentration of 40 mg / L SMX and 200 mg / L nitrate nitrogen, under the conditions of C / N of 10 and temperature of 30°C, 43.73% of nitrate nitrogen was removed and 83.65% of SMX was degraded within 48 h. The sulfamethoxazole-nitrate nitrogen dual-effect degrading strain has a strong SMX degradation ability, which greatly improves its applicability in antibiotic wastewater. Attached Figure Description

[0019] Figure 1 Phylogenetic tree of Burkholderia sp. Stardust46; Figure 2 The graph shows the concentration changes of nitrous oxide in Burkholderia sp. Stardust46 under stress from nitrate nitrogen and SMX. Figure 3 The removal efficiency of nitrate nitrogen and SMX in Burkholderia sp. Stardust46 after 72 h of culture; Figure 4 Pseudo-first-order kinetic fitting curve for the degradation of SMX by Burkholderia sp. Stardust46 over 72 h; Figure 5The pseudo-first-order kinetic fitting curve of nitrate nitrogen in Burkholderia sp. Stardust46 over 72 h is shown. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] This invention relates to a sulfamethoxazole-nitrate nitrogen dual-degrading strain, Burkholderia sp. Stardust46, which was deposited on July 22, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The strain is named Burkholderia sp. Stardust46, and its accession number is CGMCC No. 35337.

[0022] The sulfamethoxazole-nitrate nitrogen dual-effect degrading strain of the present invention is a bacterium with aerobic denitrification, which can remove SMX and nitrate nitrogen by a single strain.

[0023] The sulfamethoxazole-nitrate nitrogen dual-effect degrading strain of the present invention can use various organic matter such as sodium acetate, glucose, and sodium succinate as carbon sources and ammonia nitrogen and nitrate nitrogen as nitrogen sources to carry out heterotrophic nitrification-aerobic denitrification, thereby removing various forms of nitrogen. Furthermore, this strain possesses all the genes driving the entire denitrification process, enabling nitrate nitrogen to be ultimately removed as nitrous oxide or nitrogen gas.

[0024] The conventional pharmaceuticals involved in the following examples were purchased from Sinopharm Chemical Reagent Co., Ltd., and were of analytical grade; the mobile phase pharmaceuticals such as formic acid and methanol were of chromatographic grade and were purchased from Merck AG, Germany; the sulfamethoxazole antibiotic with a purity of >98% was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0025] Unless otherwise specified, all percentage contents in the following examples are mass percentage contents.

[0026] Example 1 Preparation method of culture medium required for isolating and culturing Burkholderia sp. Stardust46: Denitrification liquid medium: KNO3 1.0 g / L, carbon source 2.5 g / L, KH2PO4 0.1 g / L, MgSO4·7H2O 0.05 g / L, pH 7.0±0.2, trace element solution 1 mL / L (composition: EDTA 0.5 g / L, FeSO4 0.2 g / L, ZnSO4 0.01 g / L, MnCl2 0.03 g / L, H3BO3 0.3 g / L, CoCl2 0.02 g / L, CuCl2 0.001 g / L, NiCl2 0.002 g / L, NaMoO4 0.003 g / L). The pH was adjusted to 7.0±0.2 using a 20% KOH solution, and the medium was autoclaved at 121℃ for 20 min.

[0027] The carbon source is any one or more of glucose, sodium succinate, and sodium acetate; BTB denitrification solid medium: Add 1 mL of 10% bromothymol blue (BTB) aqueous solution, 40 mg SMX and 10 g agar powder to 1 L of denitrification liquid medium. Autoclave at 121°C for 20 min. When the temperature cools to about 40°C, pour into plates, let solidify, and then invert for later use.

[0028] Example 2 The domestication and isolation method for Burkholderia sp. Stardust46, which simultaneously degrades SMX and performs aerobic denitrification, is as follows: Denitrification liquid medium: KNO3 1.0 g / L, glucose 2.5 g / L, KH2PO4 0.1 g / L, MgSO4·7H2O 0.05 g / L, pH 7.0, trace element solution 1 mL / L (composition: EDTA 0.5 g / L, FeSO4 0.2 g / L, ZnSO4 0.01 g / L, MnCl2 0.03 g / L, H3BO3 0.3 g / L, CoCl2 0.02 g / L, CuCl2 0.001 g / L, NiCl2 0.002 g / L, NaMoO4 0.003 g / L). The pH was adjusted to 7.0 using a 20% KOH solution, and the medium was autoclaved at 121℃ for 20 min.

[0029] BTB denitrification solid medium: Add 1 mL of 10% bromothymol blue (BTB) aqueous solution, 40 mg SMX, and 10 g agar powder to 1 L of denitrification liquid medium. Autoclave at 121°C for 20 min. When the temperature cools to about 40°C, pour into plates, let solidify, and then invert for later use.

[0030] Using activated sludge as the inoculum, the activated sludge was centrifuged at 8000 r / min using a high-speed centrifuge, and 2g of the lower layer of bacteria was inoculated into 200ml of denitrification liquid culture medium. The SMX concentration was gradually increased in denitrification liquid medium (5→10→15→20→25→30→35→40 mg / L) in 8 stages, with each stage lasting 7 days. During the acclimatization period, the changes in nitrate nitrogen concentration (gas phase molecular absorption spectrometer) and SMX concentration (HPLC-MS) were monitored daily. Take the acclimatized bacterial culture and serially dilute it (10⁻⁶). -3 ~10 -6 After that, the bacterial solution was evenly spread on BTB denitrification solid medium and incubated at 30℃ for 3-5 days. Single colonies with a blue discoloration zone of ≥1.5cm in diameter around the colony were picked and purified by streak plating 3 times. The purified strain was inoculated into denitrification liquid medium, and its SMX concentration was adjusted to 40 mg / L. The residual concentrations of nitrate nitrogen and SMX were measured every 24 hours, and bifunctional strains with aerobic denitrification and SMX degradation capabilities were selected. The target strain was amplified by 16S rRNA gene PCR using primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3') (94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1.5 min, 30 cycles, 72℃ final extension for 10 min). After sequencing, the strain classification was confirmed by NCBI BLAST comparison. The sequencing results were compared with the NCBI database using BLAST. The results showed that the 16S rRNA gene sequence of this strain was more than 99% similar to that of closely related strains of the Burkholderia genus. Based on the morphological and physiological and biochemical characteristics, the strain was identified as Burkholderia sp. Stardust46.

[0031] Example 3 Changes in nitrous oxide concentration in Burkholderia sp. Stardust46 under nitrate nitrogen and SMX stress.

[0032] Burkholderia sp. Stardust46, a bacterium capable of simultaneously degrading SMX and performing aerobic denitrification, can completely convert nitrate nitrogen into nitrogen gas. Nitrogen production can be determined by measuring the concentration change of the intermediate product nitrous oxide. Specifically: Add 100 mL of denitrification liquid culture medium to a 300 mL brown serum bottle, and sterilize in an autoclave at 121°C for 20 min.

[0033] After cooling, strain Stardust46 was inoculated into liquid culture medium, sealed, and placed in a constant temperature shaker at 30℃ and 150rpm for 96h. During this period, the concentration of nitrous oxide in the headspace gas was determined by gas chromatography every 24h.

[0034] like Figure 2 As shown, nitrous oxide reached a peak of 171.65 ppm at 48 h, and then gradually decreased to 96.73 ppm at 72 h, indicating that Burkholderia sp. Stardust46 can further convert nitrous oxide into nitrogen gas, completing the entire denitrification process.

[0035] Example 4 Degradation capacity of Burkholderia sp. Stardust46 under different environmental factors Burkholderia sp. Stardust46 exhibited different removal efficiencies for SMX and nitrate nitrogen after being cultured in denitrification liquid medium containing different carbon sources at 30°C and 150 rpm for 48 h, but both showed good growth rates and degradation effects, indicating that strain SMX has a wide range of carbon source adaptability.

[0036] Denitrification liquid medium: KNO3 1.0 g / L, carbon source (carbon sources are glucose, sodium succinate, sodium acetate in sequence) 2.5 g / L, KH2PO4 0.1 g / L, MgSO4·7H2O 0.05 g / L, pH 7.1, trace element solution 1 mL / L (composition: EDTA 0.5 g / L, FeSO4 0.2 g / L, ZnSO4 0.01 g / L, MnCl2 0.03 g / L, H3BO3 0.3 g / L, CoCl2 0.02 g / L, CuCl2 0.001 g / L, NiCl2 0.002 g / L, NaMoO4 0.003 g / L). The pH was adjusted to 7.1 using a 20% KOH solution, and the medium was autoclaved at 121℃ for 20 min.

[0037] BTB denitrification solid medium: Add 1 mL of 10% bromothymol blue (BTB) aqueous solution, 40 mg SMX and 10 g agar powder to 1 L of denitrification liquid medium. Autoclave at 121°C for 20 min. When the temperature cools to about 40°C, pour into plates, let solidify, and then invert for later use.

[0038] Burkholderia sp. Stardust46 can grow between 25-37℃ and still maintains a good pollutant removal effect.

[0039] Burkholderia sp. Stardust46 can grow, denitrify, and degrade SMX under conditions ranging from pH 6 to 8, but its growth rate is fastest under neutral conditions (pH ≈ 7.0).

[0040] Burkholderia sp. Stardust46 grew normally and underwent aerobic denitrification at SMX concentrations ranging from 0 to 40 mg / L, indicating that the strain has a high tolerance to SMX.

[0041] Carbon-to-nitrogen ratio The SMX and denitrification efficiency of Burkholderia sp. Stardust46 varied with the carbon-to-nitrogen ratio. Due to the faster growth rate of the strain at higher carbon-to-nitrogen ratios, the removal efficiency of SMX and nitrate nitrogen was optimal when the C / N ratio was ≥10.

[0042] Example 5 Burkholderia sp. Stardust46's ability to degrade SMX and nitrate nitrogen within 72 hours Set the initial concentration of SMX in the denitrification liquid medium to 40 mg / L and the initial concentration of nitrate nitrogen to 200 mg / L. Take 200 ml of the medium and put it into a 500 ml Erlenmeyer flask.

[0043] Strains Stardust46 were picked from the slant culture medium and inoculated into the above denitrification liquid culture medium. The concentrations of residual SMX and nitrate nitrogen were measured every 24 hours.

[0044] like Figure 3 As shown, this strain exhibits the ability to simultaneously degrade SMX and perform aerobic denitrification. After 72 h of inoculation and culture, the degradation rates of SMX and nitrate nitrogen reached 81.75% and 43.73%, respectively.

[0045] During the first 0-48 hours, the bacterial concentration rises rapidly, and SMX and nitrate nitrogen are rapidly degraded, marking the rapid degradation phase.

[0046] like Figure 4 As shown, the degradation of SMX within 72 hours follows pseudo-first-order kinetics, with a degradation rate constant k reaching 0.02598 h. -1 .

[0047] like Figure 5 As shown, the degradation of nitrate nitrogen within 72 hours follows pseudo-first-order kinetics, with a degradation rate constant k reaching 0.00906 h⁻¹. -1 .

[0048] Example 6 The sulfamethoxazole-nitrate nitrogen dual-effect degrading strain of the present invention has high SMX tolerance and degradation capacity, and possesses abundant nitrogen metabolism-related genes, enabling it to completely metabolize nitrate nitrogen into nitrogen gas and nitrous oxide. When treating wastewater with high concentrations of SMX and nitrate nitrogen (such as livestock and poultry breeding wastewater), it is more efficient, energy-saving, and cost-effective than traditional denitrification processes, effectively removing SMX and reducing stress on other strains.

Claims

1. A sulfamethoxazole-nitrate nitrogen dual-effect degrading strain, characterized in that, The sulfamethoxazole-nitrate nitrogen dual-effect degrading strain is Burkholderia Stardust46, which was deposited at the China General Microbiological Culture Collection Center on July 22, 2025. The microorganism is named Burkholderia sp. Stardust46, and the accession number is CGMCCNo: 35337.

2. The method for domestication and isolation of the sulfamethoxazole-nitrate nitrogen dual-effect degrading strain as described in claim 1, characterized in that, Specifically: Activated sludge was centrifuged at high speed, and the lower layer of bacteria was inoculated into denitrification liquid medium. The SMX concentration in the denitrification liquid medium was gradually increased for acclimatization. After acclimatization, the bacterial solution was serially diluted and evenly spread on BTB denitrification solid medium. It was incubated at 30℃ for 3-5 days. Single colonies with a blue discoloration zone of ≥1.5cm in diameter around the colony were picked and purified by streak plating three times. The purified strain was inoculated into denitrification liquid medium, and its SMX concentration was adjusted to 40mg / L. The residual concentrations of nitrate nitrogen and SMX were measured every 24 hours. Bifunctional strains with aerobic denitrification and SMX degradation capabilities were selected. The target strain was amplified by 16S rRNA gene PCR, and the strain classification was confirmed by NCBI BLAST comparison. The strain was identified as Burkholderia Stardust46.

3. The method for domestication and isolation of the sulfamethoxazole-nitrate nitrogen dual-effect degrading strain as described in claim 2, characterized in that, The specific denitrification liquid culture medium is as follows: KNO3, carbon source, KH2PO4, MgSO4·7H2O, pH 7.0±0.2, trace element solution, pH adjusted to 7.0±0.2 with KOH solution, and sterilized in an autoclave. The trace element solution components include: EDTA, FeSO4, ZnSO4, MnCl2, H3BO3, CoCl2, CuCl2, NiCl2, NaMoO4; The carbon source is any one or more of glucose, sodium succinate, and sodium acetate.

4. The method for domestication and isolation of the sulfamethoxazole-nitrate nitrogen dual-effect degrading strain as described in claim 3, characterized in that, The BTB denitrification solid medium is prepared by adding bromothymol blue aqueous solution, SMX and agar powder to the denitrification liquid medium, autoclaving in an autoclave, and then cooling.

5. The application of the sulfamethoxazole-nitrate nitrogen dual-effect degrading strain as described in claim 1 in the degradation of sulfamethoxazole and nitrate nitrogen in wastewater.

6. The application as described in claim 5, characterized in that, The degradation temperature of the sulfamethoxazole-nitrate nitrogen dual-effect degrading strain is 25-37℃.

7. The application as described in claim 5, characterized in that, The degradation pH of the sulfamethoxazole-nitrate nitrogen dual-effect degrading strain was 7.

0.

8. The application as described in claim 5, characterized in that, The sulfamethoxazole-nitrate nitrogen dual-effect degrading strain has a carbon-to-nitrogen ratio of C / N ≥ 10.

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