Pseudomonas capable of degrading fluoroquinolone antibiotics, complex microbial inoculant and application of pseudomonas and complex microbial inoculant

Abstract

The invention relates to the field of microorganisms and water treatment, and discloses pseudomonas capable of degrading fluoroquinolone antibiotics, a complex microbial inoculant and application of the pseudomonas and the complex microbial inoculant. The pseudomonas is named as 11-10, the preservation number is CGMCC No.33680, the microorganism classification is named as pseudomonas sp., the preservation unit is China General Microbiological Culture Collection Center, the preservation date is February 28, 2025, and the preservation number is CGMCC No.33680, the microorganism classification is named as Pseudomonas sp., the preservation unit is China General Microbiological Culture Collection Center, and the preservation date is February 28, 2025. Firstly, the new pseudomonas strain obtained through screening has high tolerance to the fluoroquinolone antibiotics and can efficiently degrade the fluoroquinolone antibiotics; and secondly, in the composite microbial agent disclosed by the invention, the new pseudomonas species, the bacillus tequilensis and the paenibacillus deserticola can play a relatively good synergistic effect, so that the endurance capacity and the degradation capacity on the fluoroquinolone antibiotic wastewater can be further improved.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and water treatment, and particularly to Pseudomonas aeruginosa that can degrade fluoroquinolone antibiotics, compound bacterial agents, and their applications. Background Technology

[0002] Antibiotics are a class of drugs used to treat bacterial infections or inhibit the infection of pathogenic microorganisms; their discovery is considered a milestone in medical history. Antibiotics are mainly classified into quinolones, β-lactams, macrolides, and tetracyclines. Among them, fluoroquinolones (FQs), as the third generation of quinolone antibiotics, are chemically synthesized antibacterial drugs with wide applications and significant clinical value, commonly used to treat infections of the respiratory tract, urinary system, and intestines. Fluoroquinolone antibiotics mainly include norfloxacin and ofloxacin.

[0003] With the increasing demand for antibiotics, fluoroquinolone antibiotics are being synthesized in large quantities. However, only a small portion of fluoroquinolone antibiotics are metabolized and degraded in organisms, with the remainder being excreted with their original structure and biological activity. This widespread use leads to increased antibiotic residues in the environment, causing environmental problems such as antibiotic resistance and ecotoxicity. Simultaneously, the synthesis of fluoroquinolone antibiotics generates large amounts of production wastewater, containing unreacted residues, incompletely extracted products, and incompletely recovered solvents. Therefore, this type of wastewater is characterized by high concentrations of antibiotic residues, complex composition, high toxicity, and poor biodegradability, and is considered difficult-to-treat organic wastewater.

[0004] Currently, effective treatment technologies for fluoroquinolone antibiotic wastewater mainly include adsorption, photocatalytic degradation, ozone oxidation, and biological methods (e.g., CN201910052425.X, CN202411267339.8). Compared to physical and chemical methods, biological methods are a greener, more efficient, economical, and environmentally friendly approach. Recent studies have shown that certain bacteria can degrade fluoroquinolone antibiotics through enzymatic reactions. The degradation mechanisms mainly include hydroxylation, decarboxylation, ring-opening, and conjugation. For example, bacteria such as *Pseudomonas* and *Sphingomonas* can catalyze the hydroxylation and ring-opening reactions of fluoroquinolone antibiotics by producing monooxygenases and dioxygenases, ultimately converting them into low-toxicity or non-toxic small molecule compounds. Therefore, continuously screening for more strains with better degradation effects on fluoroquinolone antibiotics is of positive significance. Summary of the Invention

[0005] To address the technical problem of poor biodegradability and difficulty in efficient degradation by microorganisms of fluoroquinolone antibiotics, this invention provides a *Pseudomonas* strain capable of degrading fluoroquinolone antibiotics, a compound microbial agent, and their applications. First, the *Pseudomonas* strain screened in this invention exhibits high tolerance to fluoroquinolone antibiotics and can efficiently degrade them. Second, in the compound microbial agent of this invention, the new *Pseudomonas* strain, *Bacillus tekirae*, and *Bacillus desertis* exhibit a good synergistic effect, further enhancing the tolerance and degradation capacity of fluoroquinolone antibiotic wastewater.

[0006] The specific technical solution of the present invention includes: In a first aspect, the present invention provides a strain of Pseudomonas that can degrade fluoroquinolone antibiotics, named 11-10, with accession number CGMCC No. 33680, microbiologically classified as Pseudomonas sp., deposited at the China General Microbiological Culture Collection Center on February 28, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The strain of this invention was isolated from activated sludge used in the production of fluoroquinolone antibiotics. The 16S rRNA nucleotide sequence of this strain is shown in SEQ ID No. 1. A comparison with the EzBioCloud database shows that it is similar to *Pseudomonas hydrolytica*DSWY01. T The most similar strain was found to be 98.03% similar. A phylogenetic tree of the 16S rRNA gene was constructed, revealing that strain 11-10 was clustered on the same branch as the genus *Pseudomonas* and remained relatively stable. Therefore, strain 11-10 belongs to the genus *Pseudomonas* and was identified as a new species within the genus *Pseudomonas*.

[0008] This strain 11-10 is a Gram-negative bacterium with rod-shaped cells, measuring 1.5-2.1 μm in length and 0.4-0.6 μm in width. When cultured on LB medium at 30°C for 3 days, colonies are white, round, with a raised surface, smooth edges, and opaque, measuring 0.5-2.0 mm in size, and without spores. The strain can grow in the range of 10-40°C, with an optimum temperature of 30-37°C; it can grow in the range of pH 5.0-9.5, with an optimum pH of 7.0-8.0; it can grow in the range of NaCl 0-4%, with an optimum salinity of 0.5-2%. It is positive for oxidase, catalase, and lipase. It is negative for indole formation, methyl red test, fluorescent dye, and denitrification. It can utilize substrates such as D-xylose, D-mannose, D-fructose, D-glucose, sucrose, galactoside, cellobiose, D-salicylic acid, lactic acid, citric acid, Tween 40, glycerol, D-mannitol, maltose, L-arginine, L-serine, and L-histidine, but cannot utilize lactose, D-sorbitol, ethanol, galactose, trehalose, arabinose, L-sorbose, inositol, γ-aminobutyric acid, 2,3-butanediol, or carnosine. The main respiratory quinone is MK11. The main fatty acid is C. 18：1 ω7c / C 18：1 ω6c, C 12：0 and C 16：0 .

[0009] Further research revealed that this novel *Pseudomonas* strain exhibits high tolerance to fluoroquinolone antibiotics, demonstrating efficient degradation of these antibiotics. It also adapts to high salinity and a wide pH range, effectively degrading fluoroquinolone antibiotics even under high salinity and strong acidity / alkalinity conditions. This reduces the environmental requirements for application, simplifying water quality conditioning processes in wastewater treatment. In contrast, the ability of this new strain to degrade fluoroquinolone antibiotics has not been reported in existing technologies.

[0010] Secondly, the present invention provides a compound bacterial agent that can degrade fluoroquinolone antibiotics, comprising: the aforementioned Pseudomonas, Bacillus tekirae, and Bacillus desertis.

[0011] In compound microbial agents, complex interactions exist between various bacterial strains, affecting their growth and metabolism, and potentially exhibiting synergistic or antagonistic effects. This invention has found that combining the novel *Pseudomonas* species 11-10 with *Bacillus tekirae* and *Bacillus desertis* produces a synergistic effect. Compared to individual strains, this compound microbial agent significantly enhances the degradation of fluoroquinolone antibiotics. Furthermore, this compound microbial agent exhibits good salt tolerance, showing significantly better degradation performance against fluoroquinolone antibiotics at high salt and high fluoroquinolone antibiotic concentrations than *Pseudomonas* species 11-10 alone.

[0012] Preferably, the Bacillus tequilensis is named H21, with accession number CGMCC No.19227, microbiological classification name Bacillus tequilensis, deposited at the China General Microbiological Culture Collection Center, deposited on December 23, 2019, and located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0013] In the compound bacterial agent, Bacillus tekirae is limited to the specific Bacillus tekirae H21 screened in this invention, and the synergistic effect between strains is more obvious.

[0014] Preferably, the *Paenibacillus harenae* is named DFB2-6, with accession number CGMCC No. 20535, microbiological classification name *Paenibacillus harenae*, deposited at the China General Microbiological Culture Collection Center on August 21, 2020, and located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0015] In the compound bacterial agent, the desert sand bacillus is limited to the specific desert sand bacillus DFB2-6 screened in this invention, and the synergistic effect between strains is more obvious.

[0016] Preferably, the ratio of viable counts of *Pseudomonas*, *Bacillus tekirae*, and *Bacillus desertica* in the compound microbial agent is 1:0.2-1.5:0.2-1.5. More preferably, the ratio of viable counts of *Pseudomonas*, *Bacillus tekirae*, and *Bacillus desertica* in the compound microbial agent is 1:0.5:0.6.

[0017] The present invention has found that, under the above-mentioned preferred strain ratio, the synergistic effect among the strains in the compound microbial agent is more obvious.

[0018] Preferably, the compound microbial agent is a liquid microbial agent or a solid microbial agent.

[0019] Preferably, the compound microbial agent also includes a culture medium suitable for the growth of the strain.

[0020] Thirdly, the present invention provides a biological agent that can degrade fluoroquinolone antibiotics, comprising a whole fermentation broth or intracellular substance extract obtained from the Pseudomonas or the compound bacterial agent.

[0021] Fourthly, the present invention provides the use of the aforementioned Pseudomonas, the aforementioned compound bacterial agent, or the aforementioned biological agent in the degradation of fluoroquinolone antibiotics.

[0022] Preferably, the fluoroquinolone antibiotic is a fluoroquinolone antibiotic found in wastewater.

[0023] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention screened and obtained a new species of Pseudomonas, which has high tolerance to fluoroquinolone antibiotics and can efficiently degrade fluoroquinolone antibiotics. At the same time, it can also adapt to high salinity and a wide pH range, and can still effectively degrade fluoroquinolone antibiotics under high salinity and strong acidity / alkalinity. (2) In the compound bacterial agent of the present invention, the new species of Pseudomonas, Bacillus tekirae and Bacillus desertis can produce a synergistic effect, which can effectively improve the degradation ability of fluoroquinolone antibiotics and have high salt tolerance. Attached Figure Description

[0024] Figure 1 This is a colony photograph of Pseudomonas 11-1. Detailed Implementation

[0025] The present invention will be further described below with reference to embodiments.

[0026] General Implementation Examples Firstly, a strain of Pseudomonas that can degrade fluoroquinolone antibiotics, named 11-10, with accession number CGMCC No. 33680, microbiologically classified as Pseudomonas sp., deposited at the China General Microbiological Culture Collection Center on February 28, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0027] Secondly, a compound bacterial agent that can degrade fluoroquinolone antibiotics includes: the aforementioned Pseudomonas, Bacillus tekirae, and Bacillus desertis.

[0028] In some specific implementation cases, the Bacillus tequilensis is named H21, with accession number CGMCCNo.19227, microbiological classification name Bacillus tequilensis, deposited at the China General Microbiological Culture Collection Center, deposited on December 23, 2019, and located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0029] In some specific implementation cases, the aforementioned *Paenibacillus harenae* is named DFB2-6, with the accession number CGMCC No. 20535, the microbiological classification name is *Paenibacillus harenae*, the depositary institution is the China General Microbiological Culture Collection Center, the deposit date is August 21, 2020, and the address of the depositary institution is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0030] In some specific implementation examples, the ratio of viable counts of *Pseudomonas*, *Bacillus tekirae*, and *Bacillus desertica* in the compound microbial agent is 1:0.2-1.5:0.2-1.5. More preferably, the ratio of viable counts of *Pseudomonas*, *Bacillus tekirae*, and *Bacillus desertica* in the compound microbial agent is 1:0.5:0.6.

[0031] In some specific implementation cases, the compound microbial agent is a liquid or solid microbial agent.

[0032] In some specific implementation cases, the compound microbial agent also includes a culture medium suitable for the growth of the strain.

[0033] Thirdly, a biological agent capable of degrading fluoroquinolone antibiotics, comprising a whole fermentation broth or intracellular substance extract obtained from the aforementioned Pseudomonas or the aforementioned compound bacterial agent.

[0034] Fourthly, the application of the aforementioned Pseudomonas, the aforementioned compound bacterial agent, or the aforementioned biological agent in the degradation of fluoroquinolone antibiotics.

[0035] In some specific implementation cases, the fluoroquinolone antibiotics mentioned are fluoroquinolone antibiotics found in wastewater. Specific Implementation Example 1: Domestication of Strains (1) Preparation of culture medium The acclimatization culture medium was prepared according to the following formula: 2000 mg / L of fluoroquinolone antibiotics (mainly norfloxacin and ciprofloxacin in a 1:1 ratio), 3 g / L of KH₂PO₄, 3 g / L of K₂HPO₄, 0.2 g / L of MgSO₄·7H₂O, 0.5 g / L of NaCl, and 1 mL / L of trace element stock solution. The pH was set to 7. For solid culture media, an additional 2% agar powder was added. All the above culture media were sterilized at 121°C for 15 minutes.

[0037] The formula for the above trace element solution is as follows: FeSO4·7H2O 3.6g / L, ZnSO4·7H2O 0.3g / L, MnSO4·H2O 0.02g / L, CuSO4·5H2O 0.25g / L, CoCl2·6H2O 0.1g / L, with water as the solvent.

[0038] (2) Strain domestication Activated sludge from a fluoroquinolone antibiotic production plant in Zhejiang was taken, broken up with a glass grinder, and added to an acclimatization culture medium. The mixture was then enriched in a shaker at 30°C and 150 rpm. COD, NH3-N, and TN were monitored daily until the system became noticeably turbid, COD and TN decreased significantly, and ammonia nitrogen increased. This was considered the end of the acclimatization process.

[0039] (3) High-throughput sequencing After domestication, high-throughput sequencing was performed on the above system. The genera with a relative abundance of 1% or more are shown in Table 1. Among them, Pseudomonas had the largest proportion, accounting for 61%, followed by Sulfoxymonas, accounting for 13%.

[0040] Table 1 Generic name Chinese name Abundance Pseudomonas Pseudomonas 61％ Sulfurimonas spp. of sulfur-oxidizing monoclonal bacteria 13％ Sphingosinomonas Sphingosomalidosis 5％ Gudongella genus *Leptobacter* 4％ Marinobacterium genus *Gastromycium* 3％ Sphingobacterium Sphingosine bacillus 2％ Fluviicola genus *Aquilaria* 1％ Alcaligenes Alcaligenes 1％ Acinetobacter Acinetobacter 1％ Others other 9％ Example 2: Isolation and Obtaining of New Pseudomonas species 11-10 (1) Preparation of culture medium Luria-Bertani medium (hereinafter referred to as LB solid medium) was prepared according to the following formula: Tryptone 10 g / L, Yeast extract 5 g / L, NaCl 10 g / L. An additional 2% agar powder was added to the solid medium. All the above media were sterilized at 121°C for 15 minutes.

[0041] (2) Isolation and identification of strains The acclimatization system was isolated and cultured using LB solid medium, and a single strain was finally isolated and named 11-10.

[0042] Strains 11-10 are Gram-negative bacteria with rod-shaped cells, measuring 1.5-2.1 μm in length and 0.4-0.6 μm in width. After culturing on LB medium at 30°C for 3 days, colonies are white, round, with a raised surface, smooth edges, and opaque, measuring 0.5-2.0 mm in size, and are non-spore-forming. Figure 1The strain can grow in the range of 10-40℃, with an optimum temperature of 30-37℃; it can grow in the range of pH 5.0-9.5, with an optimum pH of 7.0-8.0; it can grow in the range of NaCl 0-4%, with an optimum salinity of 0.5-2%. It is positive for oxidase, catalase, and lipase. It is negative for indole formation, methyl red test, fluorescent dye, and denitrification. It can utilize substrates such as D-xylose, D-mannose, D-fructose, D-glucose, sucrose, galactoside, cellobiose, D-salicylic acid, lactic acid, citric acid, Tween 40, glycerol, D-mannitol, maltose, L-arginine, L-serine, and L-histidine, but cannot utilize lactose, D-sorbitol, ethanol, galactose, trehalose, arabinose, L-sorbose, inositol, γ-aminobutyric acid, 2,3-butanediol, or carnosine. The main respiratory quinone is MK11. The main fatty acid is C. 18：1 ω7c / C 18：1 ω6c, C 12：0 and C 16：0 .

[0043] The 16S rRNA gene of strain 11-10 was compared with that of Pseudomonas hydrolytica DSWY01 in the EzBioCloud database. T The most similar strain was found to be 98.03% similar. A phylogenetic tree of the 16S rRNA gene was constructed, revealing that strain 11-10 was clustered on the same branch as the genus *Pseudomonas* and remained relatively stable. Therefore, strain 11-10 likely belongs to the genus *Pseudomonas* and was identified as a suspected new species of the genus *Pseudomonas*. The 16S rRNA gene sequence of strain 11-10 is shown in SEQ ID No. 1 below: Example 3: The ability of a new species of Pseudomonas 11-10 to degrade fluoroquinolone antibiotics and influencing factors (1) Preparation of seed liquid Strain 11-10 was activated on LB solid medium and cultured at 30°C for 3 days. Single colonies were inoculated into LB liquid medium and cultured at 30°C and 150 rpm until OD600 = 1.0, which was then used as seed culture.

[0044] (2) Effect of salinity on the degradation of fluoroquinolone antibiotics: Using the same acclimatization medium as in Example 1, but with the other components and conditions unchanged, the NaCl content was changed to 0.5 g / L, 5 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L, and 50 g / L (i.e., salinities of 0.05%, 0.5%, 1.0%, 2.0%, 3.0%, 4.0%, and 5.0%, respectively) in 100 mL of acclimatization medium in a 250 mL Erlenmeyer flask. The seed culture from (1) was inoculated into the acclimatization medium at an inoculation rate of 30%, mixed well, and then placed on a shaker at 150 r / min and 30 °C for 3 days. The supernatant was then separated by centrifugation, and the COD and organic nitrogen content in the supernatant were detected. The COD and organic nitrogen removal rates were calculated, and the results are shown in Table 2.

[0045] Table 2 salinity(%) 0.05 0.5 1.0 2.0 3.0 4.0 5.0 COD removal rate (%) 75.5 74.6 72.2 66.8 43.2 30.1 18.5 Organic nitrogen removal rate (%) 43.2 42.3 40.1 35.5 26.1 20.7 9.2 The results showed that the optimal salinity for strain 11-10 to degrade fluoroquinolone antibiotics was 0.05-1.0%. When the salinity increased to 2.0%, the degradation ability of strain 11-10 for fluoroquinolone antibiotics decreased, but it could still achieve degradation, with COD and organic nitrogen removal rates reaching 66.8% and 35.5%, respectively. At a salinity of 3.0%, it maintained a COD removal rate of over 40% and an organic nitrogen removal rate of over 25%. When the salinity increased to 4.0%, the COD and organic nitrogen removal rates decreased significantly. When the salinity increased to 5.0%, the degradation effect on fluoroquinolone antibiotics was poor, possibly due to significant inhibition of strain growth.

[0046] (3) Effect of pH on the degradation of fluoroquinolone antibiotics: Using the same acclimatization medium as in Example 1, with the other components and conditions unchanged, 100 mL of acclimatization medium with pH values ​​of 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 10.0 was placed in a 250 mL Erlenmeyer flask. The seed culture from (1) was inoculated into the acclimatization medium at an inoculation rate of 30%. After mixing, the medium was placed in a shaker at 150 r / min and ℃ for 3 days. The supernatant was then separated by centrifugation, and the COD and organic nitrogen content in the supernatant were detected. The COD and organic nitrogen removal rates were calculated, and the results are shown in Table 3.

[0047] Table 3 pH 6.0 6.5 7.0 7.5 8.0 8.5 9.0 10.0 COD removal rate (%) 62.1 70.6 75.5 72.1 61.4 50.9 25.9 16.1 Organic nitrogen removal rate (%) 32.5 39.3 43.2 40.1 35.5 31.1 19.9 10.1 The results showed that the optimal pH for strain 11-10 to degrade fluoroquinolone antibiotics was 6.5-7.5. When the pH was in the range of 6.0-8.0, strain 11-10 exhibited a high capacity for degrading fluoroquinolone antibiotics, achieving a COD removal rate of over 50% and an organic nitrogen removal rate of over 30%.

[0048] (4) Effect of temperature on the degradation of fluoroquinolone antibiotics: Using the same acclimatization medium (containing 2000 mg / L of fluoroquinolone antibiotics) as in Example 1, with other components and conditions unchanged, 100 mL of acclimatization medium was placed in a 250 mL Erlenmeyer flask, and the seed liquid was inoculated into the acclimatization medium at an inoculation rate of 30%. After mixing, the flask was placed in a shaker at 150 r / min and cultured at different temperatures (10℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃) for 3 days. The supernatant was then separated by centrifugation, and the COD and organic nitrogen content in the supernatant was detected. The COD and organic nitrogen removal rates were calculated, and the results are shown in Table 4.

[0049] Table 4 Temperature (°C) 10 20 25 30 35 40 45 COD removal rate (%) 28.1 55.6 62.3 75.5 69.8 63.6 29.5 Organic nitrogen removal rate (%) 8.9 22.6 35.8 43.2 37.5 29.3 11.2 The results showed that the optimal temperature for strain 11-10 to degrade fluoroquinolone antibiotics was 30-35℃.

[0050] (5) Effect of time on the degradation of fluoroquinolone antibiotics: Using the same acclimatization medium (containing 2000 mg / L of fluoroquinolone antibiotics) as in Example 1, with other components and conditions unchanged, 100 mL of acclimatization medium was placed in a 250 mL Erlenmeyer flask, and the seed liquid was inoculated into the acclimatization medium at an inoculation rate of 30%. After mixing, the mixture was placed in a shaker at 150 r / min and 30 °C for different times (0 d, 1 d, 2 d, and 3 d, respectively). The supernatant was separated by centrifugation, and the contents of COD, NH3-N, organic nitrogen and total nitrogen (TN) in the supernatant were detected. The removal rates of COD, NH3-N, organic nitrogen and total nitrogen (TN) were calculated. The results are shown in Table 5.

[0051] Table 5 0d 1d 2d 3d COD 5091mg / L 3989mg / L 2689mg / L 1247mg / L <![CDATA[NH3-N]]> 9mg / L 33mg / L 67mg / L 127mg / L Organic nitrogen 302mg / L 276mg / L 239mg / L 172mg / L Total nitrogen 311mg / L 309mg / L 306mg / L 299mg / L The data in the table above show that experiments in a shake-flask static system demonstrate that strain 11-10 can efficiently degrade fluoroquinolone antibiotics and is adaptable to pH levels of 6.0-8.0, temperatures of 25-35℃, and salinity levels of 0.05%-2.0%. Under these conditions, COD removal rates exceeding 50% and organic nitrogen removal rates exceeding 30% can be achieved within three days. The optimal conditions for strain 11-10 to degrade fluoroquinolone antibiotics are: pH 6.5-7.5, temperature 30-35℃, and NaCl salinity of 0.05%-1.0%. Under these optimal conditions, COD removal rates can reach over 70% and organic nitrogen removal rates can reach over 40% within three days.

[0052] Example 4: Construction and optimization of a complex microbial community for degrading fluoroquinolone antibiotics (1) Preparation of culture medium Same as Example 1.

[0053] Fluoroquinolone antibiotics (mainly norfloxacin and ciprofloxacin in a 1:1 ratio) 10000 mg / L, KH2PO4 3 g / L, K2HPO4 3 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.5 g / L, trace elements 1 ml / L; pH = 7.

[0054] (2) Preparation of seed liquid New strains of Pseudomonas 11-10, Bacillus tekirae H21, and Bacillus desertis DFB2-6 were cultured on LB solid medium (same as in Example 2) at 30°C for 3 days, and single colonies were picked. The single colonies of the three strains were inoculated into LB liquid medium (same as in Example 2) and cultured at 150 rpm and 30°C to the logarithmic development phase to obtain seed culture of each strain.

[0055] (3) Synergistic effect among different strains According to the strain ratio in the table, the strain seed liquid was inoculated into the simulated wastewater culture medium at an inoculation rate of 30%, so that the initial and final concentration of the bacteria was OD600 = 0.5. After culturing at 30℃ for 5 days, the COD and organic nitrogen content in the wastewater were detected, and the COD and organic nitrogen removal rates were calculated. The results are shown in Table 6.

[0056] Table 6 strain ratio COD removal rate (%) Organic nitrogen removal rate (%) New species of Pseudomonas 11-10 75.5 43.2 Bacillus tekirulae H21 32.1 11.5 Desert sand bacillus DFB2-6 36.9 18.7 New species of Pseudomonas 11-10+ Bacillus tekirae H21 80.2 46.6 New species of Pseudomonas 11-10+ Desert Sand Bacillus DFB2-6 83.7 49.1 The results showed that when the new Pseudomonas species 11-10 was combined with Bacillus tekirae H21 and Bacillus desertis DFB2-6, the COD and organic nitrogen removal rates were higher than those of the corresponding single bacteria, indicating that the new Pseudomonas species 11-10 had a synergistic effect with A and B.

[0057] (4) Optimization of the ratio among complex microbial communities The seed cultures of the three strains were mixed according to the different volume ratios in the table, and then inoculated into the simulated wastewater culture medium at an inoculation rate of 30%, so that the initial and final concentrations of the bacteria were OD600 = 0.5. After culturing at 30℃ for 5 days, the COD and organic nitrogen content in the wastewater were detected, and the COD and organic nitrogen removal rates were calculated. The results are shown in Table 7.

[0058] Table 7 Seed liquid volume ratio COD removal rate (%) Organic nitrogen removal rate (%) 1.0：1.0：1.0 82.7 49.6 1.0：0.5：0.5 86.5 51.2 1.0：0.2：0.2 77.1 42.9 1.0：1.5：1.5 73.8 39.1 The results showed that when the seed-liquid volume ratio of the three strains was in the range of 1:0.2-1.5 or 1.5:0.2-1.5, the removal rates of COD and organic nitrogen were higher than those of a single strain, indicating that the three strains could exert a good synergistic effect and improve the treatment effect of aniline wastewater. Among them, when the seed-liquid volume ratio of the three strains was in the range of 1:0.5:0.5 (the ratio of viable bacteria was 1:0.5:0.6), the removal rates of COD and organic nitrogen were the highest.

[0059] Example 5 The ability of a complex microbial community to treat high concentrations of fluoroquinolone antibiotics under low and high salinity conditions (1) Preparation of high-concentration simulated wastewater Prepare a low-salt, high-concentration simulated wastewater according to the following formula: 10000 mg / L of fluoroquinolone antibiotics (mainly norfloxacin and ciprofloxacin in a 1:1 ratio), 3 g / L of KH2PO4, 3 g / L of K2HPO4, 0.2 g / L of MgSO4·7H2O, 0.5 g / L of NaCl, and 1 ml / L of trace elements; pH = 7.

[0060] Prepare a high-salt, high-concentration simulated wastewater according to the following formula: 10000 mg / L of fluoroquinolone antibiotics (mainly norfloxacin and ciprofloxacin in a 1:1 ratio), 3 g / L of KH2PO4, 3 g / L of K2HPO4, 0.2 g / L of MgSO4·7H2O, 20 g / L of NaCl, and 1 ml / L of trace elements; pH = 7.

[0061] (2) The preparation of seed liquid is the same as in Example 4.

[0062] (3) Simulation experiment of fluoroquinolone antibiotic wastewater under low-salt conditions The experimental setup is a continuous inlet and outlet system, comprising an inlet tank, a microbial inoculum treatment tank, and an outlet tank connected in sequence. Each tank has an effective volume of 3L. The inlet tank is used to add nutrients to adjust the water quality and to hold the incoming water; it is equipped with a stirring system at the bottom. The microbial inoculum treatment tank is a biochemical treatment tank for microorganisms, and it is equipped with an aeration system at the bottom. Wastewater in the inlet tank is pumped to the microbial inoculum treatment tank using a peristaltic pump. The outlet tank is used to collect the treated wastewater.

[0063] Initially, clean water was used for startup, supplemented with KH₂PO₄ and (NH₄)₂SO₄ as phosphorus and inorganic nitrogen sources to aid bacterial growth. Packing material was added to one-third of the volume of the bacterial treatment tank to retain the bacteria and reduce loss. Seed culture was inoculated into low-salinity simulated wastewater at a 30% inoculum rate, stirred thoroughly, and then introduced into the experimental apparatus. The dissolved oxygen (DO) content in the bacterial treatment tank was controlled at 2-4 mg / L, pH at 6.5-7.0, temperature at 30-35℃, and hydraulic retention time (HRT) at 6 days. In the continuous influent / effluent system, each tank was covered to reduce evaporation. Basic water quality tests were performed daily on the effluent. Once the effluent quality stabilized, the average values ​​of COD, NH₃-N, organic nitrogen, and total nitrogen (TN) in the effluent were calculated for two weeks after stabilization. The results are shown in Table 8.

[0064] Table 8 Water ingress Outflow (mg / L) Removal rate (%) COD 25631 4953 80.7 <![CDATA[NH3-N]]> 48 509 - Organic nitrogen 1571 731 53.5 TN 1619 1240 23.4 The results showed that the compound microbial community had good tolerance and degradation effect on high concentration (10000 mg / L) fluoroquinolone antibiotics under low salt conditions. The removal rate of COD reached 72.9% within 6 days, the removal rate of organic nitrogen reached 53.5%, and the removal rate of total nitrogen reached 23.4%.

[0065] (4) Simulation experiment of fluoroquinolone antibiotic wastewater under high salinity conditions The low-salinity simulated wastewater was replaced with high-salinity simulated wastewater, and the simulation experiment was conducted using the same method. Basic water quality tests were performed on the effluent daily. After the effluent water quality stabilized, the average values ​​of COD, NH3-N, organic nitrogen, and total nitrogen (TN) in the effluent were calculated for two weeks after stabilization. The results are shown in Table 9.

[0066] Table 9 Influent (mg / L) Outflow (mg / L) Removal rate (%) COD 25535 15347 60.1 <![CDATA[NH3-N]]> 45 306 - Organic nitrogen 1521 995 34.5 TN 1589 1301 18.1 The results showed that the compound microbial community had good tolerance and degradation effect on high concentration (10000 mg / L) fluoroquinolone antibiotics under high salt conditions. The removal rate of COD reached 60.1% within 6 days, the removal rate of organic nitrogen reached 34.5%, and the removal rate of total nitrogen reached 18.1%.

[0067] Example 6: The treatment capacity of compound bacterial agent for fluoroquinolone antibiotic wastewater (1) Wastewater Sources Wastewater from a factory in Zhejiang Province that produces fluoroquinolone antibiotics contains fluoroquinolone antibiotic substances, mainly ciprofloxacin, enrofloxacin, and clindamycin. The wastewater quality is shown in Table 10.

[0068] Table 10 COD <![CDATA[NH3-N]]> TP <![CDATA[NO2-N]]> <![CDATA[NO3-N]]> Organic nitrogen TN TDS pH 45121mg / L 152mg / L 4mg / L 0mg / L 0mg / L 2799mg / L 2951mg / L 2.1％ 8.5 (2) Wastewater nutrient adjustment Add 0.5 g / L KH2PO4 and 1 mL / L trace element solution (formulation same as in Example 1) to the raw wastewater, adjust the pH to 7.0, and complete the nutrient conditioning of the wastewater to obtain the conditioned wastewater.

[0069] (3) Preparation of compound microbial agent seed liquid Same as Example 4.

[0070] (4) Wastewater treatment test simulation To simulate engineering conditions, optimize the static test treatment effect, and explore the optimal treatment capacity of the microbial community, a continuous influent and effluent dynamic experiment was conducted. The experimental procedure was the same as in Example 5, with the influent being prepared wastewater. Basic water quality tests were performed on the effluent daily. Once the effluent water quality stabilized, the average values ​​of COD, NH4-N, organic nitrogen, and total nitrogen (TN) in the effluent were calculated for two weeks after stabilization. The results are shown in Table 11.

[0071] Table 11 Influent (mg / L) Outflow (mg / L) Removal rate (%) COD 45121 10500 72.4 <![CDATA[NH3-N]]> 152 452 - Organic nitrogen 2647 1217 54.0 TN 2799 1669 40.4 The results showed that the composite microbial community had a good treatment effect on fluoroquinolone antibiotic wastewater and could effectively degrade fluoroquinolone antibiotics such as ciprofloxacin, enrofloxacin, and clinfloxacin.

[0072] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0073] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A Pseudomonas strain capable of degrading fluoroquinolone antibiotics, characterized in that: Named 11-10, with accession number CGMCC No. 33680, and microbiologically classified as *Pseudomonas* (… Pseudomonas sp. The depositary institution is the China General Microbiological Culture Collection Center, and the deposit date is February 28, 2025.

2. A compound bacterial agent capable of degrading fluoroquinolone antibiotics, characterized in that: include: The Pseudomonas, Bacillus tekirae, and Bacillus desertis as described in claim 1.

3. The compound microbial agent according to claim 1, characterized in that: The *Bacillus tekirae* strain was named H21, with accession number CGMCC No. 19227, and its microbiological classification was *Bacillus tekirae* (…). Bacillus tequilensis The depositary institution is the China General Microbiological Culture Collection Center, and the deposit date is December 23, 2019.

4. The compound microbial agent according to claim 1, characterized in that: The *Bacillus desertis* was named DFB2-6, with accession number CGMCC No. 20535, and its microbiological classification was *Bacillus desertis* (…). Paenibacillus harenae The depositary institution is the China General Microbiological Culture Collection Center, and the deposit date is August 21, 2020.

5. The compound microbial agent according to any one of claims 2-4, characterized in that: In the compound bacterial agent, the ratio of viable bacteria of Pseudomonas, Bacillus tekirae, and Bacillus desertis is 1:0.2-1.5:0.2-1.

5.

6. The compound microbial agent according to claim 5, characterized in that: In the compound bacterial agent, the ratio of viable bacteria of Pseudomonas, Bacillus tekirae, and Bacillus desertis is 1:0.5:0.

6.

7. The compound microbial agent according to any one of claims 2-4, characterized in that: The compound microbial agent is a liquid or solid microbial agent.

8. A biological agent capable of degrading fluoroquinolone antibiotics, characterized in that: It contains a whole fermentation broth or intracellular substance extract prepared from the Pseudomonas aeruginosa of claim 1 or the compound bacterial agent of any one of claims 2-7.

9. The use of the Pseudomonas aeruginosa of claim 1, the compound bacterial agent of any one of claims 2-7, or the biological agent of claim 8 in the degradation of fluoroquinolone antibiotics.

10. The application according to claim 9, characterized in that: The fluoroquinolone antibiotics mentioned are fluoroquinolone antibiotics found in wastewater.

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