Strain of heterotrophic nitrification-aerobic denitrification pseudomonas zzu-1 capable of degrading antibiotics, microbial inoculant and application

Abstract

The present application relates to the technical field of microbial engineering, and particularly relates to a heterotrophic nitrification-aerobic denitrification Pseudomonas azurina ZZU-1 strain capable of degrading antibiotics, a microbial inoculum and application. The present application isolates and screens a Pseudomonas azurina having the ability of simultaneous denitrification and terramycin degradation from a well-operated artificial wetland sediment, and is classified and named as Pseudomonas anuradhapurensis , and is preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC No. 37130. Experimental results show that the strain not only has high efficient simultaneous denitrification ability, but also has good terramycin removal effect. The Pseudomonas azurina strain and the microbial inoculum provided by the present application are expected to be used for denitrification of wastewater containing antibiotics, and can simultaneously remove antibiotics, and have important practical significance for breaking through the biological treatment bottleneck of composite contaminated wastewater.

Description

Technical Field

[0001] This invention relates to the field of microbial engineering technology, specifically to a heterotrophic nitrifying-aerobic denitrifying Pseudomonas ZZU-1 strain that can degrade antibiotics, microbial agents, and their applications. Background Technology

[0002] High-nitrogen wastewater and antibiotic residue wastewater generated during intensive agricultural production in aquaculture, pharmaceutical manufacturing, and other industries have become a prominent challenge in water pollution control. In this type of wastewater, high concentrations of nitrogen (mainly ammonia nitrogen and nitrate nitrogen) coexist with antibiotics such as oxytetracycline, forming a complex pollution system. Oxytetracycline, a typical broad-spectrum antibiotic, has a long residual period under natural conditions and exhibits strong biotoxicity to microbial communities. It can significantly inhibit or even destroy the activity of traditional denitrifying bacteria, leading to a situation where the system suffers from "sufficient substrate but suppressed biological function."

[0003] Currently, the treatment of high-nitrogen-antibiotic complex wastewater mainly relies on stepwise processes, namely, pretreatment using physicochemical methods (such as adsorption and coagulation) followed by biological processes (such as activated sludge processes and nitrification-denitrification systems) to achieve denitrification. However, these methods have certain limitations: the process flow is lengthy, and the construction and operation costs are high; moreover, antibiotic residues continuously inhibit the functional microorganisms in the biological unit, easily leading to system instability or even failure. Although emerging technologies such as advanced oxidation and nanocatalysis have shown potential in antibiotic degradation, their simultaneous denitrification effect in complex wastewater environments is generally poor, and there are byproduct risks and cost issues.

[0004] At the microbial technology level, heterotrophic nitrifying-aerobic denitrifying bacteria have attracted much attention because they can simultaneously achieve nitrogen removal and organic matter degradation. However, most of the existing strains have poor tolerance to antibiotics such as oxytetracycline, and their activity usually decreases significantly when the concentration exceeds 1 mg / L. At the same time, strains with both high-efficiency nitrogen removal and oxytetracycline degradation capabilities are still relatively scarce, and their applicability in actual wastewater has not been verified. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a heterotrophic nitrifying-aerobic denitrifying Pseudomonas ZZU-1 strain capable of degrading antibiotics, a microbial agent, and its applications. The ZZU-1 strain provided by this invention not only achieves efficient simultaneous denitrification in nitrogen-containing wastewater but also maintains high metabolic activity under antibiotic stress and efficiently degrades tetracycline antibiotics (such as oxytetracycline), truly achieving "dual effects with one strain," and can be used in nitrogen-containing water bodies contaminated with antibiotics.

[0006] To achieve the above objectives, the specific technical solution of the present invention is as follows: The first aspect of this invention provides a heterotrophic nitrifying-aerobic denitrifying Pseudomonas ZZU-1 strain capable of degrading antibiotics, which is classified and named as follows: Pseudomonas anuradhapurensis It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 37130.

[0007] Furthermore, the bacterial characteristics of the ZZU-1 strain are as follows: Gram-negative, rod-shaped cells, no budding, and flagella; yellow colonies, round in shape, and smooth in texture; flat and dull colonies.

[0008] Furthermore, the ZZU-1 strain possesses highly efficient heterotrophic nitrification-aerobic denitrification capabilities.

[0009] A second aspect of the present invention provides a microbial inoculant containing the ZZU-1 strain.

[0010] Furthermore, the microbial agent is a liquid or a powder.

[0011] Furthermore, the effective viable count of ZZU-1 strain per milliliter or per gram of the microbial agent is ≥5.7 × 10⁻⁶. 6 CFU / mL.

[0012] A third aspect of this invention provides a method for preparing a microbial inoculant, comprising the following steps: (1) The ZZU-1 strain was inoculated into liquid culture medium and fermented at 20℃~32℃ and 120 r / min~160 r / min for 24 h~120 h to obtain fermentation broth; (2) Centrifuge the fermentation broth to obtain a bacterial precipitate, and resuspend the bacterial precipitate in sterile water to obtain a microbial agent.

[0013] Furthermore, the liquid culture medium is LB liquid culture medium or tryptone soybean broth (TSB) liquid culture medium.

[0014] The fourth aspect of this invention provides the application of ZZU-1 strain or microbial agent in nitrogen-containing water bodies with or without antibiotic contamination.

[0015] Furthermore, its application in nitrogen-containing water bodies free from antibiotic pollution involves adding ZZU-1 strain or microbial agent to nitrogen-containing water bodies free from antibiotic pollution, along with a carbon source, and culturing at 20-30℃ and 100-160 rpm for 24-72 h.

[0016] Furthermore, the nitrogen source in the nitrogen-containing water body is ammonium nitrogen and / or nitrate nitrogen.

[0017] Furthermore, the nitrogen source concentration in the nitrogen-containing water body is 20~250 mg / L, and the ZZU-1 strain or microbial agent is inoculated into the water body at a rate of 1%-5% of the total volume of the nitrogen-containing water body.

[0018] Furthermore, the carbon source is sodium citrate or sodium succinate, with a C / N ratio of 6 to 20, and the pH of the nitrogen-containing water is adjusted to 5.0 to 9.0.

[0019] Furthermore, its application in nitrogen-containing water bodies contaminated with antibiotics involves adding ZZU-1 strain or microbial agents to the nitrogen-containing water bodies contaminated with antibiotics, along with a carbon source, and culturing at 20-30℃ and 100-160 rpm for 24-72 h.

[0020] Furthermore, the antibiotic is a tetracycline antibiotic.

[0021] Furthermore, the tetracycline antibiotics include, but are not limited to, oxytetracycline, preferably oxytetracycline.

[0022] Furthermore, the nitrogen source is a mixed nitrogen source of ammonium nitrogen and nitrate nitrogen, with a concentration of 20~250 mg / L.

[0023] Furthermore, the carbon source is sodium citrate or sodium succinate, with a C / N ratio of 6 to 20, and the pH of the water is adjusted to 5.0 to 9.0.

[0024] Furthermore, when the concentration of oxytetracycline in the nitrogen-containing water body contaminated with antibiotics is 1-20 mg / L, the ZZU-1 strain or microbial agent is inoculated into the water body at a rate of 1%-5% of the total volume of the nitrogen-containing water body.

[0025] In nitrogenous water containing 15 mg / L oxytetracycline, with initial concentrations of ammonia nitrogen and nitrate nitrogen of 50 mg / L and total nitrogen concentration of 100 mg / L, the ZZU-1 strain of this invention was inoculated at a 5% (v / v) inoculum and cultured at 25℃ and 140 r / min for 32 h. The total nitrogen removal rate reached 90.2%, and the residual concentration of oxytetracycline was reduced to 1.71 μg / mL, with a removal rate of 88.6%.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The *Pseudomonas* strain ZZU-1 of this invention can simultaneously and efficiently denitrify under stress ranging from 0 to 20 mg / L oxytetracycline. After a 32-hour culture period, strain ZZU-1 achieved a 95.8% nitrate removal rate in aerobic denitrification medium (nitrate nitrogen as the sole nitrogen source); an 82% ammonia nitrogen removal rate in heterotrophic nitrification medium (ammonia nitrogen as the sole nitrogen source); and a 91.5% total nitrogen removal rate in simultaneous nitrification medium (ammonia and nitrate nitrogen as mixed nitrogen sources), with only a 1.7% accumulation of nitrite nitrogen, showing a significant improvement compared to the control (CK).

[0027] 2. The Pseudomonas ZZU-1 strain in this invention has the ability to remove antibiotics. In particular, after inoculating nitrogenous wastewater containing oxytetracycline with Pseudomonas ZZU-1, it can significantly reduce the concentration of oxytetracycline in the water while simultaneously removing nitrogen from the wastewater.

[0028] 3. The Pseudomonas ZZU-1 strain and microbial agent provided by this invention have the ability to simultaneously denitrify and degrade antibiotics in nitrogen-containing wastewater contaminated with antibiotics, and have important application potential. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a plate image of Pseudomonas ZZU-1 strain cultured on LB solid medium.

[0031] Figure 2 Phylogenetic tree of Pseudomonas ZZU-1 strain.

[0032] Figure 3 The denitrification efficiency of Pseudomonas ZZU-1 strain when ammonia nitrogen is the only nitrogen source.

[0033] Figure 4 The denitrification efficiency of Pseudomonas ZZU-1 strain when nitrate nitrogen is the only nitrogen source.

[0034] Figure 5 The denitrification efficiency of Pseudomonas ZZU-1 strain under mixed nitrogen sources.

[0035] Figure 6 This is a standard for oxytetracycline hydrochloride.

[0036] Figure 7 The curve of oxytetracycline removal by Pseudomonas ZZU-1 strain during simultaneous denitrification is shown. Detailed Implementation

[0037] The *Pseudomonas anuratus* strain ZZU-1 in this invention is classified and named as follows: Pseudomonas anuradhapurensis It was deposited on December 22, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37130. The address of the depository is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0038] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific 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. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0039] The heterotrophic nitrification medium formula in this invention is as follows: sodium citrate 4.5 g, (NH4)2SO4 0.5 g, K2HPO4 0.8 g, MgSO4·7H2O 0.5 g, NaCl 0.4 g, FeSO4·7H2O 0.05 g, CaCl2·H2O 0.06 g, distilled water 1 L, trace element solution 1 mL, and pH adjusted to 7.0-7.2.

[0040] The formulation of the aerobic denitrification medium is as follows: 5.5 g sodium citrate, 0.78 g NaNO3 (nitric acid medium) or 0.61 g NaNO2 (nitrite medium), 0.8 g K2HPO4, 0.4 g MgSO4·7H2O, 0.4 g NaCl, 0.04 g FeSO4·7H2O, 0.03 g CaCl2·H2O, 1 L distilled water, 1 mL trace element solution, and pH adjusted to 7.0-7.2.

[0041] The formulation of bromothymol blue (BTB) solid medium is as follows: 0.2 wt% bromothymol blue (1% by volume, dissolved in anhydrous ethanol) and 2 wt% agar powder are added to nitric acid medium.

[0042] The formulation of the simultaneous nitrification and denitrification medium is as follows: sodium citrate 4.6 g, (NH4)2SO4 0.25 g, NaNO3 0.32 g, K2HPO4 0.8 g, MgSO4·7H2O 0.4 g, NaCl 0.4 g, FeSO4·7H2O 0.04 g, CaCl2·H2O 0.03 g, distilled water 1 L, trace element solution 1 mL, and pH adjusted to 7.0-7.2.

[0043] The formula for the trace element solution is: H3BO3 1.5 g, ZnSO4·7H2O 0.06 g, MnSO4·H2O 0.8 g, CuSO4·5H2O 0.1 g, CoSO4·7H2O 0.01 g, (NH4)6Mo7O 24 ·4H2O 0.2 g, KI 0.18 g, distilled water 1 L.

[0044] The formula for the culture medium for acclimatizing activated sludge is as follows: sodium citrate 4.73 g, NH4Cl 0.31 g, KNO3 1 g, KH2PO4 0.15 g, MgSO4·7H2O 0.1 g, FeSO4·7H2O 0.006 g; trace elements 1 mL.

[0045] High-nitrogen and antibiotic-residual wastewater discharged from industries such as aquaculture and pharmaceuticals is one of the water treatment challenges facing my country. Antibiotics such as oxytetracycline in the wastewater significantly inhibit conventional biological treatment bacteria, creating a paradoxical situation of "sufficient substrate but functional inhibition." While existing stepwise treatment processes can partially alleviate the problem, they are lengthy, costly, and carry the risk of secondary pollution. Therefore, this invention aims to provide a heterotrophic nitrifying-aerobic denitrifying bacterial agent that maintains high metabolic activity under the aforementioned combined stress conditions, simultaneously achieving efficient denitrification and antibiotic degradation, thus potentially overcoming the bottleneck in the biological treatment of combined polluted wastewater. Example 1: Isolation, screening and identification of Pseudomonas ZZU-1 strain

[0046] S1. Obtain water samples Water samples were collected from a stable and efficient constructed wetland system.

[0047] S2. Acclimation and enrichment of microorganisms in water samples An acclimatization medium was prepared using a mixture of ammonia and nitrate nitrogen as the nitrogen source (initial total nitrogen concentration 50 mg / L), with the addition of oxytetracycline hydrochloride (5 mg / L). Water samples were inoculated into the medium at a 5% (v / v) inoculum and cultured at 25℃ and 140 rpm for 72 h with shaking to complete the initial acclimatization. Subsequently, a concentration gradient acclimatization was performed: the total nitrogen concentration (50-300 mg / L) and the oxytetracycline hydrochloride concentration (5-20 mg / L) in the medium were gradually increased. The previous generation of acclimatized bacterial culture was transferred to the new generation medium at a 5% (v / v) inoculum, and multiple subculture cycles were conducted under the same conditions. During each acclimatization cycle, the concentrations of ammonia and nitrate nitrogen were monitored regularly. Only when the degradation rate of both was ≥80% could the culture be transferred to a higher concentration medium. Acclimatization was considered successful when the bacterial population could no longer tolerate higher concentrations and the last effective acclimatization gradient of bacterial culture was preserved.

[0048]

[0049] S3. Isolation and purification of heterotrophic nitrifying-aerobic denitrifying bacteria After the bacterial suspension has been acclimatized, it was inoculated at a concentration of 5% into LB liquid medium containing sterile oxytetracycline hydrochloride solution (oxytetracycline hydrochloride concentration of 10 mg / L). The medium was shaken at 25°C and 140 r / min for 36 h. This process was repeated three times to obtain the final bacterial suspension. 1 mL of the bacterial suspension was then diluted with sterile deionized water at a concentration gradient of 10... -4 10 -5 10 -6 and 10 -7 Dilute the bacterial culture by 1:1, and evenly spread 300 μL of each diluted bacterial solution onto BTB solid medium containing oxytetracycline hydrochloride (oxytetracycline hydrochloride concentration: 10 mg / L). Invert the diluted plates and incubate at 28℃ for 36 h, observing for colony growth. After colony growth, observe the plates, select colonies with different morphological characteristics such as morphology, size, color, and texture that show a blue ring around the colony, and inoculate them onto BTB solid medium containing oxytetracycline hydrochloride using an inoculation loop. Then, isolate and purify the colonies using the streak plate method until single colonies are obtained and preserve them.

[0050] S4. Screening of heterotrophic nitrifying-aerobic denitrifying bacteria Nessler's reagent and nitrite colorimetric reagent were added to the single colony plate obtained in culture step S3 to identify whether the obtained strain had denitrification ability. One drop of Nessler's reagent and one drop of nitrite colorimetric reagent were added to the colony growth area and the blank area on the plate, respectively, and the colors were compared. Based on the color intensity and the size of the colony, a preliminary estimate of the heterotrophic nitrification-aerobic denitrification ability of the strain was made.

[0051] S5. Identification of bacterial strains Plate observation: The selected strains are cultured on LB plates, and the color, shape and other characteristics of the colonies are observed.

[0052] like Figure 1 As shown, when the strain was cultured on LB solid medium at 27°C for 32 h, its colonies were yellow in color, round in shape, and smooth in texture; the colonies were flat and dull.

[0053] 16S rDNA sequencing of strains: DNA from strains was extracted and sequenced as 16S rDNA. BLAST alignment was used to obtain sequences from related strains. Sequences with high homology were selected, and a phylogenetic tree was constructed (e.g.,...). Figure 2 (As shown).

[0054] Based on comprehensive morphological observation and 16S rDNA sequence analysis, this strain can be identified as *Pseudomonas anurata*. Pseudomonas anuradhapurensis The strain was named Pseudomonas ZZU-1. Example 2: Determination of the simultaneous denitrification capacity of Pseudomonas ZZU-1 strain

[0055] Under aseptic conditions in the laboratory, a loopful of colonies was taken from an agar plate inoculated with Pseudomonas ZZU-1 and inoculated into an Erlenmeyer flask containing 100 mL of sterilized LB broth containing 10 mg / L oxytetracycline hydrochloride. The flask was incubated at 25°C and 140 rpm with shaking until the bacterial suspension reached an OD600 of 0.8–1.0. The bacterial suspension was then inoculated at a rate of 5% (v / v) into heterotrophic nitrification medium, aerobic denitrification medium, and simultaneous nitrification-denitrification medium, using ammonia nitrogen, nitrate nitrogen as the sole nitrogen source, and mixed nitrogen sources, respectively. The corresponding medium without microorganisms served as a control. The flasks were incubated at 25°C and 140 rpm with shaking for 24–72 h, with three replicates per group. During the cultivation process, the concentrations of ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen in the culture medium were measured every 8 hours using Nessler's reagent spectrophotometry, ultraviolet spectrophotometry, and N-(1-naphthyl)-ethylenediamine spectrophotometry to observe the synchronous denitrification capacity.

[0056] The simultaneous denitrification ability of Pseudomonas ZZU-1 strain is as follows: Figure 3 , Figure 4 and Figure 5 As shown, under the condition of only 32 h of cultivation time, the removal rate of nitrate nitrogen in the aerobic denitrification medium (nitrate nitrogen as the sole nitrogen source) reached 95.8%; the removal rate of ammonia nitrogen in the heterotrophic nitrification medium (ammonia nitrogen as the sole nitrogen source) reached 82%; and the removal rate of total nitrogen in the simultaneous nitrification medium (ammonia nitrogen and nitrate nitrogen as mixed nitrogen sources) reached 91.5%, with nitrite nitrogen accumulation of only 1.7%. This demonstrates that the strain ZZU-1 of the present invention has a highly efficient simultaneous nitrogen removal capability. Example 3: Determination of the antibiotic removal ability of Pseudomonas ZZU-1 strain

[0057] To verify the efficiency of Pseudomonas ZZU-1 strain in removing antibiotics while efficiently denitrifying, this example specifically determined its ability to simultaneously remove nitrogen sources and oxytetracycline in aqueous solution. The concentration of oxytetracycline was detected using high-performance liquid chromatography-tandem mass spectrometry (LC-MS).

[0058] S1. Preparation of bacterial suspension Under aseptic conditions, a single colony of *Pseudomonas ZZU-1* strain was inoculated from a plate containing 100 mL of sterile LB broth (without any antibiotics or nitrogen source) into a 250 mL Erlenmeyer flask. The flask was incubated at 25°C and 140 rpm with shaking until the late logarithmic growth phase (OD600 approximately 0.8-1.0) to obtain a concentrated bacterial suspension. The suspension was centrifuged at 4°C and 8000 rpm for 10 min, the supernatant was discarded, and the cells were washed twice with sterile physiological saline. The suspension was then resuspended in sterile physiological saline and the OD600 was adjusted to approximately 1.0 to prepare the inoculation suspension.

[0059] S2. Experimental Design for Simultaneous Denitrification and Oxytetracycline Removal Prepare a basic inorganic salt culture medium using ammonia nitrogen and nitrate nitrogen as a mixed nitrogen source (initial concentration of 50 mg / L for both, total nitrogen 100 mg / L). Adjust the initial pH of the medium to 7.0 ± 0.2 with 1 M NaOH or 1 M HCl, and autoclave at 121°C for 20 min. Under aseptic conditions, after the medium has cooled, add sterilized oxytetracycline hydrochloride standard filtered through a 0.22 μm filter membrane. Figure 6 As shown, its initial concentration was set at 15 mg / L.

[0060] Take 100 mL of the culture base containing oxytetracycline hydrochloride and mixed nitrogen source into a 250 mL Erlenmeyer flask, inoculate it with the prepared bacterial suspension prepared in step S1 at an inoculation rate of 5% (v / v), place the Erlenmeyer flask in a constant temperature shaker, and carry out shaking culture at 25℃ and 140 r / min.

[0061] S3. Sample Collection and Pretreatment At 0 h, 8 h, 16 h, 24 h, and 32 h after the start of culture, 5 mL samples were aseptically collected. The samples were immediately centrifuged at 4 °C and 12000 r / min for 10 min, and the supernatant was collected and filtered through a 0.22 μm aqueous filter membrane for pretreatment.

[0062] Oxytetracycline sample pretreatment: The supernatant of the filtered water sample was centrifuged at 4000 rpm for 10 min at room temperature. The solid-phase extraction column was then activated, and 100 mL of the centrifuged supernatant was loaded onto the sample. The sample was eluted twice with 5 mL of methanol, and the eluates were combined. The eluates were dried under a gentle nitrogen stream, reconstituted with 1.0 mL of the initial mobile phase (95% 0.1% formic acid and 2 mM ammonium acetate aqueous solution / 5% acetonitrile), vortexed, filtered through a 0.22 μm filter membrane, and transferred to a vial for LC-MS / MS analysis. A separate sample was also prepared for the determination of total nitrogen removal.

[0063] S4. Analytical Methods Nitrogen concentration determination: Ammonia nitrogen concentration was determined by Nessler's reagent spectrophotometry, nitrite nitrogen concentration was determined by N-(1-naphthyl)-ethylenediamine spectrophotometry, and nitrate nitrogen concentration was determined by ultraviolet spectrophotometry. Total nitrogen removal rate was calculated.

[0064] Oxytetracycline concentration determination: High performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used. Chromatographic conditions were as follows: a C18 column (2.7 μm, 3.0 mm * 75 mm) was used at a column temperature of 40℃; mobile phase A consisted of 0.1% formic acid and 2 mM ammonium acetate aqueous solution, and mobile phase B consisted of acetonitrile; the flow rate was 400 μL / min; the injection volume was 5 μL; gradient elution was used with the following program: 0–0.5 min, phase A 95%; 0.5–5.5 min, phase A linearly decreased from 95% to 55%; 5.5–6.0 min, phase A maintained at 55%; 6.1–8.0 min, phase A restored to 95% and equilibrated; the total run time was 8 min. The mass spectrometry conditions were as follows: electrospray ionization (ESI) source, positive ion mode; ion source voltage 5500 V, temperature 550℃; curtain gas 35 psi, spray gas 50 psi, auxiliary heating gas 50 psi; collision gas was medium; detection mode was multiple reaction monitoring (MRM). The monitoring ion pairs and parameters for oxytetracycline hydrochloride were: quantitative ion pair m / z 461.1→426.0, declustering voltage (DP) 52 V, collision energy (CE) 29 eV; qualitative ion pair m / z 461.1→443.1, DP 52 V, CE 19 eV. Quantification was performed using the external standard method, with standard curve concentrations of: 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200 ng / mL.

[0065] S5. Results and Analysis The removal efficiency of Pseudomonas ZZU-1 strain for oxytetracycline during simultaneous denitrification is as follows: Figure 7 As shown.

[0066] During the culture period, the mixed nitrogen source (ammonia nitrogen + nitrate nitrogen) in the experimental group was efficiently removed, and the total nitrogen removal trend was consistent with the results of Example 2, reaching 90.2% at 32 h. Meanwhile, LC-MS / MS analysis showed a significant decrease in the concentration of oxytetracycline in the culture medium. The initial concentration of oxytetracycline hydrochloride was 15 mg / L, and after 32 h of culture, its residual concentration decreased to 1.71 μg / mL, achieving a removal rate of 88.6%.

[0067] The above results demonstrate that the Pseudomonas ZZU-1 strain of the present invention can not only achieve efficient and simultaneous denitrification in a system with ammonia nitrogen and nitrate nitrogen as mixed nitrogen sources, but also simultaneously and efficiently degrade oxytetracycline, a typical tetracycline antibiotic, proving its great potential for treating wastewater containing both nitrogenous pollutants and antibiotics.

[0068] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0069] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A heterotrophic nitrifying-aerobic denitrifying Pseudomonas ZZU-1 strain capable of degrading antibiotics, characterized in that, This strain is classified as... Pseudomonas anuradhapurensis It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 37130.

2. The strain according to claim 1, characterized in that, The bacterial characteristics of the ZZU-1 strain are as follows: Gram-negative, rod-shaped cells, no budding, and flagella; yellow colonies, round in shape, and smooth in texture; flat and dull colonies.

3. A microbial inoculant, characterized in that, The microbial agent contains the ZZU-1 strain.

4. The microbial agent according to claim 3, characterized in that, The effective viable count of ZZU-1 strain per milliliter or per gram of the microbial agent is ≥5.7 × 10⁻⁶. 6 CFU / mL.

5. The microbial agent according to claim 3, characterized in that, The method for preparing the microbial inoculant includes the following steps: (1) The ZZU-1 strain was inoculated into liquid culture medium and fermented at 20℃~32℃ and 120 r / min~160 r / min for 24 h~120 h to obtain fermentation broth; (2) Centrifuge the fermentation broth to obtain a bacterial precipitate, and resuspend the bacterial precipitate in sterile water to obtain a microbial agent.

6. The use of the strain described in claim 1 or 2 or the microbial agent described in any one of claims 3-5 in nitrogen-containing water bodies with or without antibiotic contamination.

7. The application according to claim 6, characterized in that, Add ZZU-1 strain or microbial agent to nitrogen-containing water bodies with or without antibiotic contamination, along with a carbon source, and incubate at 20-30℃ and 100-160 rpm for 24-72 h.

8. The application according to claim 7, characterized in that, The nitrogen source in the nitrogen-containing water body is ammonium nitrogen and / or nitrate nitrogen.

9. The application according to claim 7, characterized in that, The ZZU-1 strain or microbial agent is inoculated into the water body at a rate of 1%-5% of the total volume of nitrogen-containing water.

10. The application according to claim 7, characterized in that, The antibiotics in question are tetracycline antibiotics.

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