Antibiotic-resistant marine heterotrophic ammonia-assimilating-aerobic denitrifying bacteria and application thereof

Abstract

A strain of antibiotic-resistant marine heterotrophic ammonia assimilation-aerobic denitrifying bacteria and its application, belongs to the field of water treatment of aquaculture. The seawater antibiotic-resistant high-efficiency nitrogen removal Marinobacter nitratireducens LAN01 can use polyhydroxyalkanoate as the sole carbon source, and can realize heterotrophic ammonia assimilation and aerobic denitrification function under the stress of different concentrations of sulfamethoxazole antibiotics, and simultaneously transform phosphate. The strain has multiple resistances such as high salinity, high alkalinity and antibiotic, and can be used for seawater aquaculture effluent treatment, seawater recirculating aquaculture water treatment, high-salinity wastewater treatment and saline-alkali soil bioremediation, etc. To realize the simultaneous recovery of nitrogen, phosphorus and other nutrients in the seawater aquaculture environment.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture water treatment technology and ecological environment governance and restoration technology, specifically involving an antibiotic-resistant marine heterotrophic ammonia assimilation-aerobic denitrification bacterium and its application. Background Technology

[0002] Intensive mariculture wastewater typically contains high concentrations of inorganic nitrogen and phosphate, and is further influenced by the use of antibiotics during the aquaculture process. This not only causes eutrophication but also promotes the widespread dissemination of antibiotics and resistance genes. Sulfonamide antibiotics, represented by sulfamethoxazole (SMX), are the main antibiotics used in mariculture. They have high water solubility and a low octanol-water partition coefficient, making them more likely to remain in the aquaculture water for extended periods and pose sustained ecological risks. Biofloc technology has been proven to remove both ammonia nitrogen and phosphate under heterotrophic conditions. Biofloc technology is mainly used in in-situ aquaculture or as a component of recirculating aquaculture systems. By introducing a carbon source to adjust the carbon-to-nitrogen ratio in the water, it promotes the rapid assimilation of ammonia nitrogen into bacterial protein by heterotrophic bacteria. Ammonia assimilation does not involve the complex nitrification-denitrification process; it directly assimilates ammonia nitrogen and phosphate produced by fish into bioflocs, thereby achieving simultaneous recovery of nutrients such as nitrogen and phosphorus. This avoids the large-scale emission of greenhouse gases (N2O) during nitrification and denitrification, making it a low-carbon and environmentally friendly nitrogen recovery technology.

[0003] However, ammonia-assimilating bacteria are generally sensitive to most antibiotics, resulting in poor shock resistance and low microbial activity in practical seawater aquaculture water treatment applications, thus reducing the system's nitrogen and phosphorus removal efficiency. The salt-tolerant and antibiotic-resistant heterotrophic ammonia-assimilating bacteria screened in this patent exhibit excellent resistance to antibiotic-induced water quality shock loads and can effectively perform ammonia assimilation under various seawater aquaculture conditions, while simultaneously removing phosphates from the water, providing a new solution for seawater aquaculture water treatment. Summary of the Invention:

[0004] One aspect of this invention is to provide an antibiotic-resistant marine heterotrophic ammonia-assimilating-aerobic denitrifying marine bacterium (Marinobacter nitratireducens) LAN01, which was deposited on November 8, 2024, at the China General Microbiological Culture Collection Center (CGMCC) under the accession name LAN01 and accession number CGMCC No. 32548. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, 100101, China.

[0005] A second aspect of the present invention provides the following biological characteristics of the Marinobacter nitratireducens LAN01:

[0006] (1) After being cultured at 30°C for 48–72 h on nutrient agar medium, smooth, moist, opaque, round, milky-white colonies are formed with a diameter of 1–2 mm.

[0007] (2) Marinobacter nitratireducens LAN01 cells are Gram-negative, rod-shaped, 0.3–0.5 μm wide × 1.0–2.0 μm long. Figure 2 );

[0008] (2) It can grow on liquid culture media with one or more of NH4Cl, NaNO2 and KNO3 as nitrogen sources;

[0009] (3) The growth temperature of Marinobacter nitratireducens LAN01 is 15-40℃, the optimal growth temperature is 30℃, the required salinity is 2-4%, the required pH is 7.5-9.5, the required dissolved oxygen is 1-6 mg / L, and the rotation speed is 180-200 r / min.

[0010] (4) 16S rRNA gene sequence

[0011] Whole-genome sequencing was performed on *Marinobacter nitratireducens* LAN01, and its 16S rRNA gene sequence was extracted and BLAST compared. The results showed that the 16S rRNA gene sequence of *Marinobacter nitratireducens* LAN01 was highly similar to that of *Marinobacter nitratireducens* strain AK21. Figure 1 As shown. However, previous reports on Marinobacter nitratireducens, including Marinobacter nitratireducens strain AK21, only reported its nitrate-reducing ability, but none reported its ability to assimilate antibiotic-resistant heterotrophic ammonia, remove phosphorus, or potentially degrade antibiotics. Therefore, it can be determined that this marine bacterium (Marinobacter nitratireducens) LAN01 is a novel strain in the Marinobacter nitratireducens species.

[0012] A third aspect of this invention provides a method for screening this antibiotic-resistant, highly efficient seawater denitrification and phosphorus removal strain, the screening method comprising the following steps:

[0013] a) Isolation of strains: Bioflocs from a biofilter using polyhydroxyalkanoate (PHA) as a solid carbon source were filtered through a 200-mesh screen, and a bacterial suspension was prepared using sterile seawater and transferred to a bacterial selective medium (with SMX) for screening until the ammonia nitrogen degradation rate exceeded 80% after 24 hours. The culture was then continued for two cycles (each cycle corresponding to 24 hours of culture in the bacterial selective medium), and the strains were purified.

[0014] b) Strain purification: Take 1 mL (OD) of the selected and acclimatized bacterial culture medium. 600 =0.4), diluted with sterile PBS solution, for example, diluted 10. 3 -10 6 Then, the bacteria are streaked onto solid culture medium and cultured at a constant temperature of 30°C for 48–72 hours. After the bacteria have grown on the culture medium, single colonies are picked. The picked single colonies are purified 2–3 times. The purification process involves bacterial selection culture medium acclimatization in step a and solid culture medium culture in step b.

[0015] c) The strain purified in b) was inoculated into a bacterial enrichment medium for expansion culture;

[0016] As a preferred embodiment of the present invention, the culture medium is prepared in the following proportions:

[0017] Bacterial selective culture medium: 20 g / L PHA powder, 0.25 g / L ammonium chloride, 0.015 g / L potassium dihydrogen phosphate, 0.035 g / L dipotassium hydrogen phosphate, 0.01 g / L anhydrous manganese sulfate, 0.03 g / L ferrous sulfate heptahydrate, 10 mg / L SMX. The medium is dissolved in ultrafiltered seawater (salinity 3.0%) after autoclaving (e.g., 0.1 MPa for 20 min) and then sterilized again before use.

[0018] Bacterial solid culture medium: 15 g / L agar, 5 g / L sodium citrate dihydrate, 0.25 g / L ammonium chloride, 0.015 g / L potassium dihydrogen phosphate, 0.035 g / L dipotassium hydrogen phosphate, 0.01 g / L anhydrous manganese sulfate, 0.03 g / L ferrous sulfate heptahydrate. The medium is sterilized by autoclaving (e.g., 0.1 MPa for 20 min), then dissolved in ultrafiltered seawater (salinity 3.0%) and sterilized again before use.

[0019] Bacterial enrichment medium: 20 g / L PHA powder, 0.25 g / L ammonium chloride, 0.15 g / L potassium dihydrogen phosphate, 0.35 g / L dipotassium hydrogen phosphate, 0.01 g / L anhydrous manganese sulfate, 0.03 g / L ferrous sulfate heptahydrate, 10 mg / L SMX;

[0020] Preferably, in the application, the bacterial culture medium PHA powder has a particle size of 50 mesh to 200 mesh.

[0021] The application of Marinobacter nitratireducens LAN01 obtained in this invention is for nitrogen and phosphorus removal in marine aquaculture wastewater, especially for nitrogen and phosphorus removal in marine aquaculture wastewater containing antibiotics such as SMX. It can be used for the simultaneous recovery of nutrients such as nitrogen and phosphorus under antibiotic stress conditions; it can convert ammonia nitrogen in marine aquaculture wastewater into bioflocs, achieving efficient carbon-nitrogen coupling treatment and facilitating sedimentation and separation.

[0022] Preferably, in the application, the concentrations of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in the effluent from marine aquaculture are all below 50 mg / L.

[0023] Preferably, in the application, the concentration of phosphate in the effluent from marine aquaculture is below 20 mg / L.

[0024] Preferably, in the application, the concentration of antibiotics such as SMX in the effluent from marine aquaculture is below 20 mg / L.

[0025] Preferably, in the application, the temperature for the denitrification and phosphorus removal process of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in the effluent from marine aquaculture is between 15 and 40°C.

[0026] Preferably, in the application, the pH of the denitrification and phosphorus removal process in the effluent from marine aquaculture is between 7.5 and 9.5.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0028] 1. The present invention screens and isolates a strain of Marinobacter nitratireducens LAN01 with antibiotic-resistant heterotrophic ammonia assimilation-aerobic denitrification ability. It can exhibit good ammonia assimilation performance and assimilate phosphate under different concentrations of SMX stress. It simultaneously reduces nitrite and nitrate nitrogen through denitrification. In actual aquaculture wastewater treatment, it has stable treatment effect and high nitrogen and phosphorus removal efficiency.

[0029] 2. This strain exhibits good tolerance to the antibiotic SMX and has the potential to degrade it. The degradation rate of low concentration SMX (3.0 mg / L) is as high as 93.8%, while the degradation rate of 20 mg / L SMX can reach 29.1%. The main metabolic mechanism is bioabsorption during ammonia assimilation. The higher ammonia nitrogen / SMX ratio promotes the biotransformation of SMX.

[0030] 3. This strain simultaneously absorbs nitrogen and phosphorus via ammonia assimilation, promoting the recovery and utilization of nutrients in marine aquaculture tailwater. It also possesses excellent flocculation and sedimentation capabilities, making it easy to separate and suitable for enhancing nitrogen and phosphorus removal in marine biological filters. It has broad application prospects in the fields of marine aquaculture technology and ecological environment management and restoration. Attached image description:

[0031] Figure 1 Phylogenetic tree diagram of strain Marinobacter nitratireducens_LAN01;

[0032] Figure 2 Scanning electron microscope image of strain Marinobacter nitratireducens_LAN01;

[0033] Figure 3 The effect of strain Marinobacter nitratireducens_LAN01 on the removal of ammonia nitrogen and phosphate in culture medium and its growth curve;

[0034] Figure 4 The effect of strain Marinobacter nitratireducens_LAN01 on the removal of nitrite nitrogen, nitrate nitrogen and phosphate in culture medium and its growth curve;

[0035] Figure 5 A comparison of the ammonia nitrogen removal efficiency of strain Marinobacter nitratireducens_LAN01 under different concentrations of SMX stress.

[0036] Figure 6 Figure 1 shows the nitrogen and phosphorus removal and SMX degradation effects of strain Marinobacter nitratireducens_LAN01 in actual aquaculture wastewater treatment.

[0037] Figure 7 The denitrification pathway of strain Marinobacter nitratireducens_LAN01 predicted based on the KEGG database;

[0038] Figure 8 This is the whole genome map of strain Marinobacter nitratireducens_LAN01. Detailed implementation method:

[0039] The present invention will now be described in detail with reference to the accompanying drawings.

[0040] To more clearly illustrate the objectives, technical solutions, and advantages of this invention, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0041] Example 1

[0042] Culture Method: Bioflocs (passed through a 200-mesh filter) taken from the biofilter of marine aquaculture tailwater treatment in Li'an Port, Lingshui, Hainan (110.06E, 18.43N) were inoculated into bacterial selective medium and cultured in a shaker at 25℃ and 180 r / min. Every 24 h, the supernatant was collected, and ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, and phosphate were measured using a UV-Vis spectrophotometer. The concentration of targeted SMX was also detected using HPLC-MS. The culture continued until the ammonia nitrogen degradation rate exceeded 80% after 24 h. Two more cycles of acclimatization were then performed. 1 mL of the selected and acclimatized bacterial culture (OD200) was collected. 600 =0.4), diluted, for example, 10 with sterile PBS solution. 3 10 4 10 5 and 10 6 The culture was then streaked on solid culture medium at a constant temperature of 30°C for 48–72 hours. After the culture medium was cleared, single colonies were picked and purified 2–3 times. The colonies were then inoculated into bacterial enrichment medium for enrichment culture, and their resistance to antibiotic denitrification and phosphorus removal was comprehensively evaluated.

[0043] The measured 16S rDNA sequences were entered into the GenBank database on the NCBI website for similarity comparison, and a phylogenetic tree was drawn. Figure 1 As shown. The results showed that Marinobacter nitratireducens LAN01 had the highest similarity to Marinobacter nitratireducens. Based on physiological and biochemical characteristics, colony morphology, and heterotrophic ammonia assimilation characteristics, this strain was identified and named Marinobacter nitratireducens_LAN01.

[0044] Example 2

[0045] The following are the methods for determining the nitrogen and phosphorus metabolism physicochemical characteristics of Marinobacter nitratireducens strain LAN01.

[0046] (1) Marinobacter nitratireducens LAN01 was inoculated into a bacterial enrichment medium and cultured in a shaker at 25℃ and 180 r / min for 24 h to obtain an activated bacterial suspension. ① The obtained Marinobacter nitratireducens LAN01 activated bacterial suspension was centrifuged at 2500 rpm for 10 min; ② After removing the supernatant, the bacterial cells were resuspended and washed with sterile PBS solution; centrifuged at 2500 rpm for 10 min. ③ After resuspending the bacterial cells with PBS solution, they were inoculated at a 5% inoculum into culture media containing ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen as nitrogen sources, respectively. Samples were taken at time points to determine the utilization and transformation of various nitrogen sources by this strain.

[0047] The ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen culture medium in the above method is prepared from ultrafiltered and sterilized seawater with a salinity of 3.0%. Other components are as follows:

[0048]

[0049] like Figure 3 As shown, this strain can utilize ammonia nitrogen as a nitrogen source (corresponding to ammonia assimilation medium), and no nitrite nitrogen or nitrate nitrogen is produced during the ammonia nitrogen degradation process. It also simultaneously degrades phosphate. Even with a high concentration of the ammonia oxidation inhibitor 100 mg / L allyl thiourea (ATU) (i.e., a high concentration of ATU is added to the ammonia assimilation medium), this strain can still ensure effective ammonia nitrogen removal, proving that the denitrification mechanism of the LAN01 strain is mainly ammonia assimilation.

[0050] like Figure 4 As shown, this strain can utilize nitrite and nitrate as nitrogen sources (corresponding to denitrification medium) and simultaneously degrade phosphate. The denitrification process does not produce nitrite nitrogen or ammonia nitrogen accumulation, and the total nitrogen is converted into biomass without nitrogen loss.

[0051] Example 3

[0052] Antibiotic resistance and ammonia assimilation ability of Marinobacter nitratireducens strain LAN01:

[0053] The LAN01 strain contains SMX resistance (sul1 and sul2) genes and potential degradation (sadA and sadC) genes, such as... Figure 5 As shown, this strain can exert a high efficiency in ammonia assimilation under different stress conditions of 0-20 mg / L SMX (different concentrations of SMX were added to the ammonia assimilation medium in Example 2), and the removal rate of the initial ammonia nitrogen concentration of 45 mg / L can reach 100% within 8 h.

[0054] Example 4

[0055] Application of Marinobacter nitratireducens LAN01 strain in actual marine aquaculture wastewater treatment:

[0056] Using actual marine aquaculture wastewater as the research object, this study investigated the feasibility of using Marinobacter nitratireducens LAN01 to treat antibiotic-containing aquaculture wastewater at 25℃ and pH 7.6. The concentration of Marinobacter nitratireducens LAN01 in the antibiotic-containing aquaculture wastewater was 3 g / L. Figure 6 As shown, the concentration of SMX in the actual aquaculture effluent subjected to antibiotic shock reached as high as 2.8 mg / L, while the concentrations of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, and phosphate were 3.6 mg / L, 0.3 mg / L, 7.7 mg / L, and 1.6 mg / L, respectively. The degradation rates of ammonia nitrogen and nitrite nitrogen were 100% after 3 hours, and the degradation rate of low-concentration SMX (3.0 mg / L) was as high as 93.8%. These results indicate that strain LAN01 has a good removal effect on nitrogen, phosphorus, and antibiotics under actual marine aquaculture effluent conditions, which is superior to native microorganisms, and it has good application potential.

[0057] Example 5

[0058] Analysis of the denitrification pathway in Marinobacter nitratireducens strain LAN01:

[0059] like Figure 7 As shown, the whole genome identification results of Marinobacter nitratireducens LAN01 strain indicate that the bacterium contains a large number of ammonia assimilation (glnA and gdhA) genes, which can assimilate ammonia nitrogen in aquaculture effluent into organic nitrogen (bioflocs) to achieve ammonia nitrogen removal. However, it lacks ammonia oxidation (amoA and hao) genes and has a complete denitrification gene pathway, which proves that the denitrification mechanism of LAN01 strain is mainly heterotrophic ammonia assimilation and aerobic denitrification.

[0060] Appendix: 16S rRNA sequence of LAN01 strain

[0061]

[0062] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibiotic-resistant marine heterotrophic ammonia assimilation-aerobic denitrifying marine bacillus ( Marinobacter nitratireducens LAN01, characterized in that, It was deposited on November 8, 2024, at the China General Microbiological Culture Collection Center (CGMCC) under the accession name LAN01 and accession number CGMCC No. 32548. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, 100101, China.

2. The antibiotic-resistant marine heterotrophic ammonia assimilation-aerobic denitrifying marine bacillus described in claim 1 ( Marinobacter nitratireducens The LAN01 is used for nitrogen and phosphorus removal in marine aquaculture wastewater.

3. The antibiotic-resistant marine heterotrophic ammonia assimilation-aerobic denitrifying marine bacillus described in claim 2 ( Marinobacter nitratireducens LAN01 is used for denitrification and phosphorus removal in marine aquaculture wastewater containing the antibiotic SMX.

4. The antibiotic-resistant marine heterotrophic ammonia assimilation-aerobic denitrifying marine bacillus described in claim 3 ( Marinobacter nitratireducens The application of LAN01 is for the simultaneous recovery of nitrogen and phosphorus nutrients under antibiotic stress conditions.

5. The application according to any one of claims 2-4, wherein the concentrations of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in the marine aquaculture tailwater are all below 50 mg / L; The concentration of phosphate in the wastewater from marine aquaculture is below 20 mg / L; The concentration of antibiotic SMX in the effluent from marine aquaculture is below 20 mg / L; The temperature range for nitrogen and phosphorus removal from ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in marine aquaculture wastewater is 15-40℃. The pH value for nitrogen and phosphorus removal in marine aquaculture wastewater is between 7.5 and 9.5.

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