A tetracycline-resistant short rod pseudomonas aeruginosa denitrifying bacteria

Abstract

The present application relates to a kind of short rod Pseudomonas aeruginosa anti-tetracycline denitrifying bacteria, belong to wastewater treatment technical field, specifically including anaerobic denitrifying drug-resistant strain, the bacterial species of the bacterial strain is Pseudomonas aeruginosa, the anaerobic denitrifying drug-resistant strain is named Pseudomonas aeruginosa YF2024, and its preservation number is CGMCC NO.34912;The morphology of the anaerobic denitrifying drug-resistant strain is short rod, 1.0-1.5 μm long, 0.6-0.7 μm in diameter, gram stain is negative, VP reaction is positive, and tetracycline drug sensitivity is negative, the denitrifying bacteria provided in the present application can still maintain 98% nitrate nitrogen degradation rate under high tetracycline, high nitrogen source condition.

Description

Technical Field

[0001] This invention relates to a tetracycline-resistant short-stem Pseudomonas aeruginosa denitrifying bacterium, belonging to the field of wastewater treatment technology. Background Technology

[0002] In the field of environmental science today, nitrogen pollution has become one of the most serious challenges facing global ecosystems. With the acceleration of industrialization, the intensive development of agricultural activities, and the continuous growth of urban populations, large amounts of nitrogenous pollutants are discharged into natural water bodies through industrial wastewater, agricultural runoff, and domestic sewage. These nitrogenous pollutants, such as ammonia nitrogen and nitrate nitrogen, accumulate in water bodies, causing a series of serious environmental problems, including eutrophication, excessive algal blooms, and imbalances in aquatic ecosystems, posing a significant threat to the sustainable use of water resources and ecological security. In wastewater treatment, denitrification technology is widely used to control nitrogen pollution. Denitrification is a biochemical process in which microorganisms reduce nitrates and nitrites to nitrogen gas, thereby achieving nitrogen removal. Anaerobic denitrifying bacteria play a crucial role in this process. In wastewater treatment plants responsible for nitrogen removal, the denitrification process undertaken by anaerobic denitrifying bacteria is a vital link in wastewater treatment, and the cultivation and utilization of bacteria with high denitrification rates are of great significance in the operation of wastewater treatment plants.

[0003] Antibiotics are a class of proteins or polypeptides produced by microorganisms that have therapeutic or preventative effects against diseases. There are many types of antibiotics, with common types including macrolides, tetracyclines, sulfonamides, quinolones, aminoglycosides, chloramphenicol, and β-lactams. The inhibitory effects of antibiotics on microorganisms are mainly manifested in their ability to inhibit the synthesis of microbial cell walls, leading to bacterial cell rupture and death; their interaction with the cell membrane, thereby affecting membrane permeability and causing leakage of essential substances such as salt ions, proteins, and nucleic acids; their interference with protein synthesis; their inhibition of nucleic acid replication and transcription; and their impact on bacterial metabolic processes. With the widespread use and improper disposal of antibiotics, such as the indiscriminate discharge of pharmaceutical wastewater, aquaculture wastewater, and agricultural wastewater, antibiotic resistance has spread rapidly among pathogens. This resistance has entered the environment and induced the production of large numbers of resistant bacteria (ARBs) and antibiotic resistance genes (ARGs), becoming a major challenge in global public health and seriously threatening human health. The long-term presence of antibiotics not only harms the ecological environment, but may also selectively promote the growth of drug-resistant strains, further exacerbating the risk of the spread of resistance genes in environmental microbial communities. Therefore, drug-resistant strains that can survive in high antibiotic environments and maintain efficient denitrification capabilities have become a research hotspot for solving the dual problems of antibiotic pollution and nitrogen pollution.

[0004] Antibiotic resistance has exceeded 100 species in anaerobic denitrification systems of wastewater treatment plants, with tetracyclines dominating. Tetracyclines are widely used antibiotics and, due to their difficulty in environmental degradation, have become a typical persistent pollutant. Tetracycline pollution inhibits the activity of some microorganisms, thereby affecting the stability of the microbial community and denitrification efficiency in wastewater treatment. Therefore, screening and cultivating antibiotic-resistant strains that can maintain stable denitrification capacity in the presence of tetracyclines has significant theoretical and practical value for the simultaneous treatment of nitrogen and antibiotic pollution.

[0005] While some studies have reported the application of drug-resistant strains in wastewater treatment, most strains have limited resistance ranges and low denitrification efficiency. Furthermore, the adaptability and stability of existing strains in high-concentration antibiotic environments remain unresolved challenges. Therefore, developing an anaerobic strain with high denitrification capacity and tetracycline resistance is of significant practical importance for improving wastewater treatment technology. Summary of the Invention

[0006] To address the technical problems existing in the prior art, the present invention provides a tetracycline-resistant Pseudomonas aeruginosa denitrifying bacterium that can maintain a 98% nitrate nitrogen degradation rate even under high tetracycline and high nitrogen source conditions.

[0007] To achieve the above objectives, the technical solution adopted in this invention is a tetracycline-resistant short-stem Pseudomonas aeruginosa denitrifying bacterium, including an anaerobic denitrifying resistant strain. The strain is Pseudomonas aeruginosa YF2024, with accession number CGMCC NO.34912, deposited at the China General Microbiological Culture Collection Center on June 16, 2025.

[0008] The anaerobic denitrifying resistant strain is morphologically short rod-shaped, 1.0-1.5 μm long and 0.6-0.7 μm in diameter, Gram-negative, VP-positive, and tetracycline-sensitive.

[0009] Preferably, the anaerobic denitrifying resistant strain still has a high denitrification rate under high nitrogen source conditions, and is an anaerobic denitrifying bacterium with high denitrification capacity, which can achieve a nitrate nitrogen degradation rate of up to 98%.

[0010] Preferably, the anaerobic denitrifying resistant strain is cultured under high nitrogen source conditions using a culture medium consisting of 1 L of distilled water with the following components added.

[0011] Sodium acetate 0.623g, sodium nitrate 0.607g, magnesium chloride hexahydrate 1g, sodium chloride 0.1g, potassium dihydrogen phosphate 0.2g, dipotassium hydrogen phosphate 0.4g, minerals 4ml, biotin 2mg, thiamine 5mg, pyridoxine hydrochloride 10mg, D-pantothenic acid 5mg, lipoic acid 5mg, folic acid 2mg, riboflavin 5mg, niacin 5mg, para-aminobenzoic acid 5mg, vitamin B 12 0.1mg, MnCl2·6H2O0.1mg, ZnCl20.1mg, CuSO4·5H2O 0.03mg, Na2MoO2·2H2O 0.01mg, H3BO30.01mg.

[0012] Preferably, the anaerobic denitrifying resistant strain is a highly drug-resistant anaerobic denitrifying bacterium that can still carry out the anaerobic denitrification process even under high concentrations of drugs.

[0013] Preferably, the anaerobic denitrifying resistant strain exhibits high resistance to antibiotics.

[0014] Preferably, the anaerobic denitrifying resistant strain exhibits high resistance to the antibiotic tetracycline.

[0015] Preferably, the anaerobic denitrifying resistant strain is cultured in a culture medium containing a high concentration of the antibiotic tetracycline. This culture medium is prepared by adding the following components to 1 L of distilled water.

[0016] Sodium acetate 0.623g, sodium nitrate 0.607g, magnesium chloride hexahydrate 1g, sodium chloride 0.1g, potassium dihydrogen phosphate 0.2g, dipotassium hydrogen phosphate 0.4g, minerals 4ml, biotin 2mg, thiamine 5mg, pyridoxine hydrochloride 10mg, D-pantothenic acid 5mg, lipoic acid 5mg, folic acid 2mg, riboflavin 5mg, niacin 5mg, para-aminobenzoic acid 5mg, vitamin B 12 0.1 mg, MnCl2·6H2O 0.1 mg, ZnCl2 0.1 mg, CuSO4·5H2O 0.03 mg, Na2MoO2·2H2O 0.01 mg, H3BO3 0.01 mg, tetracycline antibiotic 512 mg.

[0017] Compared with existing technologies, the present invention has the following technical advantages: The strain provided by the present invention exhibits good growth under tetracycline (TC) antibiotic conditions and also grows well under anaerobic conditions, and can be used for the removal of anaerobic nitrate nitrogen from wastewater with high concentrations of this antibiotic. Experimental results show that the Pseudomonas aeruginosa YF2024 strain can still maintain high activity even at the minimum inhibitory concentration of the antibiotic of 512 mg / L. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the morphology of Pseudomonas aeruginosa YF2024 cells observed by electron microscopy according to the present invention.

[0019] Figure 2 This is a schematic diagram of the phylogenetic tree of the Pseudomonas aeruginosa YF2024 strain in this invention.

[0020] Figure 3 This is a schematic diagram of the growth curve of the Pseudomonas aeruginosa YF2024 strain in this invention.

[0021] Figure 4 This is a schematic diagram illustrating the nitrate nitrogen degradation rate of Pseudomonas aeruginosa YF2024 in this invention.

[0022] Figure 5 This is a schematic diagram illustrating the minimum inhibitory concentration of the antibiotic against Pseudomonas aeruginosa YF2024 in this invention. Detailed Implementation

[0023] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0024] A tetracycline-resistant short-rod Pseudomonas aeruginosa denitrifying bacterium, including an anaerobic denitrifying resistant strain, is disclosed. The strain is named Pseudomonas aeruginosa YF2024, with the accession number CGMCC NO.34912. The morphology of the anaerobic denitrifying resistant strain is short rod-shaped, 1.0-1.5 μm long and 0.6-0.7 μm in diameter. It is Gram-negative, VP-positive, and tetracycline-sensitive.

[0025] Anaerobic denitrifying resistant strains still maintain a high denitrification rate under high nitrogen source conditions, and are anaerobic denitrifying bacteria with high denitrification capacity, with a nitrate nitrogen degradation rate of up to 98%. High nitrogen source refers to a C / N ratio of 5.

[0026] Anaerobic denitrification resistant strains are highly resistant anaerobic denitrifying bacteria that can still carry out anaerobic denitrification processes even under high concentrations of antibiotics, especially tetracycline. Specifically, these anaerobic denitrification resistant strains were tested at gradients of tetracycline concentrations: 0.5 mg / L, 1 mg / L, 2 mg / L, 4 mg / L, 8 mg / L, 16 mg / L, 32 mg / L, 64 mg / L, 128 mg / L, 256 mg / L, and 512 mg / L. Even at a high concentration of 512 mg / L, they still maintained high denitrification efficiency.

[0027] A tetracycline-resistant, short-stem Pseudomonas aeruginosa denitrifying bacterium was developed using conventional microbial isolation and selective culture techniques, specifically including the following steps:

[0028] 1. Sampling

[0029] Sludge samples were taken from the Yangjiabao Wastewater Treatment Plant in Xiaodian District, Taiyuan City, Shanxi Province.

[0030] 2. Pretreatment

[0031] (1) Aerate the sludge for 1.5 hours to remove residual organic and inorganic matter in the sludge.

[0032] (2) Rinse with ultrapure water at least five times to remove unnecessary impurities from the sludge.

[0033] (3) Finally, nitrogen gas is used for aeration to remove trace amounts of oxygen in the sludge and reduce its ORP to below -120 mV.

[0034] 3. Limiting dilution

[0035] (1) Prepare a series of sterile test tubes.

[0036] (2) Take the initial diluted sample from the sample and transfer it to the first test tube.

[0037] (3) Transfer a certain amount of diluted sample to the next test tube and repeat the dilution process.

[0038] (4) Each dilution increases the sequence by an order of magnitude.

[0039] 4. Enrichment culture

[0040] (1) Experimental design: Six anaerobic bottles were set up for the experiment, of which A, B, C, and D were the experimental groups, and E and F were the blank groups, i.e., no antibiotic TC was added. The TC concentration in the enrichment medium was prepared in sequence at the ratios of 1 mg / L, 5 mg / L, and 10 mg / L, and each concentration was cultured for three cycles, for a total of nine cycles. The one with the highest denitrification capacity was selected for subculturing.

[0041] (2) Steps: Take a 1:30 volume from the final dilution and put it onto the anaerobic denitrifying bacteria enrichment medium. Purge the medium with nitrogen for 2-3 minutes to remove oxygen from the water. Incubate in a 35 ℃ constant temperature incubator for 3-4 days with a shaking speed of 150 r / min for a period of three weeks.

[0042] 5. Marking lines on a flat surface

[0043] Using a smooth, flat inoculation loop, aseptically pick up a small amount of a series of enriched cultures and streak them onto plates. After streaking, invert the plates and place them in an anaerobic bag, then incubate at 35°C for 24 hours. Select colonies with distinct blue circles and different morphologies and streak them six times for purification.

[0044] 6. Preliminary screening of bacterial strains

[0045] Single colonies from the final streak plate were transferred to 10 mL LB broth tubes for sealed anaerobic culture. The Durbin tubes were inverted, and two replicates were made for each colony. The tubes were incubated at 35°C for 3 days. Bacterial growth and gas production in the Durbin tubes were observed. The nitrogen content of strains with significant gas production was measured and inoculated onto BTB plates for verification. Finally, strains with strong denitrification ability were selected as experimental strains.

[0046] 7. Strain identification

[0047] Genomic DNA was extracted from the strain using a DNA kit from the bioengineering platform, and PCR amplification was performed using 16S rRNA gene amplification primers.

[0048] (1) PCR reaction system: Prepare PCR reaction solution in 50 μL according to the following composition: Taq PCR Mix (2X) 25 μL; Primer Mix 2 μL*; DNA 2 μL*; Ultrapure water (to a final volume of 50 μL).

[0049] (2) PCR reaction conditions: 98℃ for 2 min, 98℃ for 10 s, 60℃ for 10 s, 72℃ for 10 s, 35 cycles; 72℃ for 5 min; store at 4℃.

[0050] (3) The PCR products were subjected to agarose gel electrophoresis to check whether the amplification was successful. The target band after PCR amplification was then sent to Shanghai Meiji Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the NCBI GenBank database. Species or genera with high similarity to the sequenced sequence were selected, and a phylogenetic tree was constructed using MEGA software and the neighbor-joining method.

[0051] 8. Preservation of microbial strains

[0052] The bacterial strain is inoculated into centrifuge tubes using the glycerol preservation method and stored at -80℃ for no more than one year. Generally, a 20% aqueous solution is prepared, autoclaved at 121℃ for 20 minutes, and then aseptically stored at room temperature. When using, mix 20% glycerol and bacterial solution in a 1:1 ratio, then freeze rapidly.

[0053] The culture medium formula under high nitrogen source conditions is as follows: 0.623g sodium acetate, 0.607g sodium nitrate, 1g magnesium chloride hexahydrate, 0.1g sodium chloride, 0.2g potassium dihydrogen phosphate, 0.4g dipotassium hydrogen phosphate, 4ml minerals, 2mg biotin, 5mg thiamine, 10mg pyridoxine hydrochloride, 5mg D-pantothenic acid, 5mg lipoic acid, 2mg folic acid, 5mg riboflavin, 5mg niacin, 5mg para-aminobenzoic acid, and vitamin B12 in 1L of distilled water. 12 0.1mg, MnCl2·6H2O 0.1mg, ZnCl20.1mg, CuSO4·5H2O 0.03mg, Na2MoO2·2H2O 0.01mg, H3BO30.01mg.

[0054] The culture medium formulation under high concentrations of the antibiotic tetracycline is as follows: 1 L of distilled water containing 0.623 g sodium acetate, 0.607 g sodium nitrate, 1 g magnesium chloride hexahydrate, 0.1 g sodium chloride, 0.2 g potassium dihydrogen phosphate, 0.4 g dipotassium hydrogen phosphate, 4 ml minerals, 2 mg biotin, 5 mg thiamine, 10 mg pyridoxine hydrochloride, 5 mg D-pantothenic acid, 5 mg lipoic acid, 2 mg folic acid, 5 mg riboflavin, 5 mg niacin, 5 mg para-aminobenzoic acid, and vitamin B12. 12 0.1 mg, MnCl2·6H2O 0.1 mg, ZnCl2 0.1 mg, CuSO4·5H2O 0.03 mg, Na2MoO2·2H2O 0.01 mg, H3BO3 0.01 mg, tetracycline antibiotic 512 mg.

[0055] The above Pseudomonas aeruginosa The 16S rRNA sequence of strain YF2024 has 100% similarity to the 16S rRNA sequences of other different species of Pseudomonas aeruginosa.

[0056] Experiment Test 1: Pseudomonas aeruginosa YF2024 Physiological and Biochemical Identification

[0057] 1. Gram staining

[0058] (1) Slide preparation and fixation: Prepare smears of 18-24h culture, dry them and then fix them with a flame;

[0059] (2) Primary staining: Stain with crystal violet for 60 seconds, wash with water and spin dry;

[0060] (3) Mordant staining: stain with Gram's iodine solution for 60 seconds, wash with water and spin dry;

[0061] (4) Decolorization: Decolorize with 95% alcohol for 30-60 seconds, then wash with water and spin dry;

[0062] (5) Counterstaining: After staining with safranin for 150 seconds, wash with water and spin dry;

[0063] (6) Identification: Observe the cell color under 100x oil immersion. Purple indicates Gram-positive bacteria, and pink indicates Gram-negative bacteria.

[0064] 2. Methyl red test

[0065] Prepare fresh tryptone medium. The target bacterial strain was inoculated into the medium and cultured overnight in the experimental group. A blank control group was also included. Five drops of methyl red reagent were added to the cultured bacterial solution, and the color change of the methyl red was observed. A change from yellow to red indicated a positive result, while the change from yellow to red indicated a negative result.

[0066] 3. Sugar fermentation experiment

[0067] Prepare a fresh liquid culture medium for sugar fermentation, inoculate the target strain into the medium, and incubate at 35°C for 3 days. Observe the color change of the culture medium. If the bacterial solution turns yellow, it indicates a positive result; otherwise, it indicates a negative result.

[0068] 4. Oxygen Demand Test

[0069] Prepare a fresh anaerobic denitrification liquid culture medium, inoculate the target strain, and place them in a shaker at 30℃ for 3 days. Observe the growth of the strain. If the culture medium becomes turbid, it indicates a positive result; otherwise, it indicates a negative result.

[0070] 5. Catalase test

[0071] Prepare catalase solid culture medium in advance and put it into test tubes. Pick a single colony from the anaerobic denitrification plate containing the target strain and inoculate it onto the solid test tube. After incubating at 35°C for 3 days, observe the changes in the test tube. If black substance is produced at the bottom of the test tube, it indicates a positive result; otherwise, it indicates a negative result.

[0072] 6. Starch test

[0073] Prepare fresh starch culture medium plates in advance. Pick a single colony from the anaerobic denitrification plate containing the target strain and inoculate it onto the starch culture medium plate. Incubate overnight at 35°C. Add iodine solution to the plate and observe whether a clear zone will form. If a clear zone forms, it indicates a positive result; otherwise, it indicates a negative result.

[0074] 7. Hydrogen peroxide test

[0075] Prepare a fresh hydrogen peroxide solution. Take a small amount of the hydrogen peroxide solution onto a glass slide. Then, pick a single colony from an anaerobic denitrification plate containing the target strain and place it onto the glass slide. If bubbles are produced in the solution within half a minute, it indicates a positive result; otherwise, it indicates a negative result.

[0076] 8. Morphological observation

[0077] Single colonies were picked from plates containing the target bacterial strain and evenly distributed, and diluted to various gradients of sterile water. A 10-10 dilution was then performed. -6 and 10 -7 Two gradients were spread separately on anaerobic denitrification plates, and the culture conditions were set at 35℃ anaerobic. Colony growth was observed during the culture process. After the colonies on the plates were of moderate size and evenly distributed, the morphology of the colonies, such as shape, size, transparency, color, and whether the edges were neat, was observed, recorded, and photographed.

[0078] Test 2: Pseudomonas aeruginosa Morphological identification using scanning electron microscopy (SEM) of YF2024

[0079] Take the bacterial culture sample, centrifuge at 4000 rpm for 5 min, and discard the supernatant. Place the sample in a centrifuge tube, add 2.5% glutaraldehyde (prepared with PBS, pH=6.8), and fix at 4℃ for 1.5 h. Wash three times with 0.1 mol phosphate buffer (pH=6.8), 10 min each time. Dehydrate with 50%, 70%, 80%, and 90% ethanol solutions, 10-15 min each time, then dehydrate three times with 100% ethanol, 10-15 min each time. Prepare a 1:1 mixture of 100% ethanol and isoamyl acetate and pure isoamyl acetate for displacement, once each, 15 min each time. Pick up the displacement sample with a needle, place it in a small box made of folded filter paper, and dry in a desiccator for 8 h. Use double-sided tape to attach the sample, observation side up, to the aluminum plate of a scanning electron microscope. Deposit a 1500 nm thick metal film on the sample surface using an IB-5 (Giko) ion sputtering coating instrument.

[0080] After the culture of Example 1 and Comparative Example 1 was completed, the nitrate nitrogen content was measured, and the nitrate nitrogen removal rate was calculated. Figure 4 As shown: After approximately three generations of enrichment culture, the degradation efficiency of nitrate nitrogen under three gradient concentrations of antibiotic TC and the degradation rate of nitrate nitrogen by colonies in plates without antibiotic TC both reached 100%, indicating that... Pseudomonas aeruginosa YF2024 bacteria can still multiply normally and degrade nitrate nitrogen even under the minimum inhibitory concentration (MIC) of antibiotic TC (512 mg / L).

[0081] Test 3 Pseudomonas aeruginosa YF2024 growth curve and denitrification performance test

[0082] 1. Growth curve determination

[0083] (1) Experimental preparation

[0084] Single colonies were picked from the preserved bacterial strains and inoculated into suitable liquid culture medium for activation culture under appropriate temperature and conditions. The UV spectrophotometer was turned on and preheated for 20-30 minutes to allow the instrument to reach a stable operating state. A blank culture medium was used as a control to zero and calibrate the spectrophotometer to ensure measurement accuracy.

[0085] (2) Inoculation and culture

[0086] The activated bacterial solution was appropriately diluted, and a certain amount of the diluted bacterial solution was inoculated into sterile liquid culture medium at a volume of 1% to 5% of the culture medium volume. The inoculation time was recorded as the starting time of the growth curve. The inoculated culture medium was then placed in a constant temperature shaker at 37°C and 150 to 200 rpm for incubation.

[0087] (3) Measurement and Recording

[0088] During the cultivation process, samples are taken at predetermined time intervals. Initially, samples are taken every 1-2 hours, and later the interval can be extended to 3-4 hours. Each time a sample is taken, a certain amount of bacterial culture is drawn using a sterile pipette or tube and added to a cuvette. The cuvette is placed in a spectrophotometer, and the absorbance (OD value) of the bacterial culture is measured at a wavelength of 600 nm. The OD values ​​obtained from each measurement and the corresponding cultivation time are recorded, and a growth curve is plotted.

[0089] (4) Data processing

[0090] Plot a scatter plot on graph paper or using graphing software, with incubation time on the x-axis and OD value on the y-axis. The growth stage of the bacteria can be determined based on the trend of the scatter plot.

[0091] 2. Denitrification performance test

[0092] (1) Draw the standard curve

[0093] ① Instruments

[0094] A 50mL stoppered colorimetric tube and a UV-2201 ultraviolet spectrophotometer were used.

[0095] ② Reagents

[0096] ammonia water (ρ) 20=0.88 g / mL), acetic acid solution (1+4), ammonium aminosulfonate solution (20 g / L), thymol ethanol solution (5 g / L), silver sulfate sulfuric acid solution (10 g / L), nitrate nitrogen standard stock solution [ρ(NO3-N)=1mg / mL] (weigh 7.218g of potassium nitrate (KNO3) dried at 105~110℃ for 1 hour, dissolve in pure water, and make up to 1000mL, add 2mL of chloroform preservative), nitrate nitrogen standard working solution [ρ(NO3-N)=10 μg / mL] (pipette 5.00 mL of nitrate nitrogen standard stock solution and make up to 500 mL).

[0097] ③ Experimental steps

[0098] Pipette 0, 0.05, 0.10, 0.30, 0.50, 0.70, and 1.00 mL of nitrate nitrogen standard working solution into separate 50 mL stoppered colorimetric tubes and dilute to 1.0 mL with pure water. Add 0.1 mL of ammonium aminosulfonate solution to each tube, shake well, and let stand for 5 minutes. Then add 0.2 mL of thymol ethanol solution to each tube, shake well, add 2 mL of silver sulfate-sulfuric acid solution, mix, and let stand for 5 minutes. Add 8 mL of pure water to each tube, mix well, and then add ammonia dropwise until the solution reaches its deepest yellow color, until the silver chloride precipitate dissolves. Finally, add pure water to the 25 mL mark, mix well, and measure the absorbance at 415 nm, using a blank sample as a reference.

[0099] (2) Bacterial denitrification performance

[0100] ① Sample preparation

[0101] Take a certain amount of denitrifying bacteria culture in the logarithmic growth phase, centrifuge to collect the bacterial cells, wash the bacterial cells 2-3 times with sterile physiological saline to remove impurities and residual nutrients in the culture medium, and then resuspend the bacterial cells in sterile physiological saline to prepare a bacterial suspension of a certain concentration.

[0102] ② Reaction system setup

[0103] In a series of sterile reaction flasks, equal amounts of denitrifying bacterial suspension and denitrification medium containing nitrate were added to ensure that the initial concentrations of bacteria and nitrate in the reaction system remained consistent. The reaction flasks were then sealed with rubber stoppers or plastic wrap to prevent air from entering.

[0104] ③ Denitrification reaction

[0105] The reaction flask was placed in a constant temperature shaker and the denitrification reaction was carried out at 37°C and a slow shaking speed. During the reaction, a certain amount of reaction solution was taken out from the reaction flask at regular intervals.

[0106] ④ Absorbance measurement

[0107] The extracted reaction solution was centrifuged in a high-speed centrifuge, and the supernatant was collected. The absorbance of nitrate or nitrite in the supernatant was measured at a wavelength of 415 nm using a UV spectrophotometer.

[0108] ⑤ Plotting the standard curve (from above (1))

[0109] ⑥ Result Calculation

[0110] The denitrification rate is calculated based on the concentration of nitrate or nitrite in the reaction solution at different time points. The calculation formula is: Denitrification rate = (initial nitrate or nitrite concentration - nitrate or nitrite concentration at a certain time) / reaction time.

[0111] Test 4 Pseudomonas aeruginosa YF2024 Minimum Inhibitory Concentration Test

[0112] 1. Prepare a series of liquid culture media containing different concentrations of antibacterial drugs. The culture medium formula is as follows: 0.623 g sodium acetate, 0.607 g sodium nitrate, 1.0 g magnesium chloride hexahydrate, 0.1 g sodium chloride, 0.2 g potassium dihydrogen phosphate, 0.4 g dipotassium hydrogen phosphate, 4.0 mL minerals, 2.0 mg biotin, 5.0 mg thiamine, 10.0 mg pyridoxine hydrochloride, 5.0 mg D-pantothenic acid, 5.0 mg lipoic acid, 2.0 mg folic acid, 5.0 mg riboflavin, 5.0 mg niacin, 5.0 mg para-aminobenzoic acid, and vitamin B1 in 1 L of distilled water. 12 0.1 mg of MnCl2·6H2O, 0.1 mg of ZnCl2, 0.03 mg of CuSO4·5H2O, 0.01 mg of Na2MoO2·2H2O, and 0.01 mg of H3BO3 were added. Tetracycline concentrations were set at gradients of 0.5 mg / L, 1 mg / L, 2 mg / L, 4 mg / L, 8 mg / L, 16 mg / L, 32 mg / L, 64 mg / L, 128 mg / L, 256 mg / L, and 512 mg / L. The experiment was conducted in 96-well cell culture plates. The bacterial culture of the test strain was prepared to achieve a certain concentration, for example, 10... 6 CFU / mL.

[0113] 2. Drug dilution

[0114] The highest concentration of drug was added to the first row of wells in a 96-well plate, followed by a two-fold serial dilution. This involved adding a certain amount of drug solution from the first row of wells to the second row, mixing thoroughly, and then adding the same amount from the second row to the third row, and so on, until the last row of wells. The drug concentration in each row of wells decreased progressively.

[0115] 3. Inoculation with bacterial culture

[0116] Add an equal amount of bacterial solution to each well containing a different concentration of drug, and set up a positive control (containing only bacterial solution, without drug) and a negative control (containing only culture medium, without bacterial solution and drug).

[0117] 4. Cultivation

[0118] Place the 96-well plate in a suitable culture environment, usually a constant temperature incubator at 37°C, and incubate for 16-24 hours.

[0119] 5. Results Observation

[0120] After incubation, observe bacterial growth in each well. The minimum inhibitory concentration (MIC) is defined as the lowest drug concentration required to completely inhibit bacterial growth.

[0121] 6. Data Analysis

[0122] Based on the observation results, record the bacterial growth at each drug concentration, usually indicated by "+" for growth and "-" for no growth. Determine the MIC value, i.e., the minimum inhibitory concentration.

[0123] The results show that the above Pseudomonas aeruginosa Even with high concentrations (512 mg / L) of tetracycline, strain YF2024 was still able to carry out anaerobic denitrification.

[0124] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the scope of the present invention.

Claims

1. A strain of Pseudomonas aeruginosa ( Pseudomonas aeruginosa YF2024, characterized in that: The *Pseudomonas aeruginosa* mentioned is a tetracycline-resistant, short-stem *Pseudomonas aeruginosa* denitrifying bacterium, with the preservation number CGMCC NO.34912.

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