Pseudomonas aeruginosa capable of degrading trifloxysulfuron and application of pseudomonas aeruginosa in soil pollution control

Abstract

The invention discloses pseudomonas aeruginosa capable of degrading trifloxysulfuron and application of the pseudomonas aeruginosa in soil pollution prevention and control, and relates to the technical field of ecological restoration of environmental pollution. The invention aims to solve the problem of pollution of trifloxysulfuron in the existing soil. The pseudomonas aeruginosa is pseudomonas aeruginosa GXJ-9 and is preserved in the China Center for Type Culture Collection, and the preservation number of the pseudomonas aeruginosa is CCTCC (China Center for Type Culture Collection) NO: M 20242844. The invention relates to an application of pseudomonas aeruginosa GXJ-9, in particular to an application of the pseudomonas aeruginosa GXJ-9 in degrading soil polluted by trifloxysulfuron. According to the invention, the pseudomonas aeruginosa and the application of the pseudomonas aeruginosa can be obtained.

Description

Technical Field

[0001] The present invention relates to the field of environmental microbial remediation technology, specifically a strain of Pseudomonas aeruginosa that can degrade trifloxysulfuron and its application in soil pollution prevention and control. This strain was deposited with the China Center for Type Culture Collection (CCTCC) on December 18, 2024, with the deposit number CCTCC NO: M 20242844. Background Art

[0002] Trifloxysulfuron is a protoporphyrinogen oxidase (PPO) inhibitor herbicide and a new uracil contact herbicide developed by Fuam Hannong Co., Ltd. It is mainly used to control monocotyledonous and dicotyledonous and glyphosate-resistant weeds and is considered an alternative to glyphosate and paraquat. The chemical formula is C 19 H 18 ClF4N3O5S is a white powder. Uracil herbicides are classified as protoporphyrinogen oxidase inhibitors. Their mechanism of action is to inhibit the formation of protoporphyrinogen oxidase (PPO) during chlorophyll synthesis, resulting in the accumulation of photosensitive protoporphyrinogen IX. The resulting singlet reactive oxygen species (ROS) cause lipid peroxidation, which in turn leads to loss of membrane function in weed cells, inhibited plant growth, and even death. However, during use, trifloxysulfuron herbicides seep into the soil, causing soil pollution and even contaminating surface water, leading to chronic accumulation and exposure risks in aquatic plants and animals. Reducing the environmental impact and potential risks of trifloxysulfuron herbicides is crucial.

[0003] Biological treatment is used to eliminate pollution and repair soil, and has the advantages of being environmentally friendly, mild conditions, saving resources, and non-toxic and harmless degradation products.

[0004] Therefore, screening functional strains that can degrade trifloxysulfuron has certain scientific significance and industrial value. Summary of the Invention

[0005] The present invention aims to provide a strain of Pseudomonas aeruginosa GXJ-9 capable of degrading trifloxysulfuron, which can effectively degrade trifloxysulfuron in contaminated soil.

[0006] The detailed technical solutions adopted in this application are as follows.

[0007] In a first aspect, the present invention provides a trifloxysulfuron-degrading bacterium, which is Pseudomonas aeruginosa GXJ-9, deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M20242844.

[0008] In a second aspect, the present invention provides an application of Pseudomonas aeruginosa GXJ-9, and the application of Pseudomonas aeruginosa GXJ-9 in degrading trifloxysulfuron in contaminated soil.

[0009] Specifically, Pseudomonas aeruginosa GXJ-9 was cultured in a basic salt medium (BSM) containing trifloxysulfuron as the sole carbon and nitrogen source at 37°C overnight, and then the cells were collected by centrifugation, resuspended in sterile water, and the OD was adjusted. 600 = 1.0. Then, the bacterial solution was applied at a ratio of 1% by mass to the soil, and the degradation time for soil containing trifloxysulfuron at a concentration of 200 μg / kg was 120 hours.

[0010] The Pseudomonas aeruginosa GXJ-9 and the cultivation method of the present invention have the following beneficial effects compared with the prior art: the Pseudomonas aeruginosa GXJ-9 of the present invention has the ability to degrade trifloxysulfuron and is suitable for treating soil and water bodies contaminated by the herbicide trifloxysulfuron, without the need for dilution or additional carbon source supplementation to maintain the stability of the bacterial agent system. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The colony morphology of the strain of the present invention on BSM solid culture medium containing trifloxysulfuron as the sole carbon and nitrogen source;

[0012] Figure 2 The phylogenetic tree of strain GXJ-9 constructed based on the 16S rRNA coding gene sequence using the Neighbor-Joining method (NJ) and bootstrap iterative sampling 1000 times;

[0013] Figure 3 The liquid chromatograms for the determination of trifloxysulfuron in the present invention are as follows: (a) blank trifloxysulfuron solution (100 μg / L); (b) trifloxysulfuron (100 μg / L) added to the sample dissolution matrix; (c) acetonitrile as the liquid chromatography mobile phase blank; (d) matrix blank for sample dissolution stabilization.

[0014] Figure 4 The growth curves of the strain of the present invention at different culture temperatures were obtained by inoculating 2% of BSM medium (200 μg / L trifloxysulfuron as the sole carbon and nitrogen source).

[0015] Figure 5 This is a diagram showing the metabolic performance of the strain of the present invention when inoculated with 2% BSM medium at different culture temperatures to an initial concentration of 200 μg / L trifloxysulfuron;

[0016] Figure 6 This is a diagram showing the metabolic performance of the strain of the present invention when 2% of the BSM medium is inoculated with different initial concentrations of 50-1000 μg / L trifloxysulfuron;

[0017] Figure 7 This is a diagram showing the metabolic performance of the strain of the present invention when 2% of the BSM medium is inoculated at different initial pH values ​​to an initial concentration of 200 μg / L trifloxysulfuron;

[0018] Figure 8 The results show the application effect of the strain of the present invention at a bacterial dosage of 1% by mass in farmland soil containing 200 μg / kg trifloxystrobin. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to the examples. Unless otherwise specified, the equipment and reagents used in each example and test example can be obtained from commercial or public sources. The specific examples described herein are only used to illustrate the present invention and are not intended to limit the present invention.

[0020] Based on the information contained in this application, it will be relatively easy for those skilled in the art to make various changes and optimizations to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are encompassed within the scope of the appended claims.

[0021] In order to better understand the present invention and not to limit the scope of the present invention, all numbers used in this application to express amounts, percentages, and other numerical values ​​should be understood to be modified by the word "approximately" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different desired properties to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods. In the present invention, "about" means within 10% of a given value or range, preferably within 5%.

[0022] In the following embodiments of the present invention, unless otherwise specified, room temperature is used. Room temperature refers to the natural room temperature in all seasons without additional cooling or heating, and is generally controlled at 10-30°C, preferably 15-25°C.

[0023] Unless otherwise specified, the reagents and materials used in the embodiments of the present invention can be purchased from commercial sources.

[0024] The trifloxysulfuron-degrading strain of the present invention is Pseudomonas aeruginosa GXJ-9, which was deposited in the China Center for Type Culture Collection (CCTCC) on December 18, 2024, with the address being Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province, Postal Code 430072, and the deposit number being CCTCC NO: M 20242844.

[0025] 1. Isolation of strains

[0026] 1) Culture medium

[0027] LB liquid medium: 10.0 g / L tryptone, 5.0 g / L yeast extract powder, 10.0 g / L NaCl, adjust the pH to 7.0.

[0028] LB solid medium: LB liquid medium supplemented with 2% agar powder (wt / vol).

[0029] BSM liquid medium: Na2HPO4·12H2O 14.04 g / L, K2SO4 1.00 g / L, KH2PO4 4.00 g / L, MgCl2·6H2O 0.20 g / L, MnCl2·4H2O 0.0004 g / L, CaCl2 0.001 g / L, FeCl3·6H2O 0.001 g / L, trifloxysulfuron (final concentration 50-1000 μg / L), adjust the pH to 7.0.

[0030] BSM solid medium: BSM liquid medium supplemented with 2% agar powder (wt / vol).

[0031] 2) Separation method

[0032] Take 1 g of soil sample applied with trifloxysulfuron and add it to 20 mL of sterile water, shake and mix well, take 2 mL and add it to 100 mL of LB medium (Luria-Bertani Broth) for enrichment culture, and place it in a constant temperature shaking incubator (30°C, 180 rpm) for culture for 18 h; take 2 mL of enriched bacterial liquid and transfer it to 100 mL of BSM liquid culture medium containing 200 μg / L trifloxysulfuron, and place it in a constant temperature shaking incubator (30°C, 180 rpm) for culture.

[0033] To test the removal efficiency of candidate degrading bacteria on trifloxysulfuron, the residual content of trifloxysulfuron in the culture medium was determined by high performance liquid chromatography (HPLC). The bacteria were inoculated and subcultured in a 2% culture medium, and the above enrichment process was repeated until the trifloxysulfuron removal efficiency reached a stable level. 1 mL of the above bacterial enrichment solution was taken for gradient dilution, and the dilution factor was set to 10 based on the hemocytometer combined with the microscope count results. -4 , 10 -5 , 10 -6 150 μL of each diluted bacterial solution was spread onto a solid plate containing trifloxysulfuron (BSM) and incubated in a 30°C incubator for two days. A single colony was streaked onto a BSM plate containing trifloxysulfuron. Repeat this process 2-3 times until a single colony was isolated and named GXJ-9.

[0034] 2. Identification of strains

[0035] 1) Bacterial morphological characteristics

[0036] The strain was cultured in LB liquid at 30°C, 180 rpm, and pH 7 until the logarithmic phase. Aseptically, an inoculating loop was used to streak the GXJ-9 bacterial solution onto a solid LB plate containing 200 μg / L trifloxysulfuron at an initial concentration of 200 μg / L. The plate was then incubated at 30°C in a biochemical incubator. Observation was performed after a single colony formed on the plate. The GXJ-9 colony was round, light yellow, and smooth ( Figure 1 Gram staining and microscopic examination were performed using Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli as controls. Based on the staining results, GXJ-9 was presumed to be a Gram-negative bacterium. Subsequent physiological and biochemical tests were conducted, including catalase, oxidase, indole, methyl red, VP (Voges-Proskauer), gelatin liquefaction, and nitrate reduction. The identification results of these tests are shown in Table 1.

[0037] Table 1

[0038] Measurement items result Measurement items result Oxidase test + VP test - Catalase test + Gelatin liquefaction test + Indole test - 42℃ growth test - Methyl red test - Nitrate reduction +

[0039] 2) 16S rRNA identification

[0040] PCR amplification was performed using GXJ-9 genomic DNA extracted by the boiling method as a template, using the universal primers 27F (forward, 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (reverse, 5'-GGTTA CCTTGTTACGACTT-3') for bacterial 16S rRNA cloning. The amplification conditions on a Bio-Rad S1000 gradient PCR instrument were as follows: ① initial denaturation at 95°C for 10 min, ② subsequent denaturation at 95°C for 30 s, ③ annealing at 66-57°C for 30 s, ④ extension at 72°C for 40 s, 10 cycles of steps ②-④ (1°C decrease per cycle), ⑤ further denaturation at 95°C for 30 s, ⑥ annealing at 57°C for 30 s, ⑦ extension at 72°C for 40 s, 21 cycles of steps ⑤-⑦, ⑧ final extension at 72°C for 10 min, and ⑨ storage of the amplified product at 4°C after completion. The reaction system consisted of 10 μL of 2× Phanta Max Super-Fidelity DNA Polymerase Mix (Vazyme Biotech Co., Ltd, Nanjing, China), 1 μL each of the forward and reverse primers, 0.5 μL of 50% dimethyl sulfoxide (DMSO), 1 μL of the DNA template, and ddH₂O to a total volume of 25 μL. After amplification, the amplified product was electrophoresed on a 1.5% agarose gel and the size of the amplified product was determined by gel imaging. The PCR product was then excised and recovered for sequencing.

[0041] The amplified 16S rRNA gene nucleotide sequence of the strain was approximately 1524 bp in length (NCBI GenBank Accession No.: PV546622). Comparison analysis using the NCBI online BLAST program with known sequences showed that GXJ-9 was homologous to the 16S rRNA gene sequence of Pseudomonas aeruginosa, with a similarity exceeding 99%. The phylogenetic tree of the 16S rRNA coding gene sequence was constructed using MEGA software and the neighbor-joining method. Figure 2 In summary, based on 16S rRNA gene phylogenetic analysis and physiological and biochemical characteristics, strain GXJ-9 was inferred to be Pseudomonas aeruginosa. This strain was subsequently streaked and deposited with the China Center for Type Culture Collection (CCTCC NO: M 20242844).

[0042] 3. Chromatographic quantification of degradation of trifloxysulfuron

[0043] (1) Sample pretreatment and detection methods containing trifloxysulfuron

[0044] Weigh 5.0 g of trifloxysulfuron sample into a 50 mL centrifuge tube with a cap, add 5 mL of ultrapure water, let it stand at room temperature for 0.5 h to allow the water to fully soak the sample, then add 10 mL of acetonitrile and vortex mix it for 2 min. Then add 1.0 g of NaCl and 1.0 g of MgSO4 and vortex it for 1 min until the aqueous phase is clear. Centrifuge it at 6,000 rpm for 5 min in a desktop refrigerated centrifuge, take 1.5 mL of the upper organic phase, add 50 mg of C 18 The mixture was purified by mixing with 150 mg of anhydrous MgSO4 in a 2 mL centrifuge tube and vortexing thoroughly for 1 min. After centrifugation at 4,000 rpm for 5 min, the supernatant was filtered through a 0.22 μm organic filter membrane and placed into a chromatographic injection vial for determination of trifloxysulfuron content by high performance liquid chromatography-mass spectrometry (HPLC-MS).

[0045] The chromatographic results were analyzed using an Agilent-1260 high performance liquid chromatograph with an automatic sample injector and a Zorbax Eclipse XDB-C 18 The chromatographic column was a 4.6 mm × 150 mm, 5 μm column. The following conditions were used: column temperature, 40°C; flow rate, 1 mL / min; injection volume, 10 μL; mobile phase, acetonitrile-0.1% formic acid (vol / vol ratio, 50:50). The mass spectrometer was an Agilent-6120 single-stage quadrupole mass spectrometer. Detection conditions were: electrospray ionization (ESI) source, positive ion scan; selected ion monitoring (SIM) with a mass-to-charge ratio (m / z) of 534.1; collision-induced dissociation voltage, 170 V; drying gas (nitrogen) flow rate, 12.0 L / min; capillary voltage, ±3000 V; drying gas temperature, 350°C; gain, 10.00; nebulizer pressure, 35.0 kPa; dwell time, 290 ms; and relative dwell time, 100%. Under this detection condition, the retention time of each sample is about 5 minutes. The chromatograms of trifloxysulfuron determination, blank control, and dissolved matrix are shown in Figure 3 .

[0046] (2) Metabolic performance of the strain to an initial concentration of 200 μg / L trifloxysulfuron at different temperatures

[0047] The bacterial suspension (OD) was inoculated into a 250 mL triangular flask containing 150 mL of BSM medium at a 2% inoculation ratio. 600=0.6-0.8), cultured at different temperatures (25°C, 30°C, 37°C, 42°C), and the speed of the constant temperature shaking incubator was set to 180 rpm during the culture process. In addition, samples were taken every 24 hours during the culture process, and the absorbance value of the bacterial solution at 600 nm was measured as OD 600 The biomass was characterized, the growth curve of the strain was drawn, and the residual concentration of trifloxysulfuron in the culture medium was detected.

[0048] The growth curves of the strains at the above different culture temperatures with 200 μg / L initial concentration of trifloxysulfuron as the only carbon and nitrogen source are shown in Figure 4 The degradation efficiency of trifloxysulfuron at different temperatures is shown in Figure 5 The strain was almost unable to grow below 25°C, but could grow within the range of 30-37°C. At 25°C, the degradation rate of the strain over 120 hours was only 14.3%. At 30°C and 42°C, the degradation rates were 85.3% and 85.9%, respectively. At 37°C, the strain had the best degradation effect, reaching 95.7%.

[0049] Degradation rate (%) = (initial concentration - residual concentration) / initial concentration × 100%.

[0050] (3) Metabolic performance of strains to different initial concentrations of trifloxysulfuron

[0051] The initial concentrations of trifloxysulfuron added to the BSM medium were set to 50 μg / L, 100 μg / L, 200 μg / L, 500 μg / L, and 1000 μg / L, respectively. A 250 mL triangular flask containing 150 mL of the above medium was inoculated with the bacterial suspension (OD 600 =0.6-0.8), and cultured in a constant temperature shaking incubator at 37°C and 180 rpm.

[0052] Under different initial concentrations of trifloxysulfuron, a rotation speed of 180 rpm, a temperature of 37° C., and an initial pH of 7.0, the culture was continuously carried out for 5 days, and the residual concentration of trifloxysulfuron in the culture medium was detected every 24 hours. Figure 6 The results showed that the strain had the best degradation effect on trifloxysulfuron with an initial concentration of 200μg / L, with a degradation rate of 84.7%; the degradation effect was second best when the initial concentration was 500μg / L, with a degradation rate of 70.1%; when the initial concentrations were 50μg / L, 100μg / L and 1000μg / L, the degradation rates were 55.6%, 54.7% and 49.8%, respectively.

[0053] (4) Metabolic performance of the strain to an initial concentration of 200 μg / L trifloxysulfuron at different culture medium pH

[0054] The logarithmic phase bacterial suspension (OD 600 =0.6-0.8), and cultured at different initial pH values ​​(6, 7, 8). The temperature was set at 37°C during the culture process, and samples were taken every 24 hours to determine the residual concentration of trifloxysulfuron in the culture medium.

[0055] The results are as follows Figure 7 As shown, when the initial pH of the culture medium is 7, the degradation rate of trifloxysulfuron by the strain reaches 85.1%; when pH is 6, the degradation rate of trifloxysulfuron by the strain is 39.6%; and when pH is 8, the degradation rate of trifloxysulfuron by the strain is only 34.7%.

[0056] 4. Application test of strains in soil

[0057] A single colony of Pseudomonas aeruginosa GXJ-9 stored on a BSM solid slant or plate was inoculated into 150 mL of BSM medium containing trifloxysulfuron for acclimation and cultured overnight on a shaker at 37°C. The culture was centrifuged at 4,000 rpm for 3 minutes, the supernatant was discarded, and the suspension was resuspended in sterile water. The suspension was centrifuged again at 4,000 rpm for 3 minutes, the supernatant was discarded, and the suspension was resuspended in sterile water. The OD value of the resuspended suspension was calculated. 600 Adjust to 1.0.

[0058] Application scenario example: 20 kg of trifloxysulfuron-contaminated surface soil was collected from a horticultural experimental station in Jiangxi Province. The soil was passed through a 2 mm sieve to remove impurities. After uniform mixing and sample preparation, the initial trifloxysulfuron concentration was determined by the aforementioned HPLC-MS method to be ~200 μg / kg. The soil was divided into 6 portions, each 3 kg, and placed in plastic containers. Three of the portions were added with bacterial suspension (OD 600 ≈0.01. A certain volume of the bacterial suspension was inoculated into trifloxysulfuron-contaminated soil at a 1% weight ratio (wt / wt). Three additional volumes served as untreated controls. All reaction vessels were placed in a constant-temperature incubator protected from light at a constant temperature of 30°C. The soil moisture content was maintained at 60% (ratio to the soil's maximum water holding capacity). The soil was stirred every two days to ensure uniform aeration.

[0059] During the experiment, soil samples were collected regularly (0, 12, 24, 48, 72, 96, and 120 hours). The samples were prepared using acetonitrile extraction to detect trifloxysulfuron residues in the soil. The content was analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS), and the degradation rate was calculated. The ecological remediation effect of the bacterial agent prepared based on the functional strain GXJ-9 on contaminated soil was evaluated.

[0060] The degradation effect of strain GXJ-9 on trifloxysulfuron in soil is as follows Figure 8After 120 hours of cultivation, the degradation rate of trifloxysulfuron in the soil of the experimental group reached 89.2%, while the trifloxysulfuron content in the control group showed almost no significant change, indicating that strain GXJ-9 has a significant ability to degrade trifloxysulfuron, a pollutant in the soil.

[0061] 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 in the scope of protection of the present invention.

Claims

1. A Pseudomonas aeruginosa strain, characterized in that: The strain is Pseudomonas aeruginosa GXJ-9, deposited in China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 20242844.

2. The use of Pseudomonas aeruginosa GXJ-9 according to claim 1, characterized in that The strain is the application of Pseudomonas aeruginosa GXJ-9 in degrading the herbicide trifloxysulfuron.

3. The use of Pseudomonas aeruginosa GXJ-9 according to claim 2, characterized in that The Pseudomonas aeruginosa GXJ-9 is used to degrade the herbicide trifloxysulfuron in soil.

4. The use of Pseudomonas aeruginosa GXJ-9 according to claim 2 or 3, characterized in that Before degrading the herbicide trifloxysulfuron, the Pseudomonas aeruginosa GXJ-9 was inoculated into BSM culture medium and cultured overnight at 37° C. for activation and acclimation.

5. The use of Pseudomonas aeruginosa GXJ-9 according to claim 2, characterized in that The working concentration of Pseudomonas aeruginosa GXJ-9 was 400 μg / mL cultured in liquid until the logarithmic phase OD 600 The bacterial concentration is 1.0, and the application ratio is 1% of bacterial amount to soil mass.

6. The use of Pseudomonas aeruginosa GXJ-9 according to claim 2, characterized in that The degradation time of the herbicide trifloxysulfuron in soil containing the herbicide at a concentration of 200 μg / kg by the Pseudomonas aeruginosa GXJ-9 is 120 hours.

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