A strain of Pseudomonas aeruginosa capable of degrading triflusulfuron and its application in soil pollution control

Abstract

This invention relates to the field of environmental pollution ecological remediation technology and its application in the treatment of trifluralin-sulfuron-methyl pollution. The purpose of this invention is to address the existing problem of trifluralin-sulfuron-methyl pollution in soil. A strain of *Pseudomonas aeruginosa*, specifically *Pseudomonas aeruginosa* GXJ-9, is disclosed, deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20242844. The invention also discloses the application of *Pseudomonas aeruginosa* GXJ-9 in degrading trifluralin-sulfuron-methyl contaminated soil. This invention provides a strain of *Pseudomonas aeruginosa* and its application.

Description

Technical Field

[0001] This invention relates to the field of environmental microbial remediation technology, specifically to a strain of *Pseudomonas aeruginosa* capable of degrading trifluralin and its application in soil pollution control. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on December 18, 2024, with accession number CCTCC NO: M 20242844. Background Technology

[0002] Trifluralin is a protoporphyrinogen oxidase (PPO) inhibitor herbicide, a novel ureapyridine contact herbicide developed by Fuamuhannong Co., Ltd. It is primarily used to control monocot and dicot weeds, as well as glyphosate-resistant weeds, and is considered an alternative to glyphosate and paraquat. Its chemical formula is C1. 19 H 18 ClF4N3O5S is a white powder. Ureaprimidine herbicides belong to the protoporphyrinogen oxidase inhibitor class. Their mechanism of action is to inhibit the formation of protoporphyrinogen oxidase (PPO) during chlorophyll synthesis, leading to the accumulation of photosensitizing protoporphyrinogen IX. The resulting reactive oxygen species (ROS) cause lipid peroxidation, leading to the loss of membrane function in weed cells, inhibiting plant growth, and even causing death. However, trifluralin herbicides can seep into the soil during application, causing soil pollution and even contaminating surface water, leading to chronic accumulation and exposure risks for aquatic plants and animals. Therefore, reducing the environmental impact and potential risks of trifluralin herbicides is crucial.

[0003] Biological treatment methods for eliminating pollution and remediating soil have advantages such as being environmentally friendly, having mild conditions, saving resources, and producing non-toxic and harmless degradation products.

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

[0005] The purpose of this invention is to provide a strain of Pseudomonas aeruginosa GXJ-9 that can degrade trifluralin, effectively degrading trifluralin in contaminated soil.

[0006] The detailed technical solution adopted in this application is as follows.

[0007] In a first aspect, the present invention provides a trifluralinsulfuron-methyl degrading bacterium, wherein the strain is Pseudomonas aeruginosa GXJ-9, which is deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M20242844.

[0008] Secondly, the present invention provides an application of Pseudomonas aeruginosa GXJ-9, specifically the application of Pseudomonas aeruginosa GXJ-9 in degrading trifluralin in contaminated soil.

[0009] Specifically, *Pseudomonas aeruginosa* GXJ-9 was cultured overnight at 37°C in Basic Salt Medium (BSM) with triflusulfanilamide as the sole carbon and nitrogen source. The bacterial cells were then 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% of the soil mass, and the degradation time for soil containing triflusulfonamide at a concentration of ~200 μg / kg was 120 h.

[0010] The Pseudomonas aeruginosa GXJ-9 strain and its cultivation method of the present invention have the following advantages over the prior art: The Pseudomonas aeruginosa GXJ-9 strain of the present invention has the characteristic of degrading trifluralin, and is suitable for the treatment of soil and water bodies contaminated by the herbicide trifluralin, without the need for dilution or additional carbon source to maintain the stability of the bacterial agent system. Attached Figure Description

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

[0012] Figure 2 This invention presents a phylogenetic tree of strain GXJ-9 constructed based on the 16S rRNA coding gene sequence using the Neighbor-Joining method (NJ) and 1000 bootstrap iterations.

[0013] Figure 3 The following are liquid chromatograms for the determination of triflumethrin in this invention: (a) blank triflumethrin solution (100 μg / L); (b) sample dissolved in matrix with triflumethrin (100 μg / L); (c) acetonitrile as mobile phase blank for liquid chromatography; (d) sample dissolved in matrix blank for stabilization purposes.

[0014] Figure 4 Growth curves of the strain of the present invention inoculated at 2% BSM medium (with 200 μg / L trifluralin as the sole carbon and nitrogen source) at different culture temperatures;

[0015] Figure 5 The graph shows the metabolic performance of the strain of the present invention at an initial concentration of 200 μg / L trifluralin sulfate at different culture temperatures after 2% inoculation into BSM medium.

[0016] Figure 6 The graph shows the metabolic performance of the strain of this invention when inoculated at 2% in BSM medium with different initial concentrations of 50-1000 μg / L trifluralinsulfuron-methyl.

[0017] Figure 7 The graph shows the metabolic performance of the strain of the present invention at an initial concentration of 200 μg / L trifluralin sulfate under different initial pH values ​​after 2% inoculation into BSM medium.

[0018] Figure 8 The application effect of the strain of the present invention at a mass ratio of 1% in farmland soil containing 200 μg / kg trifluralin was investigated. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples can be obtained commercially or publicly. The specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0020] Based on the information contained in this application, those skilled in the art can readily make various changes and optimizations to the precise description of the invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the 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 invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0021] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values ​​used in this application should, in all cases, be understood to be modified by the word "about". Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods. In this invention, "about" means within 10%, preferably within 5%, of a given value or range.

[0022] Unless otherwise specified, the following embodiments of the present invention are all under normal temperature conditions. Normal temperature refers to the natural room temperature conditions in all four seasons, without additional cooling or heating treatment. Generally, the normal temperature is controlled between 10 and 30°C, preferably between 15 and 25°C.

[0023] Unless otherwise specified, the reagents and materials used in the embodiments of this invention can be purchased commercially.

[0024] The trifluralin sulfate-degrading strain of the present invention is Pseudomonas aeruginosa GXJ-9, which was deposited on December 18, 2024, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Luojia Mountain, Wuchang District, Wuhan, Hubei Province, 430072, China, with accession number CCTCC NO: M 20242844.

[0025] 1. Isolation of strains

[0026] 1) Culture medium

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

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

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

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

[0031] 2) Separation method

[0032] Take 1g of soil sample treated with trifluralin and add it to 20mL of sterile water. Shake well and add 2mL of the solution to 100mL of LB medium (Luria-Bertani Broth) for enrichment culture. Incubate the solution in a constant temperature shaking incubator (30℃, 180rpm) for 18h. Take 2mL of the enriched bacterial solution and transfer it to 100mL of BSM liquid medium containing 200μg / L trifluralin. Incubate the solution in a constant temperature shaking incubator (30℃, 180rpm).

[0033] To assess the removal efficiency of candidate degrading bacteria against triflusulfonamide, the residual triflusulfonamide content in the culture medium was determined by high-performance liquid chromatography (HPLC). The bacteria were subcultured at a 2% culture medium ratio, and the enrichment process was repeated until the triflusulfonamide removal efficiency reached a stable level. One mL of the enriched bacterial solution was then serially diluted, with the dilution factor set to 10⁻⁶ based on hemocytometer and microscopic counting results. -4 10 -5 10 -6 Take 150 μL of bacterial suspension at different dilution ratios and spread it on BSM solid plates containing triflusulfonamide. Incubate at 30℃ for two days. Pick a single colony and streak it on a BSM plate containing triflusulfonamide. Repeat 2-3 times until a single colony is finally isolated. Name this strain GXJ-9.

[0034] 2. Identification of strains

[0035] 1) Bacterial morphological characteristics

[0036] The strain was cultured in LB liquid at 30℃, 180 rpm, and pH=7 until the logarithmic growth phase. Aseptically, an inoculating loop was used to streak GXJ-9 bacterial suspension onto LB agar plates with an initial concentration of 200 μg / L trifluralin. The plates were then incubated at 30℃ in a biochemical incubator. Observation was performed after single colonies formed on the plates. GXJ-9 colonies were round, pale yellow, and smooth. Figure 1 Using Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli as controls, Gram staining and microscopic examination were performed. Based on the staining results, GXJ-9 was presumed to be a Gram-negative bacterium. Subsequent physiological and biochemical experiments were conducted, including catalase, oxidase, indole, methyl red, VP (Voges-Proskauer), gelatin liquefaction, and nitrate reduction. The identification results of the corresponding physiological and biochemical experiments 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] Using GXJ-9 genomic DNA extracted by the boiling method as a template, PCR amplification was performed using the universal primers for bacterial 16S rRNA cloning: 27F (forward, 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (reverse, 5'-GGTTA CCTTGTTACGACTT-3'). The amplification conditions on the Bio-Rad S1000 gradient PCR instrument were set as follows: ① 95℃ pre-denaturation for 10 min, ② then 95℃ denaturation for 30 s, ③ annealing at 66-57℃ for 30 s, ④ extension at 72℃ for 40 s, steps ②-④ were repeated 10 times (decreasing the temperature by 1℃ per cycle), ⑤ then 95℃ denaturation for 30 s, ⑥ annealing at 57℃ for 30 s, ⑦ extension at 72℃ for 40 s, steps ⑤-⑦ were repeated 21 times, ⑧ finally, extension at 72℃ for 10 min, ⑨ after completion, the amplified products were stored at 4℃. The reaction mixture consisted of: 10 μL of 2×Phanta Max Super-Fidelity DNA Polymerase Mix (Vazyme Biotech Co., Ltd., Nanjing, China), 1 μL each of forward and reverse primers, 0.5 μL of 50% dimethyl sulfoxide (DMSO), 1 μL of DNA template, and ddH2O to a total volume of 25 μL. After amplification, 1.5% agarose gel electrophoresis was performed, and gel imaging was used to detect the band size of the amplified products. The PCR products were then excised and recovered for sequencing.

[0041] The effective nucleotide sequence of the amplified strain's 16S rRNA gene was approximately 1524 bp (NCBI GenBank Accession No.: PV546622). Alignment 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 extracted 16S rRNA coding gene sequence, constructed using MEGA software and neighbor-joining, is detailed below. Figure 2 In summary, based on the phylogenetic analysis of the 16S rRNA gene and its physiological and biochemical characteristics, strain GXJ-9 is presumed to be *Pseudomonas aeruginosa*. The strain was subsequently streaked and deposited at the China Center for Type Culture Collection (CCTCC NO: M 20242844).

[0042] 3. Quantitative chromatographic detection of trifluralin sulfate degradation

[0043] (1) Sample pretreatment and detection methods for triflusulfuron-methyl

[0044] Weigh 5.0 g of trifluralin sulfate sample into a 50 mL capped centrifuge tube, add 5 mL of ultrapure water, and let stand at room temperature for 0.5 h to allow the water to fully wet the sample. Then add 10 mL of acetonitrile and vortex mix for 2 min. Next, add 1.0 g of NaCl and 1.0 g of MgSO4, and vortex for 1 min until the aqueous phase is clear. Centrifuge at 6,000 rpm for 5 min using a benchtop refrigerated centrifuge, and take 1.5 mL of the upper organic phase. Add 50 mg of C... 18 The solution was purified by adding 150 mg of anhydrous MgSO4 to a 2 mL centrifuge tube and vortexing thoroughly for 1 min. After centrifugation at 4,000 rpm for 5 min, the supernatant was collected, filtered through a 0.22 μm organic filter membrane, and placed into a chromatographic sample vial. The content of trifluralin was determined by high performance liquid chromatography-mass spectrometry (HPLC-MS).

[0045] Using an Agilent-1260 high-performance liquid chromatograph with an automated sampler and a Zorbax Eclipse XDB-C... 18 (4.6 mm × 150 mm, 5 μm) chromatographic column; determination conditions: column temperature 40℃; flow rate 1 mL / min; injection volume 10 μL; mobile phase acetonitrile-0.1% formic acid water (vol / vol volume ratio 50:50). Mass spectrometer: Agilent-6120 single-stage quadrupole mass spectrometer; detection conditions: electrospray ionization (ESI) ion source: positive ion scan; selected ion monitoring (SIM) mass-to-charge ratio (m / z) set to 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℃; gain 10.00; nebulizer gas pressure 35.0 kPa; residence time 290 ms; relative residence 100%. Under these detection conditions, the retention time for each sample is approximately 5 minutes. The determination of trifluralin, its blank control, and the chromatogram of the dissolved matrix are shown below. Figure 3 .

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

[0047] Inoculate the bacterial suspension (OD) at a 2% inoculation ratio into a 250 mL Erlenmeyer flask containing 150 mL of BSM medium. 600=0.6-0.8), and cultured at different temperatures (25℃, 30℃, 37℃, 42℃), with the rotation speed of the constant temperature shaking incubator set to 180 rpm during the culture process. In addition, samples were taken every 24 hours during the culture process, and the absorbance value at 600 nm was measured as OD. 600 Biomass was characterized, growth curves of the strains were plotted, and the residual concentration of trifluralin in the culture medium was detected.

[0048] The growth curves of the strains at different culture temperatures using an initial concentration of 200 μg / L trifluralin as the sole carbon and nitrogen source are shown below. Figure 4 The degradation efficiency of triflusulfonamide at different temperatures is shown in the figure. Figure 5 The strain can hardly grow below 25℃, but can grow in the range of 30-37℃. At 25℃, the degradation rate of the strain after 120 hours is only 14.3%; at 30℃ and 42℃, the degradation rates are 85.3% and 85.9%, respectively; the strain exhibits the best degradation effect at 37℃, with a degradation rate reaching 95.7%.

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

[0050] (3) Metabolic performance of strains on different initial concentrations of triflusulfonamide

[0051] The initial concentrations of trifluralin added to BSM medium were set at 50 μg / L, 100 μg / L, 200 μg / L, 500 μg / L, and 1000 μg / L. The bacterial suspension (OD) was inoculated at a 2% inoculum into 150 mL Erlenmeyer flasks containing the above medium. 600 =0.6-0.8), and cultured in a constant temperature shaking incubator at 37℃ and 180rpm.

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

[0053] (4) Metabolic performance of the strain on an initial concentration of 200 μg / L triflusulfonamide under different culture medium pH conditions

[0054] Inoculate the logarithmic phase bacterial suspension (OD) into a 250 mL Erlenmeyer flask containing 150 mL of BSM medium at a 2% inoculation ratio. 600 =0.6-0.8), and cultured at different initial pH (6, 7, 8). The temperature was set at 37℃ during the culture process, and the residual concentration of trifluralin in the culture medium was measured every 24 hours.

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

[0056] 4. Application test of the strain in soil

[0057] A single colony of *Pseudomonas aeruginosa* GXJ-9, preserved on BSM solid slants or plates, was inoculated into 150 mL of BSM medium containing trifluralin for acclimatization and activated by shaking overnight at 37°C. The overnight culture was centrifuged at 4,000 rpm for 3 min, the supernatant was discarded, and the culture was resuspended in sterile water. The culture was then centrifuged again at 4,000 rpm for 3 min, the supernatant was discarded, and the culture was resuspended in sterile water. The OD of the resuspended culture was measured. 600 Adjusted to 1.0.

[0058] Application Scenario Example: 20 kg of topsoil contaminated with trifluralin was collected from a horticultural experimental station in Jiangxi Province. The soil was sieved through a 2 mm sieve to remove impurities, and after uniform mixing, the initial trifluralin concentration was determined to be ~200 μg / kg using the aforementioned HPLC-MS method. The soil was divided into 6 portions, each weighing 3 kg, and placed in separate plastic containers. Three portions were supplemented with a bacterial suspension (OD200 of the bacterial suspension after dilution). 600 ≈0.01, a certain volume of bacterial suspension was inoculated into contaminated soil containing trifluralin at a mass ratio of 1% (wt / wt), and another 3 portions served as an untreated control group. All reaction containers were placed in a constant temperature incubator in the dark, with the temperature controlled at a constant 30℃ and the soil moisture content maintained at 60% (the proportion of the soil's maximum water holding capacity). The soil was turned over every 2 days to ensure uniform aeration.

[0059] Soil samples were collected periodically (0, 12, 24, 48, 72, 96, 120 h) during the experiment. The acetonitrile extraction method was used to prepare samples to detect trifluralin residues in the soil. The content was analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS), the degradation rate was calculated, and the ecological restoration effect of the inoculant prepared based on functional strain GXJ-9 on contaminated soil was evaluated.

[0060] The degradation effect of strain GXJ-9 on trifluralin in soil is as follows: Figure 8As shown in the figure, after 120 hours of cultivation, the degradation rate of trifluralin in the soil of the experimental group reached 89.2%, while the trifluralin content in the control group showed almost no significant change, indicating that strain GXJ-9 has a significant degradation ability for the herbicide trifluralin 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 within the protection scope of the present invention.

Claims

1. A strain of Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) strain, characterized in that, The strain is Pseudomonas aeruginosa GXJ-9, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20242844.

2. The application of Pseudomonas aeruginosa GXJ-9 as described in claim 1, characterized in that, The application of Pseudomonas aeruginosa GXJ-9 in the degradation of the herbicide trifluralin.

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

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

5. The application of Pseudomonas aeruginosa GXJ-9 according to claim 2, characterized in that, Working concentrations of Pseudomonas aeruginosa GXJ-9 were obtained by liquid culture to the logarithmic OD phase. 600 The bacterial concentration is 1.0, and it is applied at a bacterial count to soil mass ratio of 1%.

6. The application of Pseudomonas aeruginosa GXJ-9 according to claim 2, characterized in that, The *Pseudomonas aeruginosa* GXJ-9 strain degraded the herbicide trifluralin at a concentration of 200 μg / kg in soil for 120 hours.

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