Achromobacter lung SX5 and application thereof

Abstract

The invention discloses Achromobacter Pneumoniae SX5 and an application thereof. The Achromobacter Pneumobacter SX5 is classified and named as Achromobacter Pneumobacter, and is preserved in the China General Microbiological Culture Collection Center on June 6, 2025, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.34789. The Achromobacter Pneumobacter SX5 is named as Achromobacter Pneumobacter Pneumobacter Pneumobacter Pneumobacter Pneumobacter Pneumobacter Pneumobacter Pneumobacter. According to the invention, the achromobacter lung SX5 is found to have degradation activity on the bifenthrin for the first time, and can degrade the bifenthrin with the initial concentration of 25-600mg / L; under the conditions that the temperature is 27 DEG C, the pH value is 5 and the inoculum size is 4%, degradation is carried out for 5 days, and the degradation rate of 50 mg / L bifenthrin can reach 80.14%. The achromobacter lung SX5 has a wide temperature and pH application range, and the half-life period of the achromobacter lung SX5 is from 2.203 d to 3.648 d. In addition, the Achromobacter Pneumoniae SX5 has a certain broad spectrum of substrates, and can be used for degrading a plurality of other pyrethroid insecticides such as beta-cypermethrin, beta-cyhalothrin, deltamethrin and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a type of Achromobacterium pulmonaryis SX5 and its applications. Background Technology

[0002] Pyrethroids are synthetic pesticides structurally similar to natural pyrethroids. Due to their high efficiency and low toxicity, they are widely used in production and daily life. Bifenthrin is one of the most produced and used pyrethroids. Its insecticidal efficacy is related to its toxic effects on insect nerve cells; it can disrupt nerve signal transmission by controlling the insect's sodium ion channels, thus leading to insect death. Because of its high insecticidal rate, bifenthrin is widely used in agriculture, forestry, urban greening, and household spraying.

[0003] With the large-scale use of bifenthrin, its presence has been detected in farmland, rivers, and even animals and humans, posing a potential threat to the ecological environment and human health. Bifenthrin can enter the ecosystem through atmospheric deposition, surface water infiltration, agricultural drainage, and pesticide factory wastewater discharge. Due to the presence of a hydrocarbon ring in its structure, bifenthrin is highly hydrophobic, allowing it to bind strongly to soil particles and organic matter, leading to its residues seeping into deep soil and water environments, with residual periods lasting 6-75 days or even up to 8 months.

[0004] To reduce the environmental and public health risks associated with the widespread use of bifenthrin, it is necessary to develop a green and effective method to remove or reduce bifenthrin residues in the environment. Because bifenthrin is highly persistent and resistant to photodegradation, biological methods based on the degradative metabolic activity of microorganisms are the most promising and effective strategy for removing bifenthrin residues. Currently, bifenthrin-degrading bacteria are mostly identified as *Pseudomonas*, *Bacillus*, *Acinetobacter*, and *Sphingobacter*. However, there are no reports on the degradation of bifenthrin by *Achromobacterium pulmonale*, and the number of strains capable of simultaneously and efficiently degrading multiple pyrethroids is relatively limited. Therefore, it is necessary to further screen for strains that can efficiently degrade bifenthrin to expand the pyrethroid-degrading bacterial resources. Summary of the Invention

[0005] The purpose of this invention is to provide a lung achromobacterium SX5 and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides a lung achromobacter SX5, which is classified and named lung achromobacter pulmonis. It was deposited on June 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34789.

[0007] A second aspect of the present invention provides the application of the aforementioned *Achromobacterium pulmonaryis* SX5 in the degradation of pyrethroid insecticides; Furthermore, the pyrethroid insecticides include cypermethrin, deltamethrin, permethrin, cyhalothrin, fenvalerate, deltamethrin, bifenthrin, and cis-cypermethrin.

[0008] A third aspect of the present invention provides the application of the aforementioned *Achromobacterium pulmonaryis* SX5 in the preparation of a pyrethroid insecticide-degrading bacterial agent; Furthermore, the pyrethroid insecticides include cypermethrin, deltamethrin, permethrin, cyhalothrin, fenvalerate, deltamethrin, bifenthrin, and cis-cypermethrin.

[0009] A fourth aspect of the present invention provides a pyrethroid insecticide degrading bacterial agent comprising the aforementioned Achromobacterium pulmonaryis SX5; Furthermore, the preparation method of the pyrethroid insecticide degrading bacterial agent includes: inoculating the glycerol-preserved bacteria of Achromobacterium pulmonarye SX5 into LB liquid medium, and culturing overnight at 180 rpm and 30°C to obtain Achromobacterium pulmonarye SX5 seed liquid, which is the pyrethroid insecticide degrading bacterial agent.

[0010] The fifth aspect of the present invention provides a method for degrading pyrethroid insecticides, which involves inoculating the above-mentioned pyrethroid insecticide degrading bacteria into LB liquid culture medium containing pyrethroid insecticides and culturing them. Furthermore, the culture temperature is 20-40℃, the pH of the culture medium is 4-11, and the inoculation amount of the pyrethroid insecticide degrading bacteria is 1%-5%. Furthermore, the culture temperature is 27°C, the pH of the culture medium is 5, and the inoculum amount of the pyrethroid insecticide degrading bacteria is 4%.

[0011] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a *Achromobacterium pulmonaryum* SX5 strain, which is the first reported strain to possess the ability to degrade bifenthrin. On day 5, it achieved a degradation rate of 80.14% for 50 mg / L bifenthrin, exhibiting higher degradation activity compared to currently reported bifenthrin-degrading bacteria. This strain demonstrates degradation capabilities for bifenthrin concentrations ranging from 25 to 600 mg / L, with a theoretical half-life of 2.209–3.648 days. The theoretical half-life for bifenthrin concentrations of 400–600 mg / L is significantly lower than that of most bifenthrin-degrading bacteria in this concentration range. Furthermore, this strain exhibits broad substrate spectrum, capable of degrading various other pyrethroid insecticides. The isolation and identification of *Achromobacterium pulmonaryum* SX5 enriches the resource library of pyrethroid insecticide-degrading bacteria, providing a theoretical basis and technical support for the practical application of pyrethroid insecticide-degrading strains in the environment. Attached Figure Description

[0012] Figure 1 Colony morphology and electron micrograph of Achromobacterium pulmonaryis SX5.

[0013] Figure 2 Phylogenetic tree of Achromobacterium pulmonaryis SX5.

[0014] Figure 3 HPLC chromatogram of bifenthrin degradation by Achromobacterium pulmonaryis SX5.

[0015] Figure 4 Growth of Achromobacterium pulmonaryis SX5 and degradation of bifenthrin.

[0016] Figure 5 Tolerance of Achromobacterium pulmonaryis SX5 to different concentrations of bifenthrin.

[0017] Figure 6 Degradation of bifenthrin by Achromobacterium pulmonale SX5 under different single-factor conditions. (a) Degradation of bifenthrin at different culture temperatures; (b) Degradation of bifenthrin at different culture medium pH; (c) Degradation of bifenthrin at different inoculum amounts.

[0018] Figure 7 Three-dimensional response surface plots of the response surface optimization experiment of Achromobacterium pulmonaryis SX5 degrading bifenthrin. (a) The interaction between culture temperature and culture medium pH on Achromobacterium pulmonaryis SX5 degrading bifenthrin; (b) The interaction between culture temperature and inoculum size on Achromobacterium pulmonaryis SX5 degrading bifenthrin; (c) The interaction between culture medium pH and inoculum size on Achromobacterium pulmonaryis SX5 degrading bifenthrin.

[0019] Figure 8(a) Contour plots showing the effects of culture temperature and culture medium pH on bifenthrin degradation; (b) Contour plots showing the effects of culture medium pH and inoculum size on bifenthrin degradation; (c) Cubic plots showing bifenthrin degradation.

[0020] Figure 9 Degradation kinetics of bifenthrin at different initial concentrations by Achromobacterium pulmonaryis SX5.

[0021] Figure 10 Inhibition curves of different substrate concentrations on the degradation rate of Achromobacterium pulmonaryis SX5.

[0022] Figure 11 Degradation of different pyrethroid insecticides by Achromobacterium pulmonaryis SX5. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0024] 1. Experimental Materials and Methods 1.1 The materials involved in the following embodiments are as follows: Tetramethrin, β-cypermethrin, permethrin, deltamethrin, λ-cyhalothrin, and bifenthrin were purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a purity >97%. Fenvalerate and α-cypermethrin were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with a purity >99%. Fenpropathrin was purchased from Tanmo Quality Inspection Technology Co., Ltd., with a purity of 97.7%.

[0025] 1.2 The culture media involved in the implementation are as follows: LB liquid medium: NaCl 10g / L, tryptone 10g / L, yeast extract 5g / L, the remainder is water, pH is natural; sterilize at 121℃ for 20min before use.

[0026] LB solid medium: NaCl 10g / L, tryptone 10g / L, yeast extract 5g / L, plus 1.5% agar powder, the remainder is water, pH is natural; sterilize at 121℃ for 20min before use.

[0027] 1.3 The enrichment and screening steps for the target strains are as follows: (1) Gradual enrichment and domestication of microorganisms in the environment using bifenthrin as a substrate: For the first round of acclimatization, 5g of soil sample from farmland in Shaanxi Province was added to 50mL of LB liquid medium containing 50mg / L bifenthrin and cultured at 180rpm and 30℃ for 5 days to obtain the first round of enriched culture. For the second round of acclimatization, 5mL of the first round enriched culture was added to 50mL of LB liquid medium containing 100mg / L bifenthrin and cultured at 180rpm and 30℃ for 5 days to obtain the second round of enriched culture. For the third round of acclimatization, 5mL of the second round enriched culture was added to 50mL of LB liquid medium containing 200mg / L bifenthrin and cultured at 180rpm and 30℃ for 5 days to obtain the third round of enriched culture. For the fourth round of acclimatization, 5mL of the third round enriched culture was added to 50mL of LB liquid medium containing 400mg / L bifenthrin and cultured at 180rpm and 30℃ for 5 days to obtain the fourth round of enriched culture. For the fifth round of acclimatization, 5 mL of the fourth round enriched culture was added to 50 mL of LB liquid medium containing 100 mg / L bifenthrin and cultured at 180 rpm and 30 °C for 5 days to obtain the fifth round enriched culture.

[0028] (2) Isolation and purification of bifenthrin-degrading bacteria 200 μL of the fifth-round enrichment culture was diluted and spread onto LB solid medium containing 50 mg / L bifenthrin. The medium was incubated in the dark at 30°C. Single colonies with good growth were selected for streak plating purification to isolate single bacteria. The purified single bacteria were inoculated into 5 mL of LB liquid medium and incubated overnight. After mixing with an equal volume of 50% glycerol, the culture was stored at -80°C. Before use, 50 μL of the preserved culture was removed from the -80°C culture and inoculated into 5 mL of LB liquid medium. The culture was incubated overnight at 180 rpm and 30°C to serve as the seed culture for subsequent experiments.

[0029] (3) Secondary screening: The bifenthrin degradation activity of the strains obtained in the initial screening was analyzed. 1 mL of seed culture (5 vol%) was inoculated into 20 mL of LB liquid medium containing 50 mg / L bifenthrin. All experiments were performed in triplicate, with a control without seed culture. Samples were cultured at 30℃ and 180 rpm for 5 days, and then analyzed by HPLC to determine the target strain with bifenthrin degradation activity. Finally, a strain SX5 with good bifenthrin degradation activity was obtained.

[0030] 1.4 Identification of bifenthrin-degrading bacteria (1) Morphological identification of bifenthrin-degrading bacteria The strain SX5 obtained from the secondary screening was streaked on LB agar plates and incubated overnight at 37°C. Colonies were observed. Single-cell morphology was observed using scanning electron microscopy (SEM).

[0031] (2) Molecular biological identification of bifenthrin-degrading bacteria Total DNA was extracted from strain SX5 using the TIANamp Bacteria DNA Kit 50. The 16S rDNA gene of the strain was amplified by PCR using universal bacterial primers 16S(F) (5'-AGAGTTTGSTCCTGGCTCAG-3') and 16S(R) (5'-GGTTACCTTGTTACGACTT-3'). The reaction mixture consisted of 25 μL: 12.5 μL of 2×TaqMaster Mix, 1 μL of 16S(F) (10 μmol / L), 1 μL of 16S(R) (10 μmol / L), 1 μL of DNA template, and 9.5 μL of ddH2O. The reaction program was as follows: pre-denaturation at 94℃ for 3 min; denaturation at 94℃ for 3 s; annealing at 50℃ for 45 s; extension at 72℃ for 1 min 40 s, for a total of 35 cycles; and a final extension at 72℃ for 7 min. The amplified PCR products were subjected to agarose gel electrophoresis and recovered using the SanPrep Column DNA Gel Extraction Kit. After verification by 1% agarose gel electrophoresis, the recovered products were ligated with the pMD9-T Vector at 16℃ and then transformed into E. coli DH5α competent cells. The cells were plated on LB plates containing 100 μg / mL Amp and cultured for 16 h. Single colonies were picked and cultured in 1 mL of LB liquid medium (containing 100 μg / mL Amp) at 37℃ and 180 rpm for 12 h. The bacterial culture was then used for PCR verification. The amplified products were detected by 1% agarose gel electrophoresis. The bacterial cultures containing the target band were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0032] Phylogenetic tree construction: The 16S rDNA gene sequences were compared with the NCBI database, and sequences with high similarity were selected to construct a phylogenetic tree using MEGA 11.0 software.

[0033] The standard and sample processing methods involved in 1.5 are as follows: (1) Preparation of standard curves: Preparation of standard samples of high efficiency cypermethrin, cypermethrin, fenvalerate, deltamethrin and bifenthrin: Weigh a certain amount of standard powder and dissolve it in chromatographic grade methanol to prepare a stock solution with a concentration of 10 g / L. Dilute it stepwise with chromatographic grade methanol to a concentration gradient of 500 mg / L, 200 mg / L, 100 mg / L, 50 mg / L, 25 mg / L and 12.5 mg / L. After filtration through a 0.22 μm organic membrane, the samples were detected by HPLC. The peak area was taken as the abscissa and the corresponding concentration was taken as the ordinate to construct a standard curve.

[0034] (2) Sample preparation: Add an equal volume of ethyl acetate to an Erlenmeyer flask containing culture medium, sonicate for 30 min to ensure the substrate is fully dissolved in the ethyl acetate, add the mixture to a 50 mL centrifuge tube, shake thoroughly, centrifuge at 3500 rpm for 3 min, take 2 mL of the supernatant into a nitrogen blow-off tube, blow with nitrogen for 15 min until dry, then redissolve in 2 mL of chromatographic grade methanol, and filter through a 0.22 μm organic membrane. After sample preparation, store at 4℃ for testing. Calculation method for degradation rate of bifenthrin and other pyrethroids: C1 represents the concentration of bifenthrin after treatment with Achromobacterium pulmonaryis SX5, and C0 represents the concentration of bifenthrin in the blank control.

[0035] HPLC detection conditions: Column: Shimadzu C18 column (4.6*150mm; 5μm); Column temperature: 35℃; Flow rate: 0.5mL / min; Detection wavelength: 254nm; Injection volume: 10μL; Mobile phase: 95% chromatographic grade methanol; Run time: 14min.

[0036] 1.6 Pulmonary Achromobacterium SX5 Biodegradation of Bifenthrin A 5 vol% inoculum of *Achromobacterium pulmonale* SX5 seed culture was inoculated into 30 mL LB liquid medium containing 50 mg / L bifenthrin. Three replicate groups were set up (with no bacteria as a blank control). The cultures were incubated at 150 rpm and 30 °C for 0, 1, 3, 5, 7, 9, and 11 days. Bifenthrin was extracted using the complete culture medium as described in section 1.5. Bifenthrin residues were detected by HPLC, and bacterial growth was periodically monitored at 600 nm using a UV / Vis spectrophotometer. Bifenthrin degradation curves and bacterial growth curves were plotted.

[0037] A 5 vol% inoculum of *Achromobacterium pulmonale* SX5 seed culture was inoculated into 20 mL LB liquid medium containing bifenthrin at final concentrations of 25 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 400 mg / L, 600 mg / L, and 800 mg / L. Three replicates were set up (with no bacteria as a blank control). The cultures were incubated at 150 rpm and 30 °C for 5 days. Bifenthrin was extracted using the whole culture medium as described in 1.5. Bifenthrin residues were detected by HPLC, and bacterial growth was detected by microplate reader at 600 nm.

[0038] 1.7 Optimization of degradation conditions for Achromobacterium pulmonaryis SX5 Single-factor experiments were conducted to determine the optimal ranges for culture temperature, pH, and inoculum size for *Achromobacterium pulmonaryis* SX5 in degrading bifenthrin. Response surface methodology (RSM) was used to optimize the three factors, employing a Box-Behnken model. The independent variables were culture temperature, culture medium pH, and inoculum size, denoted as X1, X2, and X3, respectively, with -1, 0, and 1 representing the levels of the independent variables. Design-Expert 13 statistical software was used for design and analysis to establish a quadratic polynomial response surface regression equation between the degradation rate (Y) of 50 mg / L bifenthrin and the three independent variables. Based on the obtained regression model, the theoretical optimal degradation conditions for *Achromobacterium pulmonaryis* SX5 were predicted.

[0039] 1.8 Kinetic Study of Bifenthrin Degradation by *Achromobacterium pulmonale* SX5 at Different Concentrations: *Achromobacterium pulmonale* SX5 was inoculated into 20 mL of LB liquid medium (pH 5) containing 25 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 400 mg / L, and 600 mg / L bifenthrin, respectively, at 27℃ and 150 rpm for 0, 1, 3, 5, 7, 9, 11, and 13 days. Bifenthrin was extracted using the whole culture medium as described in 1.5. The degradation rate of bifenthrin by *Achromobacterium pulmonale* SX5 at each concentration was determined, and bifenthrin degradation kinetic curves were plotted. The Andrews equation was used to analyze the kinetics of the biodegradation process of bifenthrin at different concentrations.

[0040] 1.9 Broad-spectrum degradation of Achromobacterium pulmonaryis SX5: Achromobacterium pulmonaryis SX5 seed culture was inoculated at a 5 vol% inoculum into 20 mL LB liquid medium containing 50 mg / L of cypermethrin, lambda-cyhalothrin, permethrin, cyhalothrin, fenvalerate, deltamethrin, bifenthrin, and cis-cypermethrin. The culture was incubated at 30 °C and 150 rpm for 5 days. Subsequently, the samples were extracted, and the degradation ability of this bifenthrin-degrading bacterium for different pyrethroids was detected by HPLC.

[0041] 2 Results and Analysis 2.1 Identification of the bifenthrin-degrading bacterium SX5 After isolation, purification, and secondary screening verification, a strain SX5 with bifenthrin degradation activity was obtained. The colony morphology of strain SX5 on LB agar plates is as follows. Figure 1 As shown in a, its single-celled form is round, pale yellow, with a raised center and neat edges. Figure 1b shows the single-cell morphology of strain SX5 under a scanning electron microscope. It is a cylindrical rod-shaped bacterium, occurring in short or long chains, with a wrinkled surface and no flagella. 16S rDNA sequencing and homology comparison using NCBI revealed that strain SX5 had a 94% coverage rate with *Achromobacter pulmonis* (pulmonary achromobacter), with a 99.80% identity. A phylogenetic tree was constructed... Figure 2 The strain SX5 was found to be in the same clade as *Achromobacter pulmonis*, with a spread value of 97, and was therefore identified as *Achromobacter pulmonis*. Strain SX5 is the first *Achromobacter pulmonis* strain to be found with bifenthrin-degrading capabilities. The 16S rDNA gene sequence of this strain has the accession number PV436819 in the GenBank database.

[0042] Based on the above identification results, the strain SX5 isolated in this invention was identified as *Achromobacter pulmonis*. *Achromobacter pulmonis* SX5 was deposited on June 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34789.

[0043] 2.2 Degradation of bifenthrin by Achromobacterium pulmonaryis SX5 The degradation of bifenthrin by Achromobacterium pulmonaryis SX5 was analyzed by high performance liquid chromatography, and the results are as follows: Figure 3 As shown, compared with the blank control, the absorption peak of bifenthrin treated with strain SX5 (150 rpm, 30 ℃) for 5 days was significantly reduced, and new substance peaks appeared, indicating that Achromobacterium pulmonaryis SX5 has degradation activity against bifenthrin.

[0044] The growth and bifenthrin degradation curves of strain SX5 in LB liquid medium containing 50 mg / L bifenthrin (150 rpm, 30℃) are shown below. Figure 4 As shown, the growth of strain SX5 increased when the concentration of bifenthrin decreased. Strain SX5 experienced rapid growth in the first 3 days, which was its rapid growth phase; at day 1, the degradation rate of bifenthrin reached 67.81%; at day 3, strain SX5 entered the stationary phase, at which point the OD of strain SX5... 600The absorbance was 4.92, indicating that the degradation rate of bifenthrin increased to 73.77%. At 5 days, the degradation rate reached 80.14%, at which point the growth of strain SX5 plateaued, and the degradation rate of bifenthrin also decreased. At 11 days, the degradation rate was 95.72%, and by 13 days, bifenthrin was almost completely degraded. These results demonstrate that strain SX5 has a high efficiency in degrading bifenthrin and grows well in LB liquid medium containing bifenthrin.

[0045] The degradation of different concentrations of bifenthrin by strain SX5 over 5 days at 150 rpm and 30℃ is as follows: Figure 5 As shown, *Achromobacterium pulmonale* SX5 exhibited a certain degradation effect on bifenthrin at concentrations ranging from 20 to 600 mg / L. Strain SX5 showed the best degradation effect on bifenthrin at concentrations of 20 mg / L and 50 mg / L, with degradation rates of 90.56% and 80.41% respectively on day 5. Furthermore, strain SX5 showed a superior growth rate at this time, with a relatively high OD value. 600 The absorbance values ​​were all greater than 4.0; as the concentration of bifenthrin increased, the degradation rate of bifenthrin by strain SX5 gradually decreased, and the OD... 600 The absorbance value also gradually decreased; Achromobacterium pulmonaryis SX5 still has a certain degradation ability for bifenthrin at a concentration as high as 600 mg / L.

[0046] 2.3 Optimization of Response Surface Methodology for Bifenthrin Degradation by Achromobacterium pulmonaryis SX5 Based on the results of single-factor experiments on strain SX5, such as Figure 6 As shown, strain SX5 maintained over 80% bifenthrin degradation activity at culture temperatures of 20-30℃, over 60% at 35-40℃, and over 35% at 50℃, indicating a wide temperature adaptability. Strain SX5 exhibited bifenthrin degradation activity within a culture medium pH range of 2-11, demonstrating its acid and alkali tolerance, making it suitable for use in more extreme environments. It showed high bifenthrin degradation activity within a pH range of 4-8, with degradation rates exceeding 60%, with the best degradation effect observed at pH 5. In contrast, currently reported bifenthrin-degrading strains generally showed better degradation at pH 7. When the inoculum size was 3 vol%, strain SX5 exhibited the best bifenthrin degradation effect, and the OD... 600The values ​​were also relatively high. Response surface methodology was used to optimize three key factors affecting the ability of *Achromobacterium pulmonale* SX5 to degrade bifenthrin. Culture temperature (X1), culture medium pH (X2), and inoculum size (X3) were used as independent variables, with -1, 0, and 1 representing the levels of these three variables, respectively. The degradation rate (Y) of strain SX5 on day 5 in LB liquid medium containing 50 mg / L bifenthrin was used as the response value. Using a Box-Behnken design, 15 experimental schemes were randomly obtained, as shown in Table 1. The results were further analyzed using response surface methodology and fitted to a quadratic polynomial equation, yielding the following formula: Table 1. Results of Box-Behnken design experiment using response surface methodology for strain SX5 The analysis of variance for the fitted model is shown in Table 2. Model RV 2 The P-value was 0.9775, indicating that the equation can effectively predict the optimal degradation conditions for bifenthrin. Meanwhile, the model's P-value was 0.0013, less than 0.05, indicating that the model terms were significant. Regression analysis in Table 2 shows that the pH of the culture medium, inoculum size, culture temperature, pH interaction term, pH-inoculum interaction term, culture temperature square term, and pH square term have significant effects on the degradation of bifenthrin by strain SX5.

[0047] Table 2. Analysis of Variance in Response Surface Experiment for Strains SX5 The optimized three-dimensional response surface plot of bifenthrin degradation is shown below. Figure 7 As shown, (a): 3D diagram of the interaction between temperature and pH on the degradation of bifenthrin; (b): 3D diagram of the interaction between temperature and inoculum size on the degradation of bifenthrin; (c): 3D diagram of the interaction between pH and inoculum size on the degradation of bifenthrin.

[0048] The contour plot and cubic optimization diagram of bifenthrin degradation are as follows: Figure 8 As shown, through further analysis using cubic diagram optimization, three-dimensional response surface modeling, and quadratic polynomial equations, the response surface model successfully predicted the optimal degradation conditions for 50 mg / L bifenthrin by strain SX5: a culture temperature of 27℃, a culture medium pH of 5, and an inoculum size of 4 vol%. Under these conditions, the maximum theoretical degradation rate of bifenthrin by *Achromobacterium pulmonarye* SX5 was 81.90%. Experimental verification showed that when *Achromobacterium pulmonarye* SX5 seed culture was inoculated into 30 mL of LB liquid medium (pH = 5) containing 50 mg / L bifenthrin at a 4 vol% inoculum size and cultured at 150 rpm and 27℃ for 5 days, the degradation rate of bifenthrin by *Achromobacterium pulmonarye* SX5 was 80.14%. This degradation rate is close to the theoretically predicted maximum degradation rate (81.90%), reflecting the reliability of the optimized degradation conditions determined by the model.

[0049] 2.4 Degradation kinetics of different concentrations of bifenthrin by Achromobacterium pulmonaryis SX5 The degradation process of bifenthrin by strain SX5 at different initial concentrations is as follows: Figure 9 As shown in the figure. Under the conditions of culture temperature 27℃, culture medium pH 5, and inoculum size 4 vol%, *Achromobacterium pulmonale* SX5 exhibited rapid degradation of 25 mg / L and 50 mg / L bifenthrin, with degradation rates exceeding 50% on day 3. On day 9, strain SX5 showed a degradation efficiency exceeding 90% for both 25 mg / L and 50 mg / L bifenthrin, demonstrating good degradation performance. On day 11, it degraded 91.6% and 95.72% of 25 mg / L and 50 mg / L bifenthrin, respectively. Strain SX5 showed slower degradation efficiency for bifenthrin at concentrations of 100-600 mg / L, and the degradation rate gradually decreased with increasing concentration.

[0050] Table 3 shows the primary degradation kinetic parameters of bifenthrin at different concentrations, and the correlation coefficient R. 2 The range of 0.7594–0.9814 indicates that the degradation of bifenthrin follows first-order degradation kinetics. Overall, except for the degradation rate constant k, which gradually decreases with increasing bifenthrin concentration at an initial concentration of 200 mg / L, the half-life, ranging from 2.209 d to 3.648 d, also generally increases with increasing concentration.

[0051] Table 3. Kinetic parameters of primary degradation of bifenthrin at different concentrations The kinetics of the biodegradation process of bifenthrin at different concentrations were analyzed using the Andrews equation, and the results are as follows: Figure 10 As shown. The theoretical values ​​of the model established by the Andrews equations agree well with the actual values ​​(R0). 2 =0.8475), the maximum specific degradation rate of bifenthrin (q) max The value is 0.5430d. -1 Half-rate constant (K) s The concentration was 20.0022 mg / L, and the inhibition coefficient (K) was... i The concentration was 40.7909 mg / L. By differentiating the nonlinear fitting equation, the concentration at q was found to be... max The corresponding concentration (S) max The concentration was 28.56 mg / L, which is the optimal predicted concentration for the degradation of bifenthrin by Achromobacterium pulmonaryis SX5.

[0052] 2.6 Degradation of different pyrethroid insecticides by *Achromobacterium pulmonale* SX5 The degradation effect of *Achromobacterium pulmonale* SX5 on different pyrethroid insecticides, such as... Figure 11As shown, after culturing in LB medium containing 50 mg / L of different pyrethroids at 30℃ and 150 rpm for 5 days, strain SX5 exhibited a certain degradation effect on all nine pyrethroid insecticides. The best degradation effect was observed against lambda-cyhalothrin, with a degradation rate of 89.33% within 5 days; followed by lambda-cyhalothrin at 84.45%. The degradation effects against deltamethrin, cis-cyhalothrin, and bifenthrin were all greater than 70%, at 80.57%, 70.1%, and 80.14%, respectively. The degradation effects against permethrin and fenvalerate were also greater than 60%. The worst degradation effect was against cypermethrin, with a degradation rate of only 47.69% within 5 days. In summary, strain SX5 demonstrated a significant broad-spectrum degradation ability against pyrethroid insecticides. Currently, strains capable of simultaneously degrading multiple pyrethroid insecticides have been reported. In this invention, Achromobacterium pulmonaryis SX5 can degrade a wider variety of pyrethroid insecticides than most of the previously reported strains.

Claims

1. A type of Achromobacterium pulmonaryis SX5, characterized in that: The classification name of the lung achromobacterium SX5 is lung achromobacterium ( Achromobacter pulmonis It was deposited on June 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34789.

2. The application of Achromobacterium pulmonaryis SX5 as described in claim 1 in the degradation of pyrethroid insecticides.

3. The application according to claim 2, characterized in that: The pyrethroid insecticides include methamidophos, lambda-cyhalothrin, permethrin, lambda-cyhalothrin, cypermethrin, deltamethrin, bifenthrin, and cis-cyhalothrin.

4. The application of Achromobacterium pulmonaryis SX5 as described in claim 1 in the preparation of pyrethroid insecticide degrading bacterial agents.

5. The application according to claim 4, characterized in that: The pyrethroid insecticides include methamidophos, lambda-cyhalothrin, permethrin, lambda-cyhalothrin, cypermethrin, deltamethrin, bifenthrin, and cis-cyhalothrin.

6. A pyrethroid insecticide-degrading microbial agent, characterized in that: It includes the pulmonary achromobacterium SX5 as described in claim 1.

7. The pyrethroid insecticide degrading microbial agent according to claim 6, characterized in that: The preparation method of the pyrethroid insecticide degrading bacterial agent includes: inoculating the glycerol-preserved bacteria of Achromobacterium pulmonaryis SX5 into LB liquid medium and culturing overnight at 180 rpm and 30°C to obtain Achromobacterium pulmonaryis SX5 seed liquid, which is the pyrethroid insecticide degrading bacterial agent.

8. A method for degrading pyrethroid insecticides, characterized in that: The pyrethroid insecticide degrading bacteria agent according to any one of claims 7 to 8 is inoculated into LB liquid medium containing pyrethroid insecticides and cultured.

9. The method according to claim 8, characterized in that: The culture temperature is 20-40℃, the pH of the culture medium is 4-11, and the inoculation amount of the pyrethroid insecticide degrading bacteria is 1%-5%.

10. The method according to claim 8, characterized in that: The culture temperature was 27°C, the pH of the culture medium was 5, and the inoculum amount of the pyrethroid insecticide degrading bacteria was 4%.

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