Bifunctional insecticide-degrading bacterial strain D35 and application thereof
By screening and optimizing the degradation conditions of Pseudomonas D35, the efficient degradation of neonicotinoids and benzoylurea insecticides was achieved, solving the problem that it is difficult to degrade these two types of insecticides simultaneously in existing technologies, and significantly improving the pollution status of soil and water environments.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
- Filing Date
- 2023-03-23
- Publication Date
- 2026-07-21
AI Technical Summary
The lack of existing microbial strains capable of simultaneously and efficiently degrading neonicotinoids and benzoylurea pesticides makes it difficult to effectively remediate soil and water pollution.
A Pseudomonas sp. strain D35 was screened and identified. Its degradation conditions were optimized using response surface methodology to achieve efficient degradation of the neonicotinoid insecticide acetamiprid and the benzoylurea insecticide flufenoxuron, with degradation rates of 80.21% and 91.61%, respectively.
Within 72 hours, strain D35 was able to significantly degrade the neonicotinoid insecticide acetamiprid and the benzoylurea insecticide flufenoxuron in soil or water, with degradation rates of over 80% and over 90%, respectively, effectively solving the pollution problems in soil and water environments.
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Figure CN116376764B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the microbial remediation of environmental pollution, specifically to a bifunctional insecticide-degrading strain D35 and its applications. Background Technology
[0002] Neonicotinic insecticides are a class of compounds with pharmacodynamic groups such as nitromimine, nitrosimine, and tricyanacetyl. They paralyze and eventually kill insects by blocking the normal transmission of the central nervous system. Major types include imidacloprid, acetamiprid, thiamethoxam, thiamethoxam, dinotefuran, and acetamiprid. They are widely used in the control of various agricultural pests, accounting for about one-third of the global insecticide market. Acetamiprid, a neonicotinic insecticide, is acid-resistant, photostable, and environmentally persistent. Repeated use leads to accumulation, with a half-life in soil exceeding one year. It also has high leaching and runoff potential, accumulating in soil and entering surface water and groundwater via surface runoff or infiltration. Acetamiprid residues can be detected in most rivers worldwide.
[0003] Benzoylurea insecticides belong to the chitinous insecticide inhibitor class and mainly include flufenoxuron, diflubenzuron, and tebufenozide. They are used to control various pests such as armyworms, beet armyworms, tussock moths, cabbage caterpillars, diamondback moths, and rice leaf rollers. However, benzoylurea insecticides have a slow effect in soil and a long residual period after application. Their decomposition products are also highly toxic. Residual mixtures on vegetables can cause serious diseases such as carcinogenicity and teratogenicity in mice. Their decomposition products can also migrate from the soil into groundwater, causing pollution of agricultural soil and drinking water sources. Microorganisms are the main factor in eliminating pesticide residues in soil. For contaminated soil and water, microorganisms can easily come into contact with pesticide residues and thus exert their degradation function. Among them, soil bacteria have the characteristics of rapid growth and direct promotion of crop growth. They can utilize their natural growth and reproduction to degrade neonicotinoid and benzoylurea insecticide residues in the soil or water environment, ultimately achieving the purpose of soil remediation.
[0004] Currently, there are few reports both domestically and internationally on the screening and research of bacteria that degrade neonicotinoid and benzoylurea insecticides. Existing strains have been shown to degrade only a single type of insecticide, and there are no reports on the research and application of high-yield, high-activity strains capable of simultaneously degrading both types of insecticides. Therefore, screening for bacteria capable of simultaneously degrading neonicotinoid and benzoylurea insecticides is of great significance. Summary of the Invention
[0005] Purpose of the invention: To address the problems existing in the prior art, the present invention provides a bifunctional insecticide-degrading strain D35, which can simultaneously degrade neonicotinoid insecticides and benzoylurea insecticides.
[0006] Another object of the present invention is to provide a degradation agent produced by the degradation strain and its application.
[0007] Technical solution: In order to achieve the above objectives, the present invention provides a bifunctional insecticide-degrading strain D35, which has been identified as Pseudomonas sp. This strain has been deposited at the China Center for Type Culture Collection, with accession number CCTCC NO:M 2022982 and deposit date of June 27, 2022.
[0008] The application of the bifunctional insecticide-degrading strain D35 described in this invention in the simultaneous degradation of neonicotinoid insecticides and benzoylurea insecticides.
[0009] The application of the bifunctional insecticide-degrading strain D35 in simultaneously degrading neonicotinoid and benzoylurea insecticides in soil or water environments.
[0010] The neonicotinoid insecticide is acetamiprid, and the benzoylurea insecticide is flufenoxuron.
[0011] The degradation conditions are: pH 6-8 and temperature 28-37℃.
[0012] Preferably, the degradation rate optimization of strain D35 using response surface methodology provides an optimization method for the biodegradation of neonicotinoid insecticides in soil or aquatic environments.
[0013] The optimization using response surface methodology includes the following steps:
[0014] Steps: 1) Plackett-Burman design experiment
[0015] The Plackett-Burman assay was used to screen key factors affecting the degradation of acetamiprid by strain D35. Five environmental conditions that may affect the degradation ability of the strain were selected at low (-1) and high (1) levels: tryptone concentration (0.5%, 1.0%), temperature (30, 37℃), pH (7.0, 8.0), inoculum amount (2%, 5%), and initial pesticide concentration (50, 100 mg / L). The degradation rate of acetamiprid by the strain at 72 h was used as the response value for evaluation.
[0016] 2) Steepest Climb Test
[0017] Based on the experimental results in 1), the significance ranking of each factor and the first-order fitting equation were obtained. The direction and step size of the change of the main influencing factors were designed to find the maximum response region.
[0018] 3) Box-Behnken Design Experiment
[0019] Based on the results of the steepest climbing test, after approaching the maximum response area, the high (1), medium (0), and low (-1) levels of three significant factors were determined. The Box-Behnken Design in Minitab 19 was used to conduct the experiment, optimize the levels of significant factors, find the optimal environmental conditions for strain degradation, and verify them.
[0020] Finally, the optimal culture medium and degradation conditions for acetamiprid were selected: basal salt liquid medium with tryptone added at a concentration of 10.19 g / L, pH = 7.0, initial acetamiprid concentration of 50 mg / L, inoculum amount of 5.24% (inoculated by volume ratio with D35 bacterial culture cultured to the logarithmic phase), and culture temperature of 30℃. Thus, this invention proposes a method for the efficient degradation of acetamiprid using strain D35.
[0021] The present invention relates to a degrading agent produced by the bifunctional insecticide degrading strain D35.
[0022] The fermentation of the microbial agent includes the following steps:
[0023] 1) Inoculate the bacterial culture of strain D35 into the fermentation medium and culture with shaking until the logarithmic phase to obtain the fermentation strain;
[0024] 2) Inoculate the above-cultured fermentation strain into the culture medium of the seed tank and culture until the logarithmic growth phase to obtain the seed liquid;
[0025] 3) Inoculate the seed culture into the culture medium in the production tank for cultivation; after fermentation, the culture medium is discharged from the tank and packaged into liquid form.
[0026] The application of the degrading microbial agent described in this invention in the simultaneous degradation of neonicotinoid insecticides and benzoylurea insecticides.
[0027] The application of the degrading microbial agent in simultaneously degrading neonicotinoid insecticides and benzoylurea insecticides in soil or water environments.
[0028] This invention, through the domestication and treatment of earthworms, screens and isolates highly efficient degrading strains from earthworm larvae, and performs response surface optimization analysis on the degradation conditions of acetamiprid. This provides resource security and scientific basis for using degrading bacteria to alleviate the pollution of neonicotinoid insecticide acetamiprid and benzoylurea insecticide flufenoxuron in soil and water environments.
[0029] Beneficial effects: Compared with the prior art, the present invention has the following advantages:
[0030] This invention isolates and screens a novel strain of Pseudomonas sp. D35 from earthworms, which can be used to simultaneously degrade the neonicotinoid insecticide acetamiprid and the benzoylurea insecticide flufenoxuron. This strain achieves a maximum degradation rate of 80.21% and 91.61% for acetamiprid and flufenoxuron in basal salt culture medium, respectively, within 72 hours. The degrading bacterium D35 of this invention can be used to degrade residual neonicotinoid insecticides acetamiprid and flufenoxuron in soil or aquatic environments. It can achieve a degradation rate of over 80% for residual neonicotinoid insecticide acetamiprid in soil or aquatic environments within a short time, and a degradation rate of over 90% for flufenoxuron within 3 days. This invention effectively utilizes biological methods to solve the pollution residue problem caused by neonicotinoid and benzoylurea insecticides in soil or aquatic environments.
[0031] This invention uses Design-Expert 8.0.5 software to perform response surface methodology optimization analysis on the degradation of acetamiprid by strain D35. Experimental results show that the model has high precision and small error, and can predict the degradation rate well.
[0032] The strain D35 provided by this invention belongs to the genus *Pseudomonas*, and features low production cost, ease of use, and high activity. It can efficiently degrade the neonicotinoid insecticide acetamiprid and the benzoylurea insecticide flufenoxuron. Furthermore, this invention aligns with the concept of green and safe environmental protection, maximizing the utilization of soil microbial resources, and is of great significance for the production of pollution-free vegetables and green food. Attached Figure Description
[0033] Figure 1 Photograph of bacterial colonies of strain D35;
[0034] Figure 2 Electron micrograph of strain D35;
[0035] Figure 3 This is a phylogenetic tree diagram of strain D35;
[0036] Figure 4 Effects of different culture conditions on the degradation of acetamiprid by strain D35 (A: nitrogen source; B: temperature; C: pH; D: inoculum size; E: initial acetamiprid concentration)
[0037] Figure 5 Effects of tryptone, temperature and inoculum size on the degradation rate of acetamiprid (A: tryptone and temperature, B: temperature and inoculum size, C: tryptone and inoculum size);
[0038] Figure 6 HPLC chromatograms of acetamiprid degradation by strain D35 after response surface methodology optimization (A: CK; B: treatment);
[0039] Figure 7 HPLC chromatograms of flufenoxuron degradation by strain D35 (A: CK; B: treatment). Detailed Implementation
[0040] The present invention will be further described below with reference to the accompanying drawings and embodiments.
[0041] Unless otherwise specified, all raw materials and reagents used in this invention are commercially available.
[0042] Example 1
[0043] Screening and separation of D35:
[0044] The degrading strains selected for enrichment in the experiment were derived from earthworms sourced from the Tropical Ecological Agriculture Base of the Chinese Academy of Tropical Agricultural Sciences. The soil used for earthworm domestication was collected from Chengmai County, Haikou City. Before the experiment, the earthworms were washed with sterile water and placed on moist filter paper in the dark for 24 hours to cleanse their intestines. Afterward, the earthworms were placed in an environment containing acetamiprid at a concentration of 1.5 mg / L. -1 They were grown in the soil, and on day 28, 5g of substrate was weighed from the earthworm enclosure and added to the soil containing 80mg·L⁻¹. -1 In an inorganic salt medium with acetamiprid as the sole carbon source, the culture was incubated at 30°C and 150 rpm for 72 h. The culture was then transferred to the same medium at an inoculum volume of 5%, and this transfer was repeated three times. Finally, the enrichment solution was serially diluted, and 10 μL of each solution was collected. -3 ~10 -6 0.02 mL of the enrichment solution at dilution was spread onto a substrate containing 80 mg·L⁻¹ -1 On solid agar plates with acetamiprid as the sole carbon source, after 72 hours of incubation at 30°C, single colonies grown on the plates were picked and inoculated onto plates containing 80 mg·L⁻¹ carbon. -1 In an inorganic salt medium with acetamiprid as the sole carbon source, the degradation effect of the medium on acetamiprid and flufenoxuron was verified by high performance liquid chromatography (HPLC) after incubation at 30°C and 150 rpm for 72 h. The purified medium was then identified. The basic salt medium formula (1 L) was: 1.0 g NH4NO3, 1.0 g NaCl, 1.5 g K2HPO4, 0.5 g KH2PO4, 0.2 g MgSO4·7H2O; pH 7.0; 18.0 g agar was added to the solid medium.
[0045] Verification method for the degradation effect of acetamiprid: After the reaction, dichloromethane was added to the culture medium at a 1:1 volume ratio, and the mixture was vigorously shaken and allowed to stand. The upper aqueous phase was removed, and anhydrous Na2SO4 was added to remove a small amount of water. 1 mL of the organic phase was evaporated, and an equal volume of methanol was added to dissolve it. After filtration through 0.22 μm, the solution was analyzed by high-performance liquid chromatography (HPLC). The HPLC conditions were as follows: mobile phase: water:methanol (60:40, volume ratio); C18-WP column (5 μm × 4.6 mm × 250 mm); column temperature: room temperature; detection wavelength: 270 nm; injection volume: 10 μL; flow rate: 1.0 mL·min. -1 External standard method: quantification based on peak area.
[0046] Verification method for flufenoxuron degradation effect: Add an equal volume of dichloromethane to the culture medium, shake vigorously, and allow to stand for layering. Take 1 mL of the lower layer of dichloromethane, and after complete evaporation, add 1 mL of methanol (chromatographic grade) to dissolve it. Filter through a 0.22 μm filter membrane. The content of flufenoxuron in the extract was determined by high performance liquid chromatography (HPLC). HPLC conditions: methanol:water (80:20, V / V), C18-WP reversed-phase column (5 μm × 4.6 mm × 250 mm), column temperature: room temperature, measurement wavelength: 254 nm, injection volume: 10 μL, flow rate: 1.0 mL·min⁻¹ -1 External standard method for quantification based on peak area. The experiment was repeated three times, and the results were averaged.
[0047] An aerobic bacterium capable of degrading acetamiprid and flufenoxuron was isolated from the enrichment solution and named D35. This bacterium showed that it could degrade acetamiprid and flufenoxuron at a concentration of 50 mg·L⁻¹ within 72 hours. -1 The degradation rates of flufenoxuron and acetamiprid reached over 91% and 55%, respectively.
[0048] The bacterial colonies are milky white, moist, and translucent. Figure 1 Gram-negative. Under a transmission microscope, the bacterial cells are rod-shaped, flagellated, and do not form spores. Figure 2 ).
[0049] Using the genomic DNA of strain D35 as a template, PCR amplification was performed using universal primers for bacterial 16S rRNA gene sequencing, yielding a 16S rDNA gene sequence of approximately 1495 bp, as shown in SEQ ID No: 1, GenBank accession number OP288121. Blast analysis in the EzBioCloud database (www.EzBioCloud.net) showed that strain D35 was most homologous to strains of the genus *Pseudomonas*, and most closely related to *Pseudomonas yangonensis* MY50. T(MK907288) is most closely related, with 99.10% 16S rDNA gene similarity. A phylogenetic tree was constructed. Figure 3 Based on morphological and physiological-biochemical characteristics, strain D35 was preliminarily identified as *Pseudomonas* sp. and named *Pseudomonas sp. D35*. This strain was deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2022982 and deposit date of June 27, 2022.
[0050] Example 2
[0051] To optimize the degradation of acetamiprid by strain D35, response surface methodology was used to rapidly optimize culture conditions and improve the degradation rate of the neonicotinoid insecticide acetamiprid by strain D35.
[0052] The specific steps are as follows:
[0053] (1) Single-factor experiment:
[0054] The optimal environmental conditions for the degradation of acetamiprid by strain D35 were investigated by varying the composition and culture conditions of the basal salt liquid culture medium. 50 mL of strain D35 cultured to the logarithmic growth phase was washed and resuspended with an equal volume of sterile water, and then inoculated into 100 mL of nitrogen-free liquid basal salt medium at a 5% (v / v) inoculum. Various nitrogen sources (peptone, yeast extract, ammonium nitrate, potassium nitrate, ammonium sulfate, and urea) were added at 0.5% (w / w) respectively. The medium was cultured for 72 h at an initial acetamiprid concentration of 50 mg / L and at 30℃ and 150 rpm. The degradation rate of each sample was measured, with three replicates for each gradient.
[0055] Other single-factor experiments: 400 mL of strain D35 cultured to the logarithmic phase was washed and resuspended with the same volume of sterile water. Except for the inoculum size experiment, 5% by volume was inoculated into 100 mL of liquid basal salt medium containing 0.5% tryptone. Temperature (20, 25, 30, 37, 42℃), pH (5.0, 6.0, 7.0, 7.5, 8.0, 9.0), inoculum size (0.5%, 1%, 2%, 5%, 8%, 10%), and initial pesticide concentration (10, 50, 100, 200, 300, 400 mg / L) were set and cultured at 150 rpm for 72 h. The degradation rate was measured, and each gradient was performed in triplicate.
[0056] The results are as follows Figure 4As shown in the figure. The results of the single-factor experiment showed that the optimal nitrogen source for the degradation of acetamiprid by strain D35 was tryptone, the optimal temperature was 30℃, the optimal degradation pH was 7.0, and the optimal inoculum size was 5%. When the initial concentration of acetamiprid was 200 mg / L or below, the initial concentration had almost no effect on the degradation of D35.
[0057] (2) Plackett-Burman Design Experiment
[0058] Based on the results of single-factor experiments, the Plackett-Burman assay was used to screen the key influencing factors of acetamiprid degradation by strain D35. Five environmental conditions that might affect the degradation ability of the strain were selected at low (-1) and high (1) levels: peptone concentration (0.5%, 1.0%), temperature (30, 37℃), pH (7.0, 8.0), inoculum size (2%, 5%), and initial pesticide concentration (50, 100 mg / L). The degradation rate of acetamiprid by the strain at 72 h was used as the response value for evaluation.
[0059] The experimental design and degradation rates for each treatment are shown in Table 1, and the statistical analysis is shown in Table 2. The results show that the model's multivariate correlation coefficient R0 is [value missing]. 2 =0.9935, Adjustment value Adj R 2 =0.9835, P value <0.001, reaching a highly significant level, indicating that the model has good correlation and strong correlation among factors. Temperature, tryptone content, and initial inoculum size have a significant impact on the degradation rate of the strain (P <0.01); the order of influence is temperature > tryptone content > initial inoculum size. Among them, tryptone content and initial inoculum size are positively correlated with the degradation rate of acetamiprid by the strain, while temperature is negatively correlated with the degradation rate of the strain. With pH 7.0 and initial acetamiprid concentration of 50 mg / L as the main factors, the step size and direction of the steepest climbing experiment were designed.
[0060] Table 1. Plackett-Burman Experimental Design and Results
[0061]
[0062]
[0063] Table 2 Statistical Analysis of the Plackett-Burman Trial
[0064]
[0065] Note: * indicates a significant difference (p<0.05), ** indicates an extremely significant difference (p<0.01).
[0066] (3) Steepest Climb Test
[0067] Based on the Plackett-Burman design experiment results, the significance ranking of each factor and the first-order fitting equation were obtained. The direction and step size of the change of the main influencing factors were designed to find the maximum response region.
[0068] Gradually increase the tryptone content and initial inoculum size, and decrease the culture temperature to conduct a scaling-up experiment. The experimental design and results are shown in Table 3. As shown in Table 3, the degradation of the strain initially increases and then decreases, reaching its highest value in group 3. Therefore, the experimental conditions of group 3 were selected as the central point for response surface methodology optimization.
[0069] Table 3. Experimental Design and Results for the Steepest Climb
[0070]
[0071] (4) Box-Behnken Design Experiment
[0072] Based on the results of the steepest climbing test, after approaching the maximum response area, the high (1), medium (0), and low (-1) levels of three significant factors were determined. The Box-Behnken Design in Minitab 19 was used to conduct the experiment, optimize the levels of significant factors, find the optimal environmental conditions for strain degradation, and verify them.
[0073] Using the culture conditions of treatment 3 as the center point of the BBD experiment, a response surface methodology was designed to optimize the experiment. The experimental design and results are shown in Table 4, and the analysis is shown in Table 5. A ternary quadratic regression equation was obtained for tryptone concentration, temperature, and inoculum size: Acetamiprid degradation rate Y = -1370.9 + 64.20A + 69.83B + 33.80D - 3.192A 2 -1.1810B 2 -2.727D 2 +0.0687AB - 0.225AD - 0.0987BD. The multiple correlation coefficient R0 of the regression equation. 2 =0.9958, Adj R 2 =0.9904, the response surface regression model reached the highly significant level (P<0.001), the lack-of-fit term P>0.05, the difference was not significant. Therefore, it can be seen that the model has a high degree of fit and can reflect the relationship between each factor and the response value well, and can be applied to the prediction of strain degradation rate.
[0074] Table 4 Box-Behnken Design Experimental Design and Results
[0075]
[0076]
[0077] Table 5 Statistical Analysis of Box-Behnken Design Experiments
[0078]
[0079] Note: * indicates a significant difference (p<0.05), ** indicates an extremely significant difference (p<0.01).
[0080] Using Design-Expert 8.0.5, a response surface plot of the interactions of various factors was drawn, and the results are as follows. Figure 5 As shown in the figure. Optimal value analysis of the regression equation revealed that the optimal degradation conditions were a tryptone concentration of 10.19 g / L, a temperature of 29.64℃, and an inoculum volume ratio of 5.24%, under which the predicted degradation rate of acetamiprid by the strain was 79.85%. To verify these optimal conditions, considering practical operational scenarios, the optimized conditions were modified as follows: basal salt liquid medium with tryptone at a concentration of 10.19 g / L, pH = 7.0, initial acetamiprid concentration of 50 mg / L, inoculum volume ratio of 5.24% (logarithmic phase bacterial culture), culture temperature of 30℃, and rotation speed of 150 rpm. The degradation liquid phase profile of the strain under these conditions is shown in the figure. Figure 6 As shown, the strain achieved a degradation rate of 80.21% for acetamiprid in 72 hours, which is relatively small compared with the theoretical value, indicating that the response surface methodology was reasonable and effective in optimizing the degradation conditions of the strain.
[0081] Example 3
[0082] The degradation effect of strain D35 on acetamiprid in basal salt medium:
[0083] Add 50 mg·L⁻¹ of tryptone to the basal salt medium (added to a final concentration of 10.19 g / L in Example 1, pH = 7.0) to a final concentration of 50 mg·L⁻¹. -1 Acetamiprid was inoculated into bacterial culture of strain D35 in the logarithmic growth phase at an inoculum rate of 5.24% (v / v). Simultaneously, acetamiprid was added to the basal salt medium (containing 1.019% tryptone in Example 1) to a final concentration of 50 mg / L. -1 Acetamiprid was inoculated at a 5.24% (v / v) inactivated strain D35 as a control. The cultures were incubated at 30°C with shaking at 150 rpm. Samples were taken periodically, and the degradation of acetamiprid by the strain was detected by high-performance liquid chromatography using the method described in Example 1. Strain D35 achieved a degradation rate of over 80% for acetamiprid within 72 hours.
[0084] Example 4
[0085] The degradation effect of strain D35 on flufenoxuron in basal salt medium:
[0086] Add 50 mg·L⁻¹ to the basal salt medium (same as in Example 1, pH = 7.0) to achieve a final concentration of 50 mg·L⁻¹. -1 Flufenoxuron was inoculated into bacterial culture of strain D35 in the logarithmic growth phase at a 1% (v / v) inoculum. Simultaneously, 50 mg·L⁻¹ of flufenoxuron was added to the basal salt medium (same as in Example 1). -1 Flufenoxuron was inoculated at a 1% (v / v) inactivated strain D35 as a control. The culture was carried out at 30°C with shaking at 150 rpm. Samples were taken periodically, and the degradation of flufenoxuron by the strain was detected by high-performance liquid chromatography using the method described in Example 1 for verifying the degradation effect. The results are as follows: Figure 7 As shown, strain D35 can achieve a degradation rate of over 91% for flufenoxuron within 72 hours.
[0087] Example 5
[0088] Preparation method of strain D35 degrading agent
[0089] 1) Inoculate the test tube solution of strain D35 at a volume ratio of 1% into the fermentation medium, shake and culture until the logarithmic phase to obtain the fermentation strain;
[0090] 2) The above-cultured fermentation strains were inoculated into a 500-liter seed tank with a liquid volume of 70% (based on the volume of the fermenter, the same below) at an inoculation rate of 5% (v / v, based on the volume of the culture medium) and cultured until the logarithmic growth phase to obtain the seed liquid.
[0091] 3) Inoculate the seed culture into the culture medium of the production tank with a volume of 70% at an inoculation rate of 5% (v / v, based on the volume of the culture medium, the same below).
[0092] 4) During the cultivation process in the seed tank and production tank, the sterile air ventilation ratio was 1:0.8 vvm, the stirring speed was 180 rpm, the cultivation temperature was 28℃, and the entire cultivation process took 48 hours. After fermentation, the culture medium was directly dispensed into liquid form using packaging bottles.
[0093] The fermentation medium, the seed tank medium, and the production tank medium have the same formula: 0.1 wt% glucose, 1.0 wt% NaCl, 0.5 wt% peptone, 0.25 wt% yeast extract, and pH 7.2.
[0094] Example 6
[0095] Fermentation of degrading microbial agents:
[0096] 1) Inoculate the test tube solution of strain D35 at a volume ratio of 1.5% into the fermentation medium, shake and culture until the logarithmic phase to obtain the fermentation strain;
[0097] 2) The above-cultured fermentation strains were inoculated into a 500-liter seed tank with a liquid volume of 70% (based on the volume of the fermenter) at an inoculation rate of 8% (v / v, based on the volume of the culture medium) and cultured until the logarithmic growth phase to obtain the seed liquid.
[0098] 3) Inoculate the seed culture into the culture medium of the production tank with a volume of 70% at an inoculation rate of 8% (v / v, based on the volume of the culture medium, the same below).
[0099] 4) During the cultivation process in the seed tank and production tank, the sterile air ventilation ratio was 1:0.8 vvm, the stirring speed was 220 rpm, the cultivation temperature was 32℃, and the entire cultivation process took 36 hours. After fermentation, the culture medium was directly dispensed into liquid form using plastic packaging containers.
[0100] The fermentation medium, the seed tank medium, and the production tank medium have the same formula: 0.1 wt% glucose, 1.0 wt% NaCl, 0.5 wt% peptone, 0.25 wt% yeast extract, and pH 7.5.
[0101] Example 7
[0102] Preparation method of degrading bacterial agent
[0103] The preparation method of the degradation agent in Example 7 is basically the same as that in Example 6, except that in step 4), the ventilation rate of sterile air in the seed tank and production tank is 1:0.9vvm, the stirring speed is 200rpm, the culture temperature is 30℃, and the culture time of the whole process is 44 hours.
[0104] Example 8
[0105] Degradation test of acetamiprid and flufenoxuron by strain D35 in soil
[0106] Soil samples from vegetable fields free of acetamiprid and flufenoxuron were used as test samples. The soil samples were sieved through a 2mm sieve. Certain amounts of acetamiprid and flufenoxuron were then evenly mixed into 500g of soil, respectively, to achieve a final concentration of 15 mg / kg for both acetamiprid and flufenoxuron in the soil. -1 Fresh D35 bacterial culture in logarithmic phase was cultured, and the bacterial cells were collected, washed three times with sterile deionized water, and then resuspended in sterile water to adjust the cell concentration to approximately 1.0 × 10⁻⁶. 9 cfu·mL -1The inoculum was 5% by volume inoculated into the soil and cultured in a 30°C dark incubator. Uninoculated soil was used as a control. The soil moisture content was maintained at 60% during the incubation period. After 10 days and 3 days of incubation, the residual amount was determined by high performance liquid chromatography. The results showed that strain D35 achieved a degradation rate of 80.13% for acetamiprid in the soil within 10 days and a degradation rate of 90.32% for flufenoxuron within 3 days.
Claims
1. A strain of Pseudomonas ( Pseudomonas sp.)D35, characterized in that, It has been deposited at the China Center for Type Culture Collection on June 27, 2022, with accession number CCTCC NO: M 2022982.
2. The use of the Pseudomonas D35 of claim 1 in the simultaneous degradation of acetamiprid and flufenoxuron in soil or water environments.
3. The application according to claim 2, characterized in that, The degradation conditions are: pH 6-8, temperature 28-37℃.
4. A degrading agent produced using Pseudomonas D35 as described in claim 1.
5. The degrading microbial agent according to claim 4, characterized in that, The fermentation of the microbial agent includes the following steps: 1) Inoculate the bacterial culture of strain D35 into the fermentation medium and culture with shaking until the logarithmic phase to obtain the fermentation strain; 2) Inoculate the above-cultured fermentation strains into the culture medium of the seed tank and culture until the logarithmic growth phase to obtain the seed liquid; 3) Inoculate the seed culture into the culture medium in the production tank for cultivation; after fermentation, the culture medium is discharged from the tank and packaged into liquid form.
6. The application of the degrading microbial agent according to claim 4 in the simultaneous degradation of acetamiprid and flufenoxuron in soil or water environments.