Bacillus velezensis FL-5 and application thereof
By the Bacillus Bacillus FL-5 isolated from the soil of rapeseed fields, the persistent pollution of flulorin in the soil and the harm to soil microorganisms were solved, and the efficient degradation of flulorin was achieved, with a degradation rate of 81.97%.
Patent Information
- Application Number
- CN202411890046.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-20
- Publication Date
- 2025-04-18
AI Technical Summary
As a widely used herbicide, although Flulorin has an efficient herbicide effect, its large-scale use causes soil pollution and is harmful to soil microorganisms and is difficult to effectively degrade.
A Bacillus Bacillus FL-5 was isolated from the soil of the rapeseed field where FL-5 was applied by enrichment and domestication separation. This strain was able to efficiently degrade FL-5 under optimized conditions.
The degradation rate of FL-5 reached 81.97% in 14 days, solving the problem of persistent pollution of FL-5 in the soil and significantly reducing the harm to soil microorganisms.
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Figure CN119842522A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a bacillus, in particular to a Bacillus Velez FL-5 and an application thereof. Background Art
[0002] Trifluralin, chemical name is 2,6-dinitro-N,N-dipropyl-4-trifluoromethylaniline, molecular formula is C 13 H 16 F3N3O4 is a dinitroaniline selective pre-emergence herbicide that is currently widely used.
[0003] In actual agricultural production, fluroxyproline is generally made into an emulsifiable concentrate product. In addition to being widely used in farmland, it is also widely used in aquaculture. Fluroxyproline has the advantages of safety, high efficiency, and a broad spectrum of weed control, and is widely used in farmland production. Fluroxyproline is easily soluble in most organic solvents such as n-hexane, ethanol, acetonitrile, ethyl acetate, xylene, acetone, etc. Fluroxyproline is volatile and photolyzed under natural conditions. In the soil, fluroxyproline is usually fixed to the soil due to the adsorption of soil colloids, making it difficult to move and volatilize.
[0004] The mode of action of fluramide in herbicides is that it enters the weed cells through the bud sheath structure of grass weeds, while some dicotyledonous weeds absorb fluramide through the hypocotyl. Tubulin in plants can polymerize and participate in cell division. After entering the cells, fluramide first inhibits the development of tubulin tissue in plant cells, destroys the normal metabolic activities of weed cells, and hinders the mitotic process of weed cells, thereby killing weeds. It is worth noting that due to the poor leaching solubility of fluramide, it is easy to interact with the active groups in soil organic matter in the soil to form bound compounds that are difficult to measure, and it is not easy to degrade, thereby causing delayed pollution to the soil and endangering the growth and development of subsequent crops. Fluramide can also poison and affect the genes of mammals, and its carcinogenicity is classified as Class C. The U.S. Environmental Protection Agency classifies fluramide as a persistent bioaccumulative toxic substance. Some European countries have restricted or even banned the use of fluramide, but fluramide is cost-effective, so it is still a commonly used agricultural herbicide in my country.
[0005] The use of pesticides has significantly improved the efficiency of agricultural production in my country. Pimentel et al. (Amounts of pesticides reaching target pests: Environmental impacts and ethics [J]. Journal of Agricultural & Environmental Ethics, 1995, 8 (1): 17-29) studied the fate of pesticides in the environment and found that only a small amount of pesticides can be used directly to control weeds, while more than 99% of pesticides will be released directly or indirectly into the natural environment. These pesticides released into the natural environment will be collected into surface water through atmospheric circulation, water circulation, etc., affecting the ecological environment and even posing a threat to the safety of domestic water. Among them, a large part of the pesticides will be adsorbed in the soil by soil colloids and degraded and transformed by various factors such as microorganisms in the soil. Trifluralin plays a vital role in my country's agricultural production, but the large-scale use of trifluralin has become one of the important causes of soil pollution in my country. The impact of trifluralin on soil microorganisms cannot be ignored. Specifically, it affects the number and distribution of soil microorganisms, changes the community structure of soil microorganisms, reduces the metabolic activity of soil microorganisms, and inhibits the activity of certain enzymes in the soil. The impact of trifluralin as an external pollutant on soil microorganisms has been confirmed by many scholars, and there are also many studies on the impact of trifluralin on the N cycle in the soil.
[0006] Flufluralin can be degraded through physical, chemical or biological reactions. Under experimental conditions, the main degradation pathways of flufluralin in soil are photolysis, volatilization, leaching, etc. Photolysis and volatilization of flufluralin are two important degradation pathways. However, flufluralin is difficult to photolyze and volatilize at a soil depth of less than 0.2m, and mainly relies on the degradation of microorganisms. Microbial degradation is the main degradation pathway of flufluralin. At present, several strains of trifluralin-degrading bacteria have been studied and reported at home and abroad. For example, Bellinaso (Biodegradation of the herbicide trifluralin by bacteria isolated from soil [J]. Fems Microbiology Ecology, 2003, 43 (2): 191-194) screened and isolated 8 trifluralin-degrading strains from soil where trifluralin had been continuously applied for 40 years and tested the degradation ability of these strains on trifluralin. Finally, 4 strains with more significant trifluralin degradation effects were screened out, namely Klebsiella sp., Herbaspirillum sp. and two Bacillus strains, with degradation rates of 24.60%, 16.40%, 25.00% and 18.40% respectively. Sato Yoko et al. (Degradation of Trifluralin by Bacteria Isolated from Soil [J]. Weed Research, 1992, 37 (3): 213-219) isolated two trifluralin-degrading bacteria from soil and tested their ability to degrade trifluralin. Ni Haiyan (Isolation and Identification of Pendimethalin-Degrading Strains, Analysis of Degradation Pathways and Cloning of Nitroreductase Gene [D]. Nanjing Agricultural University. 2016) isolated a strain of Bacillus subtilis Y3 from soil and extracted the functional gene PNR from it, verifying that this gene can degrade trifluralin. Ji Li et al. (Isolation and Identification of Trifluralin-Degrading Strains and Study on Degradation Characteristics [J]. Jiangsu Agricultural Sciences. 2016, (3): 390-393) isolated a strain of Leucobacter that can degrade trifluralin from aquaculture water. Erguven et al. (The capacity of some newly bacteria and fungi for biodegradation of herbicide trifluralin under agiated culture media[J]. Cellular and Molecular Biology(Noisy-le-Grand,France),2016,62(6):74) isolated 11 species of bacteria and fungi that can degrade trifluralin.
[0007] Currently, there are few studies on the degradation characteristics of trifluralin-degrading strains. Summary of the invention
[0008] The invention aims to provide a Bacillus Velezii FL-5 and an application thereof, which can effectively degrade trifluralin.
[0009] In order to achieve the above-mentioned object, the present invention provides a Bacillus velezensis FL-5, which is deposited in Guangdong Provincial Microbiological Culture Collection Center, with a deposit date of December 18, 2023, a deposit number of GDMCC No: 64156, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0010] Another object of the present invention is to provide the use of the Bacillus Velez FL-5 in degrading trifluralin.
[0011] Preferably, the seed liquid of Bacillus Velez FL-5 is used to degrade trifluralin.
[0012] Preferably, the OD of the seed solution of Bacillus Velez FL-5 is 600 Above 1.0.
[0013] Preferably, the seed solution of Bacillus Velez FL-5 is used to degrade trifluralin at a temperature of 25-35° C., a pH of 7-9, an inoculation amount of 4-5%, a trifluralin concentration of 0.1-1 mg / L, and a degradation time of more than 7 days.
[0014] Another object of the present invention is to provide a degradation agent for degrading trifluralin containing the Bacillus Velez FL-5.
[0015] The Bacillus Velez FL-5 of the present invention and its application have the following advantages:
[0016] The invention separates the rapeseed field soil to which trifluralin is applied by an enrichment and acclimation separation method, and obtains trifluralin-efficient degrading bacteria FL-5. Through morphological and molecular identification, FL-5 is identified as Bacillus Velezii. The degradation rate of trifluralin by FL-5 at 14 days is 51.18%. Through optimization of degradation conditions, the optimal degradation conditions of FL-5 are 35° C., pH 7, and inoculation amount of 5%. The degradation rate of trifluralin by the degradation strain at 14 days reaches 81.97%. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 This is the colony morphology of the FL-5 degradation strain of the present invention.
[0018] Figure 2This is the Gram staining result of the FL-5 degradation strain of the present invention.
[0019] Figure 3 This is a phylogenetic tree diagram of the FL-5 degradation strain of the present invention.
[0020] Figure 4 This is the growth curve of the FL-5 degradation strain of the present invention.
[0021] Figure 5 This is the effect of different culture temperatures on the degradation ability of the FL-5 degradation strain of the present invention.
[0022] Figure 6 This is the effect of different pH on the degradation ability of the FL-5 degradation strain of the present invention.
[0023] Figure 7 This is the effect of different inoculation amounts on the degradation ability of the FL-5 degradation strain of the present invention.
[0024] Figure 8 This is the effect of different substrate concentrations on the degradation ability of the FL-5 degradation strain of the present invention.
[0025] Fig. 9 This is the effect of different culture times on the degradation ability of the FL-5 degradation strain of the present invention. DETAILED DESCRIPTION
[0026] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0027] Reagents and culture media used in the following experiments:
[0028] The purity of trifluralin standard product was 99.3% (Pesticide Testing Laboratory, Shenyang Institute of Chemical Industry); the soil sample came from the rapeseed field where trifluralin was applied at Qinghai Academy of Agriculture and Forestry Sciences.
[0029] LB medium: 3 g meat extract, 10 g peptone, 5 g lactose and 0.024 g bromothymol blue, pH = 7.0, sterilized at 115°C for 30 min; 1.5% agar was added to the solid medium.
[0030] Improved Martin medium: 2 g yeast extract powder, 20 g glucose, 5 g peptone, 1 g dipotassium hydrogen phosphate and 0.5 g magnesium sulfate, pH = 6.4 ± 0.2, sterilized at 121°C for 30 min; 1.5% agar was added to the solid culture medium.
[0031] Gao's medium No. 1: 0.5 g sodium chloride, 20 g soluble starch, 1 g potassium nitrate, 0.5 g dipotassium hydrogen phosphate, 0.01 g ferrous sulfate and 0.244 g anhydrous magnesium sulfate, sterilized at 121°C for 30 min; 1.5% agar was added to the solid culture medium.
[0032] Inorganic salt culture medium: 0.4 g ammonium sulfate, 1.2 g ammonium nitrate, 0.5 g potassium dihydrogen phosphate, 1.5 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate, 0.5 g sodium chloride and 0.05 g yeast extract, pH 7.0, sterilized at 121°C for 30 min; 1.5% agar was added to the solid culture medium.
[0033] Experimental Example 1 Screening of degradation strains
[0034] 1. Screening of degradation strains
[0035] Weigh 1g of soil enriched with trifluralin and add 9mL of sterile water to make a soil suspension, and dilute it with sterile water to get 10 -4 ~10 -1 0.1 mL of soil suspension of different concentrations was taken and spread on three solid culture media (LB, modified Martin, Gao's No. 1) containing 1 mg / L trifluralin, three parallels for each culture medium, and cultured at 30°C for 3 days, and then the growth of colonies on the culture medium plates was observed. Single colonies were picked from the plates and repeatedly streaked and isolated for multiple times until pure strains and single colonies appeared, and each colony was numbered and stored in the corresponding solid slant culture medium.
[0036] The domesticated strains with the potential to degrade trifluralin were inoculated into three liquid culture media (LB, modified Martin, Gao's No. 1) and cultured for 3 days as seed liquid. The seed liquid was inoculated with 1% of the inoculation amount into three liquid culture media with a concentration of 1 mg / L trifluralin, and cultured at 30°C and 180r / min with shaking. Samples were taken every 1d, 3d, 5d, 7d and 14d, and the culture media with the same concentration of trifluralin but no bacteria were added were used as controls. After being treated according to the culture medium pretreatment method, the degradation rate of trifluralin by each strain was detected and analyzed by GC-ECD method.
[0037] The above-mentioned culture medium pretreatment method is as follows: 10 mL of LB, modified Martin, and Gao's No. 1 culture medium is measured accurately to (0.01 mL), 10 mL of n-hexane is added, and vortexed for 3 minutes to mix, 4 g of MgSO4 and 1 g of NaCl are added, and vortexed for 1 minute. Centrifuge at 5000 r / min for 5 minutes, take 1.5 mL of supernatant and place it in a purification centrifuge tube (containing 100 mg of MgSO4 and 100 mg of PSA), vortex for 1 minute, centrifuge at 12000 r / min for 2 minutes, and take the supernatant through a 0.22 μm filter membrane for GC-ECD detection.
[0038] The degradation rate calculation formula is as follows:
[0039] Q(%)=(C0-C t ) / C0×100%
[0040] Where, Q is the degradation rate of trifluralin (%); C0 is the initial concentration of trifluralin (mg / L); C t is the residual trifluralin content in the culture medium after the culture is completed (mg / L).
[0041] A total of 22 strains were screened in the experiment, among which strains FL-3, FL-5, and FL-6 showed significant differences from their corresponding blank control groups, indicating that these three strains had a degradation effect on trifluralin. Among them, the degradation rates of strain FL-5 at 1d, 3d, 5d, 7d, and 14d were 4.68%, 15.37%, 37.91%, 47.05%, and 51.18%, respectively. The concentration of trifluralin in each culture medium showed a gradual decrease with the extension of treatment time. The concentration of trifluralin in the culture medium of strain FL-5 decreased rapidly within 7d, from the initial concentration of 1mg / L to 0.49mg / L, but as the treatment time was extended to 14d, the concentration of trifluralin only decreased by 0.04mg / L.
[0042] 2. Identification of degradation strains
[0043] (1) Morphological identification
[0044] The isolated and purified strains were streaked on solid plates, cultured at 30°C, and the morphology of the colonies growing on the plates was observed. The strain FL-5 was cultured on LB medium for 24 hours, and sterile water was applied to the slides. A few spores were picked up with a needle and evenly and dispersed in the water. After fixation, Gram staining was performed, and the morphology of the strain was observed under an optical microscope at 100× using the oil lens of the microscope.
[0045] The colony morphology of strain FL-5 is as follows Figure 1 As shown, the FL-5 colony is grayish white with an irregular, rough, and wrinkled surface.
[0046] like Figure 2 As shown, the Gram staining result of FL-5 is purple, indicating that FL-5 is a Gram-positive bacterium, and both bacteria are short rod-shaped.
[0047] (2) Molecular identification
[0048] The genomic DNA of strain FL-5 was extracted and PCR amplified using bacterial universal primers 27F / 1492R, gyrA, gyrB, and rpoB (Table 1). The amplified products were sequenced, and after obtaining the sequence, the NCBI website was logged in to select the Blast window, and the obtained gene sequence was input for homology sequence alignment, and the joint phylogenetic tree of each gene was constructed using Phylosuite software.
[0049] Table 1 is the primer information table
[0050]
[0051] The bacterial universal primers 27F / 1492R, gyrA, gyrB, and rpoB were used to amplify the strain FL-5, and nucleotide fragments of 1434 bp, 998 bp, 1115 bp, and 1159 bp were obtained. The obtained sequences were uploaded to the GenBank database for BLAST comparison analysis, and the strains with higher homology were selected for phylogenetic tree analysis. The results showed that FL-5 and Bacillus velezensis Q6 (OQ181207.1) were clustered into the same branch ( Figure 3 ), combined with morphological and molecular biological identification, it was determined that strain FL-5 was Bacillus velezensis.
[0052] The above-mentioned strain FL-5 (Bacillus velezensis FL-5) is deposited in Guangdong Provincial Microbiological Culture Collection Center, with a deposit date of December 18, 2023, a deposit number of GDMCC No: 64156, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0053] 3. Strain growth curve
[0054] The selected FL-5 was picked up with an inoculation loop and inoculated into LB liquid medium, and cultured at 30°C and 180r / min for 12h as seed solution. 1% of the seed solution was inoculated into new LB liquid medium, and cultured at 30°C and 180r / min. The OD of the fermentation liquid was measured every 2h. 600 The growth curve of the strain was drawn with blank LB liquid medium as the control.
[0055] like Figure 4 As shown, FL-5 is in a delayed phase from 0 to 8 hours, with slow growth; it enters a rapid growth phase from 8 to 10 hours, enters a stable growth phase from 12 to 36 hours, and enters a decline phase from 36 to 48 hours.
[0056] Experimental Example 2 Single Factor Degradation Characteristics
[0057] The strain FL-5 was picked up with an inoculation loop and inoculated into 250 mL LB liquid medium, and cultured at 28°C and 180 r / min for 12 h as seed solution. 1% of the seed solution was inoculated into new LB liquid medium, and cultured at 30°C and 180 r / min until the logarithmic phase (OD 600 =1.0) as seed solution, and its degradation characteristics were studied as follows:
[0058] 1. Effect of temperature on the degradation of trifluralin by strains
[0059] The seed liquid of strain FL-5 was inoculated at 3% into LB liquid culture medium containing 1 mg / L fluralin culture medium, the initial pH was adjusted to 7.0, and the culture temperatures were set at 20°C, 25°C, 30°C, 35°C, and 40°C, respectively. Each treatment was repeated 3 times, and the culture was carried out in a shaker at 180 r / min in a light-shielded shaking incubator for 14 days. The degradation of fluralin at 1, 3, 5, 7, and 14 days was measured, respectively.
[0060] The results are as follows Figure 5 As shown in the figure, after the strain was inoculated, the degradation rate of fluralin by the strain increased with the increase of temperature, and the degradation rate of FL-5 was the lowest at 40℃, which may be because the lower or higher temperature affected the growth of the bacteria, resulting in low enzyme activity, affecting the degradation ability of the strain to fluralin. The degradation rate of the strain was the fastest at 1 to 7 days, and the degradation rate of the strain slowed down significantly after 7 days. The degradation rate of FL-5 at 35℃ for 7 days was 59.84%; at 7 to 14 days, the degradation rate of FL-5 at 35℃ for 14 days was 70.63%.
[0061] 2. Effect of pH on the degradation of trifluralin by strains
[0062] The seed liquid of strain FL-5 was inoculated at 3% into LB liquid culture medium containing 1 mg / L fluralin culture medium, the culture temperature was set at 30°C, the initial pH of the liquid culture medium was adjusted to 4, 5, 6, 7, 8, and 9, each treatment was repeated 3 times, and the culture was carried out in a shaker at 30°C and 180 r / min for 14 days in a light-shielded shaking incubator. The degradation rates of fluralin at 1, 3, 5, 7, and 14 days were measured, respectively.
[0063] The results are as follows Figure 6 As shown in the figure, 14 days after inoculation, the degradation rate of fluralin by the three strains increased with the increase of pH, and the degradation rate of the strains was the fastest at 1 to 7 days. The effect of pH on the degradation rate of FL-5 was not significant. As the degradation days increased, the degradation rate of fluralin by the strains gradually slowed down. At 14 days, FL-5 had the best degradation effect on fluralin at pH 7 and 8, with degradation rates reaching 71.12% and 72.08%, respectively. It was preliminarily concluded that the degradation effect of fluralin-degrading strains was best under neutral or alkaline conditions.
[0064] 3. Effect of inoculation size on the degradation of trifluralin by strains
[0065] The seed liquid of strain FL-5 was inoculated into LB liquid culture medium containing 1 mg / L fluralin culture medium at an inoculum rate of 1%, 2%, 3%, 4% and 5%, respectively. The initial pH was adjusted to 7.0. 5% sterile water was added to the control group. The culture was incubated in a shaker at 30°C and 180 r / min for 14 days in a light-shielded manner. The degradation rates of fluralin at 1, 3, 5, 7 and 14 days were measured, respectively.
[0066] The results are as follows Figure 7 As shown in the figure, the degradation rate of FL-5 on trifluralin increased with the increase of inoculation amount. At 7 days, the degradation effect was best when the inoculation amount was 5%, and the degradation rate reached 70.02%. As the degradation time increased, the degradation rate of the strain gradually slowed down. At 14 days, FL-5 had the best degradation effect when the inoculation amount was 5%, and the degradation rate reached 77.54%. It can be seen from the test results that too high or too low inoculation amount affects the degradation ability of the strain. When the inoculation amount is small, the growth cycle of the strain is long; when the inoculation amount is too large, the number of strains gradually increases with the metabolic process, resulting in competition between microorganisms for nutrients and oxygen and other reaction substances, resulting in nutrient deficiency during the degradation process, affecting the growth and reproduction of the strain.
[0067] 4. Effect of substrate concentration on the degradation of trifluralin by strains
[0068] The seed liquid of strain FL-5 was inoculated at a 3% inoculum into LB liquid culture medium containing 0.1 mg / L, 1 mg / L, and 10 mg / L fluralin culture medium, and the initial pH was adjusted to 7.0. Each treatment was repeated 3 times, and the culture was incubated in a shaker at 30°C and 180 r / min for 14 days in a light-shielded manner. The degradation rates of fluralin at 1, 3, 5, 7, and 14 days were measured, respectively.
[0069] The results are as follows Figure 8 As shown in the figure, the degradation ability of LF-5 at 0.1 and 1 mg / L concentrations was not significantly different. As the concentration increased to 10 mg / L, the degradation effect of the three strains on trifluralin decreased significantly. When the concentration of trifluralin was 0.1 mg / L, the degradation rate of FL-5 was 76.62% in 14 days; when the concentration increased to 1 mg / L, the degradation rate of FL-5 was 78.01% in 14 days, and when the substrate concentration was 10 mg / L, the degradation rate of FL-5 was 28.73% in 14 days. Therefore, it was preliminarily concluded that the strain had a better degradation efficiency between 0.1 and 1 mg / L concentrations.
[0070] 5. Effect of culture time on the degradation of trifluralin by strains
[0071] The seed liquid of strain FL-5 was inoculated at a 3% inoculum into LB liquid culture medium containing 1 mg / L fluralin culture medium, and the initial pH was adjusted to 7.0. 5% sterile water was added to the control group. Each treatment was repeated 3 times and cultured in a shaker at 30°C and 180 r / min with light shielding for 1, 3, 5, 7, 14, 21, and 28 days. The residual amount of fluralin was determined after the culture was completed.
[0072] The results are as follows Fig. 9 As shown in the figure, the degradation rate of fluralin by strain FL-5 increased with the extension of time, and the three degrading bacteria showed a faster degradation trend from 1 to 7 days, and then the degradation rate decreased from 7 to 28 days. The degradation rates of fluralin by FL-5 at 1, 3, 5, 7, 14, and 28 days were 14.63%, 25.24%, 57.86%, 68.43%, 77.93%, 85.04%, and 89.17%, respectively. At 28 days of degradation culture, the degradation rate of FL-5 was 77.88% higher than that of CK.
[0073] 6. Response Surface Optimization
[0074] On the basis of single factor experiment, degradation temperature (℃), pH and inoculation size (%) were selected as independent variables, and the response value was the degradation rate of trifluralin. A Box-Behnken three-factor three-level experiment was designed. A total of 17 test points were designed to explore the optimal degradation conditions for trifluralin-degrading strains.
[0075] (1) Equation analysis of response surface regression equation
[0076] On the basis of the single factor test, the temperature, pH and inoculation size were selected for the Box-Behnken test. The FL-5 response value test design and results are shown in Tables 2 and 3. The degradation rate of trifluralin in the table was fitted, and the equation of the conditional regression model of FL-5 degradation of trifluralin was:
[0077] Y=64.92+1.61A+1.42B+1.13C-2.10AB+3.5AC-4.58BC+10.87A 2 +2.71B 2 +1.
[0078] 53C 2
[0079] It can be concluded from the formula that the effects of the three factors on the degradation of trifluralin during the degradation process of FL-5 are not conventional linear relationships, indicating that there are interactive effects among the factors during the degradation process of the strain.
[0080] The FL-5 regression equation was subjected to variance analysis, and the results are shown in Table 4. The F value of the model was 34.75, P < 0.0001, indicating that the model has high reliability. The P value of the lack-of-fit term was 0.0973 > 0.05, indicating that the lack-of-fit term was not significant and there was no lack-of-fit factor. The linear terms A, B and the quadratic term A 2 , B 2 The P values of the interaction terms AB, AC, and BC are all less than 0.05, indicating that each factor has a significant impact on the response value results. 2 The P values of all are > 0.05, indicating that the results have no significant impact. Through variance analysis, it can be concluded that the order of influence of the three factors on the degradation rate of trifluralin is A>B>C, that is, temperature>pH value>inoculation amount. Table 5 is the fitting result of the response data. It can be seen from Table 5 that the R of the regression equation 2 It is 0.9781, indicating that the model can explain 97.81% of the response value variation. 2 It is 0.9500, which means that in the response surface quadratic model, more than 95.00% of the change in the degradation rate of trifluralin comes from the influence of temperature, pH value and inoculation amount, indicating that the regression equation model has a good fit. CV is 2.17%<10%, indicating that the credibility and accuracy of the test statement are high. Adeq Precision is 17.9965>4, which once again proves that the model is reasonable and reliable. Predicted R 2 With Adjusted R 2 The two values are relatively high, indicating that the model can fully simulate the test process.
[0081] Table 2 is the Box-Behnken test factors and level design for optimizing the degradation conditions of trifluralin by FL-5
[0082]
[0083] Table 3 shows the optimized Box-Behnken test design and results of the degradation of trifluralin by FL-5
[0084]
[0085] Table 4 is the variance analysis of the FL-5 response surface test results
[0086]
[0087]
[0088] Table 5 shows the fitting results of FL-5 response data.
[0089]
[0090] Through the above analysis of the response surface model, it can be concluded that the optimal conditions for FL-5 to degrade trifluralin are: 35℃, pH 7.1931, inoculation amount 5%, and the predicted degradation rate is 83.6%. The degradation conditions of FL-5 were revised to 35℃, pH 7, inoculation amount 5%. According to this degradation condition, the degradation time of the strain was 14 days, and the actual degradation rate of trifluralin was 81.97%.
[0091] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.
Claims
1. A Bacillus velezensis FL-5, characterized in that The Bacillus Velez FL-5 is deposited in the Guangdong Provincial Microbiological Culture Collection Center, with a deposit date of December 18, 2023, a deposit number of GDMCC No: 64156, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
2. Use of the Bacillus Velez FL-5 as claimed in claim 1 in degrading trifluralin.
3. The use according to claim 2, characterized in that: The seed liquid of the Bacillus Velez FL-5 is used to degrade trifluralin.
4. The use according to claim 3, characterized in that: The OD of the seed solution of Bacillus velez FL-5 600 Above 1.
0.
5. The use according to claim 3, characterized in that: The seed liquid of the Velez Bacillus FL-5 is used to degrade trifluralin at a temperature of 25-35°C, a pH of 7-9, an inoculation amount of 4-5%, a trifluralin concentration of 0.1-1 mg / L, and a degradation time of more than 7 days.
6. A degradation agent for degrading trifluralin containing the Bacillus Velez FL-5 as claimed in claim 1.