A strain capable of degrading quizalofop-ethyl and its application
By optimizing the culture conditions of strain MJ-8, the problem of low efficiency of microbial degradation of quizalofop-p-ethyl was solved, efficient degradation was achieved and environmental pollution was reduced, providing an environmentally friendly bioremediation tool.
Patent Information
- Application Number
- CN202411999076.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-10-03
- Estimated Expiration
- 2044-12-31
AI Technical Summary
Existing microorganisms have low efficiency in degrading quizalofop-p-ethyl, making it difficult to effectively degrade the herbicide in actual environments, and traditional methods may cause secondary pollution.
The strain MJ-8 was used, and its culture conditions (pH 6-7, temperature 25-35℃, inoculation amount 1%-9%) were optimized. Through shaking culture and centrifugal washing treatment, the strain MJ-8 that can efficiently degrade quizalofop-p-ethyl was obtained and applied to degradation in soil and water.
Under optimized conditions, the MJ-8 strain can effectively degrade quizalofop-p-ethyl, produce low-toxicity products, reduce environmental residues, and exhibit high degradation activity against other AOPP herbicides, providing an environmentally friendly bioremediation solution.
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Figure CN119955647B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of microorganisms, and particularly relates to a strain for degrading quizalofop-ethyl and its application. Background Art
[0002] Chemical herbicides play a vital role in modern agriculture, particularly chiral herbicides, such as aryloxyphenoxypropionates (AOPPs), which have garnered widespread attention for their highly effective herbicidal activity. AOPP herbicides achieve weed control by inhibiting the activity of acetyl-CoA carboxylase (ACCase), thereby blocking the biosynthesis of fatty acids in weeds. Quizalofop-p-ethyl is a representative AOPP herbicide, primarily used to control grass weeds in broadleaf crop fields. However, its use poses a threat to the environment and human health. It is strictly regulated in several countries and has even been listed as a possible human carcinogen. Reports indicate that quizalofop-p-ethyl may cause liver damage, and studies have found it may contribute to obesity. Furthermore, quizalofop-p-ethyl has severe effects on the embryonic development and endocrine system of aquatic organisms, such as zebrafish, and has also been shown to be toxic to earthworms. Experimental studies have also shown that quizalofop-p-ethyl is cytotoxic and genotoxic, inhibiting cell division and causing chromosomal aberrations. Other studies have shown that quizalofop-p-ethyl inhibits pollen germination and pollen tube growth.
[0003] To mitigate the ecological impact of quizalofop-p-ethyl and its metabolites, studying their degradation processes in the environment is crucial. Currently, methods for degrading pesticide residues include photodegradation, phytodegradation, and microbial degradation. Microbial degradation has garnered significant attention due to its key role in environmental remediation, particularly in reducing pesticide loads in soil and water. Compared to physical and chemical methods, microbial degradation offers advantages such as no secondary pollution, self-reproduction, and continuous degradation, reducing the cost and labor of repeated additions. However, the efficiency of microbial degradation of quizalofop-p-ethyl can be affected by various factors, such as ambient temperature, humidity, and competition from indigenous microorganisms, which may limit its degradation efficiency in practical applications. Furthermore, highly efficient degrading bacteria screened or engineered under laboratory conditions may have limited adaptability and colonization in natural environments, making it difficult to achieve the expected degradation efficiency in contaminated soils. Therefore, despite the significant advantages of microbial degradation, these challenges still need to be overcome in practical applications to improve the efficiency and effectiveness of microbial degradation of quizalofop-p-ethyl. Summary of the Invention
[0004] The present invention provides a strain for degrading quizalofop-p-ethyl and its application, aiming to solve the technical problem in the prior art that microorganisms have poor degradation effects of quizalofop-p-ethyl.
[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:
[0006] In a first aspect, the present invention provides a strain for degrading Quizalofop-P-ethyl, with a biological preservation number of CGMCC No. 31610; the strain is named MJ-8; the MJ-8 strain belongs to Prioccolumbia gigantea.
[0007] In a preferred embodiment, the conditions for the degradation of Quizalofop-P-ethyl by the MJ-8 strain are: pH 6-7, temperature 25-35° C., and inoculation amount 1%-9%.
[0008] Based on the above scheme, under the conditions, the metabolic activity and degradation ability of the MJ-8 strain reached the best, so that quizalofop-p-ethyl can be more effectively degraded and its residues in the environment can be reduced.
[0009] Preferably, the cultivation method of the MJ-8 strain is:
[0010] S1. Cultivate the strain: inoculate the strain MJ-8 into the culture medium and culture at a temperature of 28-32°C and a shaking speed of 150-170 rpm until the strain reaches the logarithmic growth phase;
[0011] S2. Collect the strain: Centrifuge the cultured strain for 8-12 minutes, discard the supernatant, and wash the bacteria at least three times with MSM medium to obtain the MJ-8 strain.
[0012] Based on the above scheme, controlling the temperature and shaker speed helps maintain the oxygen supply and uniform distribution of nutrients required for bacterial growth, thereby ensuring healthy growth and activity. The strain is cultured until the logarithmic growth phase, as bacteria in this phase have strong metabolic activity and are suitable for subsequent degradation experiments. Centrifugal washing removes unnecessary impurities and components from the culture medium, obtaining a pure MJ-8 strain.
[0013] Preferably, in step S1, the culture medium is NB culture medium.
[0014] Based on the above scheme, NB medium can provide nitrogen source, B vitamins, amino acids and other growth factors for the growth of MJ-8 strain, and is a medium suitable for bacterial growth.
[0015] In a second aspect, the present invention provides use of the MJ-8 strain described in the first aspect in preparing a degradable herbicide Quizalofop-P-ethyl.
[0016] A preferred embodiment is the use of the strain in degrading the quizalofop-ethyl herbicide in soil.
[0017] Based on the above scheme, the MJ-8 strain can effectively degrade quizalofop-p-ethyl in the soil and reduce its residue in the environment. The application of the MJ-8 strain in degrading the quizalofop-p-ethyl herbicide in the soil not only helps to control environmental pollution, but also promotes the healthy development of soil microorganisms.
[0018] The beneficial effects of the present invention are:
[0019] The present invention provides a strain and application for degrading quizalofop-p-ethyl. Experiments have shown that the MJ-8 strain can effectively degrade quizalofop-p-ethyl, producing products with low or no toxicity. Furthermore, the MJ-8 strain can grow in a culture medium containing quizalofop-p-ethyl, indicating that it can adapt to the presence of quizalofop-p-ethyl and can metabolize it using quizalofop-p-ethyl as a carbon source. This ability gives the MJ-8 strain enormous potential for practical application in the field of bioremediation. Compared with traditional physical and chemical degradation methods, using the MJ-8 strain for bioremediation can avoid the generation of secondary pollution, providing a more environmentally friendly solution. Furthermore, the MJ-8 strain has a certain ability to degrade other AOPP herbicides. Experiments have shown that the MJ-8 strain exhibits high degradation activity against clodinafop-butyl, fluazifop-butyl, cyhalofop-butyl, fluazifop-p-ethyl, and diclofop-p-ethyl, with degradation rates exceeding 90%. It also has a certain degradation effect on fenoxaprop-p-butyl and oxadiazol-butyl, providing a basis for the application of the MJ-8 strain in the remediation of agricultural soil contamination. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.
[0021] Figure 1 It is the degradation of quizalofop-p-ethyl during the growth of strain MJ-8.
[0022] Figure 2 It is the degradation ability of strain MJ-8 on quizalofop-p-ethyl under different culture conditions.
[0023] Figure 3 This is the colony morphology of strain MJ-8.
[0024] Figure 4 This is an electron micrograph of strain MJ-8. DETAILED DESCRIPTION
[0025] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.
[0026] Example 1:
[0027] This embodiment provides a strain for degrading quizalofop-ethyl, with a biological deposit number of CGMCC No. 31610; deposit unit: General Microbiology Center of China Culture Collection Administration; deposit date: August 6, 2024; classification name: Priestia megaterium; the strain is named MJ-8; the MJ-8 strain belongs to Priestia megaterium.
[0028] The conditions for the degradation of quizalofop-ethyl by the MJ-8 strain are: pH 6-7, temperature 25-35° C., and an inoculation amount of at least 1%-9%.
[0029] Specifically, strain MJ-8 has a high degradation efficiency under weakly acidic and neutral conditions. The optimum temperature for strain MJ-8 to degrade quizalofop-p-ethyl is 30°C, and the inoculation amount ranges from 1% to 9%. With the increase of inoculation amount and the extension of time, the degradation rate gradually increases. However, when the inoculation amount is too high, the strain grows rapidly, which may lead to insufficient substrate for growth and metabolism. Experiments have shown that the most appropriate ratio of the concentration of MJ-8 strain to the concentration of quizalofop-p-ethyl is maintained between 1:10 and 1:9.
[0030] The cultivation method of the MJ-8 strain is:
[0031] S1. Cultivate the strain: inoculate the strain MJ-8 into the culture medium and culture at a temperature of 28-32°C and a shaking speed of 150-170 rpm until the strain reaches the logarithmic growth phase;
[0032] S2. Collect the strain: Centrifuge the cultured strain for 8-12 minutes, discard the supernatant, and wash the bacteria at least three times with MSM medium to obtain the MJ-8 strain.
[0033] Specifically, S1. Cultivating the strain: First, inoculate the strain MJ-8 into NB medium and culture at a temperature of 30°C and a shaking speed of 160 rpm until the strain reaches the logarithmic growth phase;
[0034] S2. Collect the strain: Centrifuge the cultured strain at 5000 rpm for 10 minutes, then discard the supernatant; wash the bacteria three times with fresh MSM medium;
[0035] Next, the OD600 of the bacterial suspension was adjusted to 1.0 to standardize the bacterial concentration; 4-6 parts by mass of the bacterial suspension were inoculated into 100 parts by mass of MSM medium containing 18-20 mg / L quizalofop-p-ethyl, and the reaction was carried out at 30 ° C and 160 rpm for 7 days. The degradation of quizalofop-p-ethyl was monitored every 12 hours by a UV spectrophotometer.
[0036] Example 2:
[0037] This example provides the use of the MJ-8 strain described in Example 1 in the preparation of a degradable herbicide, Quizalofop-P-ethyl.
[0038] The MJ-8 strain is used to degrade the quizalofop-ethyl herbicide in soil.
[0039] Specifically, the MJ-8 strain can be used to degrade the residues of the herbicide quizalofop-p-ethyl, reduce pesticide pollution in farmland soil and water, and can also be used as a bioremediation agent to treat soil and water contaminated by quizalofop-p-ethyl, degrade organic pollutants in the environment through its metabolic activities, and improve environmental quality.
[0040] The present invention will be further explained below in conjunction with experiments:
[0041] 1. Materials and Methods
[0042] 1.1 Chemicals and culture media
[0043] Quizalofop-p-ethyl (purity 94.4%), cyhalofop-butyl (purity 96.44%), clodinafop-butyl (purity 95%), fluazifop-butyl (purity 99.1%), oxadiazol-butyl (purity 97.8%), oxadiazol-butyl (purity 98%), fluazifop-butyl (purity 97%), and diclofop-butyl (purity 96%) were purchased from Hunan Xinchangshan Agricultural Development Co., Ltd. All pesticide technicals were dissolved in methanol (HPLC grade) to prepare stock solutions (1×10 4 mg / L) and sterilized using a filter with a pore size of 0.22 μm.
[0044] In this study, bacteria were cultured using beef extract peptone broth (NB), Luria-Bertani (LB) medium, and mineral salts medium (MSM).
[0045] 1.2 Isolation and identification of bacteria that degrade the herbicide quizalofop-p-ethyl
[0046] The test samples were collected from the soil of a cotton field in Baoguo Village, Canggang Town, Hanshou County, Changde City, Hunan Province (N: 28°57′6", E: 111°52′46"), where quizalofop-ethyl is used all year round. The soil samples were added to MSM (basal salt medium) containing 20 mg / L quizalofop-ethyl. After incubation at 30°C and 160 rpm for 7 days, the enrichment solution was diluted and spread on an MSM agar plate supplemented with 20 mg / L quizalofop-ethyl. After culturing for 5 days, colonies were picked and streaked on the MSM plate until a single colony was obtained. The obtained strain was inoculated into an MSM medium containing 20 mg / L quizalofop-ethyl and cultured for 7 days. Its degradation ability was detected by HPLC. The strain with the highest degradation ability was selected and named MJ-8.
[0047] 1.3 Chemical analysis
[0048] Strain MJ-8 was inoculated into MSM medium, with a blank control containing no inoculation. An equal volume of acetonitrile was added to the culture medium (HPLC), and the mixture was centrifuged at 10,000 rpm for 5 min. The supernatant was filtered through a 0.22 μm membrane and analyzed by high-performance liquid chromatography (HPLC) equipped with a C18 column (Athena C18-WP, 5 μm, 4.6 × 250 mm) (Agilent Technologies, USA). A mobile phase consisting of acetonitrile:water (85:15 v / v) was used at a flow rate of 1.0 mL / min, with an injection volume of 20 μL. The column temperature was maintained at 25°C, and the UV detector was set at 234 nm. The concentration of quizalofop-ethyl was determined based on the peak area of the standard curve. To detect the metabolic degradation products of quizalofop-ethyl, samples were obtained using the above method and analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOFMS). Gradient elution was used, and MS analysis was performed using an electrospray ionization source in positive polarity mode.
[0049] 1.4 Investigating the effects of cell growth and culture conditions on the degradation of the herbicide quizalofop-p-ethyl by the MJ-8 strain
[0050] (1) Strain culture: The MJ-8 strain was inoculated into NB medium and cultured at 30°C and a shaking speed of 160 rpm until the logarithmic growth phase.
[0051] (2) Bacterial collection and washing: The cultured strain was centrifuged at 5000 rpm for 10 minutes, the supernatant was discarded, and the bacteria were washed three times with fresh MSM medium. The OD value of the bacterial suspension was calculated. 600 Adjusted to 1.0.
[0052] (3) Inoculation and incubation: The bacterial suspension was inoculated into 20 mL of MSM medium containing 20 mg / L quizalofop-p-ethyl at a ratio of 5% (v / v) and incubated at 30°C and 160 rpm for 7 days.
[0053] (4) Growth monitoring: Use an ultraviolet spectrophotometer to monitor bacterial growth every 12 hours, and set up samples without inoculation of strains as a control group.
[0054] (5) Optimization of culture conditions: Strain MJ-8 was inoculated in MSM medium containing 20 mg / L quizalofop-p-ethyl. The effects of different temperatures (20-40°C), medium pH (5-9), inoculum size (1-9%, v / v), and initial quizalofop-p-ethyl concentration (20-100 mg / L) on the degradation effect were investigated.
[0055] At the same time, the present invention also explored the degradation of other members of the AOPP (cyhalofop-butyl, clodinafop-butyl, fluazifop-butyl, diclofop-butyl, fluazifop-butyl, fenoxaprop-butyl and oxadiazol-butyl) by strain MJ-8. The residual amount was determined using the method described in Section 1.3.
[0056] 2. Results and Analysis
[0057] See Figure 3 and Figure 4 , Figure 3 The growth of the MJ-8 strain on the plate is shown, in which the colonies are clearly visible and evenly distributed, indicating that the MJ-8 strain has vigorous growth vitality and good reproduction ability. Figure 4 Electron microscopy images revealed the microstructure of the MJ-8 strain. High-resolution electron microscopy images clearly show the strain's intact cell morphology, with both its cell wall and cell membrane structures showing good integrity and stability.
[0058] Degradation dynamics of quizalofop-ethyl by strain MJ-8 in MSM liquid medium. Figure 1 The MJ-8 strain was inoculated into MSM medium containing 20 mg / L quizalofop-p-ethyl at pH 7.0 and 30°C. After 96 hours, the cell concentration reached its maximum. Compared with the control group, the residual rate of quizalofop-p-ethyl decreased with increasing cell concentration. The growth curve of the MJ-8 strain was positively correlated with the degradation rate of quizalofop-p-ethyl.
[0059] In order to evaluate the potential of strain MJ-8 to degrade the herbicide quizalofop-p-ethyl, the effects of different culture conditions on the degradation effect were investigated. Figure 2 These culture conditions include pH level, culture temperature, inoculum size and substrate concentration. The test results show that pH level has a significant effect on the degradation of quizalofop-p-ethyl by MJ-8 strain. Figure 2A, Under pH 6-7 conditions, the degradation rates of quizalofop-p-ethyl reached 83.56% and 82.55% after 120 hours, respectively. However, under pH 5, 8, and 9 conditions, the degradation rates of quizalofop-p-ethyl dropped to 49.25%, 62.74%, and 53.36% after 120 hours, respectively, indicating that lower or higher pH values severely hindered the growth and degradation efficiency of the MJ-8 strain.
[0060] In addition, the culture temperature is also a key factor affecting the degradation performance of Quizalofop-Phthiop-ethyl. Figure 2 B. Tests show that the optimal temperature for quizalofop-p-ethyl degradation by the MJ-8 strain is 30°C, but its degradation efficiency is low at lower temperatures. This is because low temperatures restrict the growth of the strain, thereby affecting degradation efficiency. When the temperature is within the 35-40°C range, the MJ-8 strain exhibits higher degradation ability as the culture time increases. This is because the MJ-8 strain is a Bacillus, and its bacteria and the degrading enzymes it produces are relatively resistant to high temperatures, so the degradation efficiency is not significantly affected at higher temperatures.
[0061] Inoculum size is also an important factor affecting the degradation rate of Quizalofop-PhD. Figure 2 C. In the experiment, the inoculation amount ranged from 1% to 9%. The results showed that as the inoculation amount increased, the degradation rate also gradually increased. The appropriate inoculation amount is conducive to the degradation of quizalofop-ethyl by microorganisms. Too little inoculation amount will lead to slow growth of the strain and affect cell survival rate. When the inoculation amount is too large, the strain will grow rapidly, which may lead to insufficient substrate for growth and metabolism. Figure 2 D. Substrate concentration also affects the degradation efficiency of quizalofop-p-ethyl. As the quizalofop-p-ethyl concentration increases, its degradation rate gradually decreases, and the degradation velocity also decreases. This indicates that quizalofop-p-ethyl has a certain degree of toxicity. The MJ-8 strain can effectively degrade quizalofop-p-ethyl within a certain range, but once the quizalofop-p-ethyl concentration exceeds a certain limit, it will seriously harm the growth of the strain, thereby affecting the degradation efficiency.
[0062] In addition, see Figure 2 The E. MJ-8 strain also exhibited high catalytic activity against other AOPP herbicides. Among the seven AOPP herbicides tested, clodinafop-butyl was completely degraded, while the degradation rates for fluazifop-butyl, cyhalofop-butyl, fluazifop-butyl, and diclofop-butyl were 99.63%, 91.71%, 99.6%, and 99.99%, respectively. The degradation rates for fenoxaprop-butyl and oxadiazon were lower, at 46.99% and 28.13%, respectively.
[0063] 3. Test conclusion
[0064] This experiment investigated the degradation efficiency of quizalofop-p-ethyl by the MJ-8 strain under different culture conditions. The following are the main findings and conclusions of the experiment:
[0065] (1) Strain growth and quizalofop-p-ethyl degradation: The MJ-8 strain has a good degradation ability for quizalofop-p-ethyl. Under the conditions of pH 7.0 and 30°C, the strain growth is positively correlated with the degradation of quizalofop-p-ethyl. The cell concentration reaches the maximum after 96 hours, and the residual rate of quizalofop-p-ethyl is significantly reduced.
[0066] (2) Effect of culture conditions on degradation efficiency: The degradation efficiency of the MJ-8 strain was the highest under conditions of pH 6 and 7, while the degradation efficiency decreased significantly at pH 5, 8, and 9. 30°C is the optimal temperature for the degradation of quizalofop-p-ethyl by the MJ-8 strain, and low temperature conditions limit the growth and degradation efficiency of the strain. Increasing the inoculation amount can improve the degradation efficiency of quizalofop-p-ethyl by the MJ-8 strain, but if the inoculation amount is too large, the growth and degradation ability of the strain may be limited due to insufficient substrate supply. At the same time, as the concentration of quizalofop-p-ethyl increases, the degradation rate and degradation rate are observed to decrease. Therefore, in order to ensure the effectiveness of the degradation process, the inoculation amount needs to be adjusted according to the concentration of quizalofop-p-ethyl, and the ratio of the strain concentration to the quizalofop-p-ethyl concentration should be kept between 1:9 and 1:10. This ensures that the strain has sufficient substrate for degradation while maintaining a high degradation activity, thereby achieving more effective biodegradation effects at different quizalofop-p-ethyl concentrations.
[0067] (3) Degradation ability of other AOPP herbicides: The MJ-8 strain showed high degradation activity against clodinafop-butyl, fluazifop-butyl, cyhalofop-butyl, fluazifop-butyl and diclofop-butyl, with degradation rates exceeding 90%. It also had a certain degradation effect on fenoxaprop-butyl and oxadiazon, which indicated that the MJ-8 strain had a certain degradation ability against AOPP herbicides.
[0068] (4) Environmental remediation potential: The MJ-8 strain has a high degradation efficiency of quizalofop-p-ethyl under weakly acidic and neutral conditions, indicating its potential for practical application in environmental remediation. The strain's temperature tolerance and ability to degrade quizalofop-p-ethyl make it a promising bioremediation tool.
[0069] In summary, the MJ-8 strain can effectively degrade the herbicide quizalofop-p-ethyl and has a certain ability to degrade other AOPP herbicides. These results provide a basis for the application of the MJ-8 strain in the remediation of agricultural soil and water pollution.
[0070] The present invention is not limited to the above-mentioned optional implementation modes. Anyone can derive other forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, any technical solution that falls within the scope defined by the claims of the present invention falls within the scope of protection of the present invention.
Claims
1. A strain for degrading quizalofop-ethyl, characterized by: The biological deposit number is CGMCC No.31610; the strain is named MJ-8; the MJ-8 strain belongs to Priesteria gigantea ( Priestia megaterium ).
2. The quizalofop-ethyl degrading strain according to claim 1, characterized in that: The conditions for the degradation of quizalofop-ethyl by the MJ-8 strain are: pH 6-7, temperature 25-35°C, and inoculation amount 1%-9%.
3. The quizalofop-ethyl degrading strain according to claim 1, characterized in that: The cultivation method of the MJ-8 strain is: S1. Cultivate the strain: inoculate the strain MJ-8 into the culture medium and culture at a temperature of 28-32°C and a shaking speed of 150-170 rpm until the strain reaches the logarithmic growth phase; S2. Collect the strain: Centrifuge the cultured strain for 8-12 minutes, discard the supernatant, and wash the bacteria at least three times with MSM medium to obtain the MJ-8 strain.
4. The quizalofop-ethyl degrading strain according to claim 3, characterized in that: In step S1, the culture medium is NB culture medium.
5. A use of the strain according to any one of claims 1 to 4, characterized in that: The MJ-8 strain is used in the preparation of a quizalofop-ethyl herbicide for degradation.
6. The use of the strain according to claim 5, characterized in that: The MJ-8 strain is used to degrade the quizalofop-ethyl herbicide in soil and water.