A microbial agent for degrading quizalofop-p-ethyl and its preparation method

Abstract

This invention relates to a microbial agent for degrading quizalofop-P-ethyl and its preparation method. The microbial agent is a liquid microbial agent obtained by fermentation culture of *Stenotrophomonas* sp. G1, a species of *Stenotrophomonas*, which was deposited on March 3, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2023239. This invention further provides a method for preparing the aforementioned quizalofop-P-ethyl degrading microbial agent. This agent exhibits high biological activity and a good degradation effect on quizalofop-P-ethyl. Its production is characterized by a short cycle and low cost, and the agent also has good environmental compatibility.

Description

Technical Field

[0001] This invention belongs to the field of microbial remediation of organic pollution technology, specifically, it relates to a microbial agent for degrading the herbicide quizalofop-P-ethyl and its preparation method. Background Technology

[0002] Quizalofop-P-ethyl is a systemic herbicide that inhibits acetyl-CoA carboxylase and is a widely used herbicide for legumes. However, it also inhibits the growth of gramineous crops such as corn, rice, and wheat. With the increasing need for weed control, the use of quizalofop-P-ethyl has been rising. Quizalofop-P-ethyl's byproduct, quizalofop-P-ethyl acid, has good water solubility and easily enters surface water, expanding its pollution. Quizalofop-P-ethyl is now widely present in soil, water environments, and related crops. The Ministry of Agriculture and Rural Affairs of China has set a maximum residue limit of 0.2 mg / L for quizalofop-P-ethyl in vegetables and other crops. Therefore, it is urgent to treat quizalofop-P-ethyl contaminated land and crops. Currently, the remediation of quizalofop-P-ethyl pollution mainly includes physical remediation, chemical remediation, and bioremediation. Physical and chemical remediation are not widely used in practice due to their drawbacks such as incomplete remediation, potential for secondary pollution, and high remediation costs. Bioremediation technology, due to its low cost, thorough remediation, and relatively low environmental risk, is an environmentally friendly remediation technology. With increasing emphasis on food and ecological security, bioremediation has become an important strategy for achieving sustainable development in modern agriculture. Summary of the Invention

[0003] To effectively remediate soil and water bodies contaminated or exceeding the standards for quizalofop-P-ethyl, this invention, through extensive research and screening, discovered a strain of Oligotrophozoites that is highly efficient at degrading quizalofop-P-ethyl herbicide, thus obtaining the technical solution of this invention.

[0004] An oligotrophomonas sp. G1 that degrades the herbicide quizalofop-P-ethyl, was deposited on March 3, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2023239.

[0005] Application of Stenotrophomonas G1 in the degradation of the herbicide quizalofop-P-ethyl, wherein the Stenotrophomonas G1 (S. G1) was deposited on March 3, 2023 at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2023239.

[0006] The application of a microbial agent in the degradation of the herbicide quizalofop-P-ethyl, wherein the microbial agent comprises Stenotrophomonas sp. G1 or its metabolites.

[0007] In one aspect, the present invention provides a microbial inoculant comprising Stenotrophomonas sp. G1 or its metabolites, wherein the inoculant is one of a liquid preparation, a lyophilized powder, a wettable powder, a suspension, an emulsion, or granules. The bacterial cells are prepared from Stenotrophomonas sp. G1 through strain activation, seed culture, and fermentation culture; the metabolites are derived from the supernatant obtained by centrifugation after fermentation of Stenotrophomonas G1. Optionally, the microbial inoculant may further contain one or more of the following: a carbon source, a nitrogen source, a lyophilization protectant, an oil, or a surfactant, which are beneficial to microbial growth or fixation.

[0008] The application of a microbial inoculant in the degradation of the herbicide quizalofop-P-ethyl, wherein the microbial inoculant comprises Stenotrophomonas sp. G1 or its metabolites, and the microbial inoculant is sprayed onto soil, water bodies, or crop surfaces requiring bioremediation. Preferably, the inoculum amount of Stenotrophomonas G1 is 2-5%, the effective pH is 6.0-8.0, and the effective temperature is 37-42°C.

[0009] In one aspect, the present invention provides a method for preparing a microbial agent for degrading the herbicide quizalofop-P-ethyl, comprising the following steps:

[0010] (1) Activation of strain: The Stenotrophomonas sp. G1 strain stored at low temperature was inoculated onto a solid culture medium and cultured at 30℃ for 24h. Single colonies were picked and placed in a 3mL seed culture medium test tube and cultured at 30℃ and 150rpm for 24h as inoculum for later use.

[0011] (2) Seed culture: Inoculate the seed culture medium with the seed liquid prepared in step (1) at an inoculation rate of 1%, stir at 180-200 rpm, and culture for 24 hours to obtain the seed liquid;

[0012] (3) Fermentation culture: Inoculate the seed liquid prepared in step (2) into the fermentation medium at an inoculation rate of 8%-10%, with a liquid volume of 80.872 mL / L, a temperature of 29.010℃, a stirring speed of 180 rpm-200 rpm, and a culture time of 24 h to obtain the fermentation broth, namely the oligotrophomonas liquid inoculum.

[0013] The seed culture medium in steps (1) and (2) of this invention consists of: 5 g / L yeast extract, 10 g / L peptone, and 10 g / L NaCl.

[0014] The fermentation medium in step (3) consists of: 10 g / L yeast extract, 3% maltose, 1 g / L NaCl, 0.2 g / L MgSO4, 1.5 g / L K2HPO4, 1.0 g / L NH4Cl, 0.5 g / L KH2PO4, and pH 6.308.

[0015] 3. Beneficial effects

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] The microbial agent of this invention, *Stenotrophomonas* sp. G1, possesses a highly efficient ability to degrade quizalofop-p-ethyl. Liquid fermentation is convenient and efficient, allowing for greater contact with the soil to be remediated, and effectively degrades quizalofop-p-ethyl. At an inoculum level of 2%, the liquid agent achieves a degradation rate of over 86% for 25 mg / L quizalofop-p-ethyl within 6 hours. The microbial agent of this invention has low production costs and can be directly applied to the soil, showing broad application prospects. Attached Figure Description

[0018] Figure 1 The colony morphology and scanning electron microscope image of the strain of this invention are shown.

[0019] Figure 2 This is a diagram showing the degradation effect of the bacterial agent of the present invention on quizalofop-p-ethyl;

[0020] Figure 3 This is a phylogenetic tree of the 16S rRNA of strain G1 of the present invention;

[0021] Figure 4 The graph shows the effect of different temperatures on the degradation of quizalofop-p-ethyl by the inoculant of this invention.

[0022] Figure 5 The graph shows the effect of different pH values ​​on the degradation of quizalofop-p-ethyl by the bacterial agent of this invention.

[0023] Figure 6 The graph shows the effect of different substrate concentrations on the degradation of quizalofop-p-ethyl by the bacterial agent of this invention.

[0024] Figure 7 The graph shows the effect of different inoculum amounts on the degradation of quizalofop-p-ethyl by the inoculum agent of this invention. Detailed Implementation

[0025] Example 1

[0026] Isolation and screening of Stenotrophomonas sp. G1 strains:

[0027] a. Soil sampling: Samples were taken from a farmland in Chizhou City, Anhui Province, where quizalofop-p-ethyl had been applied for a long period of time.

[0028] b. Preparation of culture medium:

[0029] MSM liquid medium: K₂HPO₄ 1.5 g / L, KH₂PO₄ 0.5 g / L, NaCl 1.0 g / L, NH₄Cl 1.0 g / L, MgSO₄·7H₂O 0.2 g / L, adjust pH to 7.0. For solid medium, add 1.5% agar powder.

[0030] c. Weigh 5g of soil sample into 100mL of MSM medium containing 20mg / L quizalofop-P-ethyl, and culture with shaking at 30℃ and 150rpm. Then, inoculate with 2% (v / v) of the enrichment culture solution into MSM medium containing 20mg / L quizalofop-P-ethyl, and culture for 7 days at 30℃ and 150rpm. Repeat this enrichment culture three times. 10g of the enrichment culture solution showing degradation effect... -3 -10 -9 The samples were serially diluted and spread onto solid MSM medium containing 20 mg / L quizalofop-p-ethyl, and then inverted in a 30°C incubator.

[0031] d. After colonies have grown, single colonies of different morphology, size, and color are selected from the plates and streaked multiple times for purification. HPLC is used to detect and screen single bacteria with quizalofop-p-ethyl degradation effects. Through repeated testing and screening, strains capable of efficiently degrading quizalofop-p-ethyl are obtained.

[0032] e. from Figure 2 It can be seen that the selected G1 strain can efficiently degrade 20 mg / L quizalofop-p-ethyl within 8 hours.

[0033] Example 2

[0034] Identification of G1 strain:

[0035] a. Microbiological characteristics: The G1 strain has rod-shaped cells, without flagella, measuring 10-20 μm × 3.5-4 μm. After incubation on MSM solid medium at 30°C for 24 h, the colonies are round, slightly yellow, with a raised surface, neat edges, and are dry and opaque.

[0036] b. Physiological and biochemical characteristics:

[0037]

[0038]

[0039] Note: "+" indicates a positive reaction, and "-" indicates a negative reaction.

[0040] c. Molecular biological identification:

[0041] The 16S rRNA amplified sequence of strain G1 was compared and analyzed in the NCBI database. It showed 99.65% homology with *Stenotrophomonas* sp. Based on the strain's morphological and physiological / biochemical characteristics, strain G1 was preliminarily identified as belonging to the genus *Oligotrophomonas*. The phylogenetic tree of the 16S rRNA of strain G1 is shown below. Figure 3 As shown. The G1 strain was named Stenotrophomonas sp. G1 and deposited on March 3, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2023239.

[0042] Example 3

[0043] Preparation of liquid inoculum of Stenotrophomonas sp. G1:

[0044] a. Strain activation:

[0045] The low-temperature preserved Stenotrophomonas sp. G1 strain was inoculated onto a solid culture medium and cultured at 30℃ for 24 h. Single colonies were picked and placed in 3 mL seed culture medium test tubes and cultured at 30℃ and 150 rpm for 24 h as inoculum for later use.

[0046] b. Seed culture:

[0047] The seed solution prepared in step (1) was inoculated into the seed culture medium at an inoculation rate of 1%, the stirring speed was 180-200 rpm, and the culture time was 24 h to obtain the seed solution.

[0048] c. Fermentation culture:

[0049] The seed liquid prepared in step (2) was inoculated into the fermentation medium at an inoculation rate of 8%-10%, with a liquid volume of 80.872 mL / L, a temperature of 29.010℃, a stirring speed of 180 rpm-200 rpm, and a culture time of 24 h to obtain the fermentation broth, namely the Oligotrophic Monotrophic Bacterium liquid inoculum.

[0050] The seed culture medium in steps a and b consists of: 5 g / L yeast extract, 10 g / L peptone, and 10 g / L NaCl.

[0051] The fermentation medium in step c consists of: 10 g / L yeast extract, 3% maltose, 1 g / L NaCl, 0.2 g / L MgSO4, 1.5 g / L K2HPO4, 0.5 g / L KH2PO4, 1.0 g / L NH4Cl, and pH 6.308.

[0052] Example 4

[0053] Effects of different temperatures on the degradation of quizalofop-p-ethyl by liquid inoculum of Stenotrophomonas sp. G1:

[0054] a. Preparation of bacterial suspension:

[0055] The liquid bacterial culture was centrifuged at 8000 rpm for 5 min to collect the bacterial cells. The cells were washed twice with 20 mL of sterile MSM, resuspended, and the OD was adjusted. 600 nm Up to version 2.0.

[0056] b. Experimental treatment: The above bacterial suspension was inoculated into 10 mL of MSM liquid medium at an inoculation rate of 2%, and quizalofop-P-ethyl was added to make the final concentration 20 mg / L. The culture was carried out at different temperatures from 16 to 42 °C with shaking at 150 rpm. The blank control group was replaced with an equal volume of sterile water instead of bacterial suspension. All other conditions were the same as those of the experimental group. Samples were taken at regular intervals, and the concentration of quizalofop-P-ethyl in the samples was detected by HPLC. The degradation rate of quizalofop-P-ethyl by the liquid bacterial agent was calculated. All treatments were repeated in triplicate.

[0057] c. Experimental results:

[0058] The effect of temperature on the degradation of quizalofop-p-ethyl by liquid inoculants, such as Figure 4 As shown, the optimal temperature for the degradation of quizalofop-P-ethyl by liquid bacterial agent is 37℃-42℃. It can completely degrade 20 mg / L of quizalofop-P-ethyl within 2 hours. At 30℃, the degradation rate of liquid bacterial agent decreases slightly. After 6 hours, 99% of quizalofop-P-ethyl can be degraded. At 25℃, the degradation rate of quizalofop-P-ethyl by liquid bacterial agent can reach 100% after 8 hours. When the temperature is 16℃, the degradation ability of liquid bacterial agent is inhibited. After 10 hours, approximately 75% of quizalofop-P-ethyl can be degraded.

[0059] Example 5

[0060] Effects of different pH values ​​on the degradation of quizalofop-p-ethyl by liquid inoculum of Stenotrophomonas sp. G1:

[0061] a. Preparation of bacterial suspension:

[0062] The liquid bacterial culture was centrifuged at 8000 rpm for 5 min to collect the bacterial cells. The cells were washed twice with 20 mL of sterile MSM, resuspended, and the OD was adjusted. 600 nm Up to version 2.0.

[0063] b. Experimental treatment: The initial pH of MSM medium was adjusted to 5.0, 6.0, 7.0 and 8.0. 2% of the bacterial suspension of strain G1 was inoculated into 10 mL of MSM medium at different pH values. A final concentration of 20 mg / L quizalofop-P-ethyl was added. The medium was cultured at 30℃ with shaking at 150 r / min. The blank control group was replaced with an equal volume of sterile water instead of the bacterial suspension. All other conditions were the same as the experimental group. Samples were taken at regular intervals, and the content of quizalofop-P-ethyl in the samples was detected by HPLC. The degradation rate of quizalofop-P-ethyl by the liquid bacterial agent was calculated. All treatments were replicated in triplicate.

[0064] c. Experimental results:

[0065] The effect of pH on the degradation of quizalofop-p-ethyl by liquid bacterial agents, such as Figure 5 As shown, the optimal pH for the degradation of quizalofop-P-ethyl by liquid bacterial agent is 8.0, and the liquid bacterial agent can degrade more than 99% of 20 mg / L quizalofop-P-ethyl within 6 hours at pH 6.0-8.0.

[0066] Example 6

[0067] Effects of different substrate concentrations on the degradation of quizalofop-p-ethyl by liquid inoculum of Stenotrophomonas sp. G1:

[0068] a. Preparation of bacterial suspension:

[0069] The liquid bacterial culture was centrifuged at 8000 rpm for 5 min to collect the bacterial cells. The cells were washed twice with 20 mL of sterile MSM, resuspended, and the OD was adjusted. 600 nm Up to version 2.0.

[0070] b. Experimental treatment: The above bacterial suspension was inoculated into 10 mL of MSM liquid medium at an inoculation rate of 2%, and quizalofop-P-ethyl was added to make final concentrations of 5 mg / L, 10 mg / L, 30 mg / L, 50 mg / L and 1000 mg / L, respectively. The culture was carried out at 30℃ and shaken at 150 rpm. The blank control group was replaced with an equal volume of sterile water instead of bacterial suspension. All other conditions were the same as those of the experimental group. Samples were taken at regular intervals, and the concentration of quizalofop-P-ethyl in the samples was detected by HPLC. The degradation rate of quizalofop-P-ethyl by the liquid bacterial agent was calculated. All treatments were replicated in triplicate.

[0071] c. Experimental Results: The effect of different substrate concentrations on the degradation of quizalofop-p-ethyl by liquid inoculants, as follows: Figure 6 As shown, the liquid bacterial agent can degrade 5 mg / L and 10 mg / L quizalofop-P-ethyl 100% within 2 hours, achieve 100% degradation of 30 mg / L quizalofop-P-ethyl 100% at 12 hours, completely degrade 50 mg / L quizalofop-P-ethyl at 30 hours, and achieve a degradation rate of over 85% of 100 mg / L quizalofop-P-ethyl at 42 hours.

[0072] Example 7

[0073] Effects of different inoculum amounts on the degradation of quizalofop-p-ethyl by liquid inoculum of Stenotrophomonas sp. G1:

[0074] a. Preparation of bacterial suspension:

[0075] The liquid bacterial culture was centrifuged at 8000 rpm for 5 min to collect the bacterial cells. The cells were washed twice with 20 mL of sterile MSM, resuspended, and the OD was adjusted. 600 nm Up to version 2.0.

[0076] b. Experimental treatment: The above bacterial suspension was inoculated into 10 mL of MSM liquid medium at inoculation rates of 1%, 2%, 3%, 4%, and 5% (v / v), respectively. Quizalofop-P-ethyl was added to bring the final concentration to 25 mg / L. The culture was carried out at 30 °C with shaking at 150 r / min. The blank control group was replaced with an equal volume of sterile water instead of bacterial suspension. All other conditions were the same as those of the experimental group. Samples were taken at regular intervals, and the concentration of quizalofop-P-ethyl in the samples was detected by HPLC. The degradation rate of quizalofop-P-ethyl by the liquid bacterial agent was calculated. All treatments were replicated in triplicate.

[0077] c. Experimental Results: The effect of different inoculum amounts on the degradation of quizalofop-p-ethyl by liquid inoculum, as follows: Figure 7 As shown, the results indicate that the optimal inoculum size is 5%. After 3 hours, the liquid inoculum can degrade 100% of 25 mg / L quizalofop-P-ethyl. The degradation rate is higher when the inoculum size is between 3% and 5%, and it can degrade 25 mg / L quizalofop-P-ethyl within 5 hours. When the inoculum size is 2%, the degradation rate of 25 mg / L quizalofop-P-ethyl by the liquid inoculum reaches more than 86% within 6 hours. When the inoculum size is 1%, the degradation rate of 25 mg / L quizalofop-P-ethyl by the liquid inoculum within 6 hours is only 55%.

[0078] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. An oligotrophic bacterium G1 that degrades the herbicide quizalofop-P-ethyl, characterized in that, The oligotrophomonas ( Stenotrophomonas sp G1 was deposited on March 3, 2023, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2023239.

2. The application of the oligotrophomonas G1 according to claim 1 in the degradation of the herbicide quizalofop-P-ethyl.

3. The application of the oligotrophomonas G1 according to claim 1 in the preparation of microbial agents that degrade the herbicide quizalofop-P-ethyl.

4. The application according to claim 3, characterized in that, The microbial agent includes Oligotrophomonas G1; the agent is one of the following: liquid preparation, lyophilized powder, wettable powder, suspension, emulsion, or granules.

5. In the application according to claim 4, the microbial agent further contains one or more of the following: carbon source, nitrogen source, freeze-drying protectant, oil, and surfactant, which are beneficial to the growth or fixation of microorganisms.

6. The application of a microbial agent in the degradation of the herbicide quizalofop-P-ethyl, said microbial agent comprising the oligotrophic monoclonal bacteria as described in claim 1 (… Stenotrophomonas sp . )G1; Spray the microbial agent onto the surface of soil, water, and crops that require bioremediation.

7. The application according to claim 6, characterized in that, The inoculum size of the oligotrophomonas G1 was 2-5%, the pH was 6.0-8.0, and the incubation temperature was 37-42 ℃.

8. A method for preparing a microbial agent for degrading the herbicide quizalofop-P-ethyl, characterized in that, The method includes the following steps: (1) Activation of strain: The oligotrophomonas G1 of claim 1, which is stored at low temperature, is inoculated on a solid culture medium and cultured at 30 °C for 24 h. Single colonies are picked and placed in a 3 mL seed culture medium test tube and cultured at 30 °C and 150 rpm for 24 h as inoculum for later use. (2) Seed culture: Inoculate the seed culture medium with the seed liquid prepared in step (1) at an inoculation rate of 1%, stir at 180-200 rpm, and culture for 24 h to obtain the seed liquid; (3) Fermentation culture: The seed liquid prepared in step (2) is inoculated into the fermentation medium at an inoculation rate of 8%-10%, with a liquid volume of 80.872 mL / L, a temperature of 29.010 ℃, a stirring speed of 180 rpm-200 rpm, and a culture time of 24 h to obtain the fermentation broth, namely the oligotrophomonas liquid inoculant.

9. The preparation method according to claim 8, characterized in that, The seed culture medium in steps (1) and (2) consists of: 5 g / L yeast extract, 10 g / L peptone, and 10 g / L NaCl.

10. The preparation method according to claim 8, characterized in that, The fermentation medium in step (3) consists of: 10 g / L yeast extract, 3% maltose, 1 g / L NaCl, 0.2 g / L MgSO4, 1.5 g / L K2HPO4, 1.0 g / L NH4Cl, 0.5 g / L KH2PO4, and pH 6.308.

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