strain Aureimonas ureilytica L12 and its application
By isolating and culturing the strain Aureimonas ureilytica L12 from glyphosate-resistant Amaranth plants, the problem of inconsistent degradation effects of existing strains has been solved, achieving efficient degradation of glyphosate, reducing environmental residues, and protecting ecological health.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HEBEI UNIVERSITY
- Filing Date
- 2025-02-06
- Publication Date
- 2026-05-15
AI Technical Summary
Existing strains show inconsistent performance in degrading glyphosate and exhibit degradation issues, leading to glyphosate residues in the soil that harm ecosystems and human health. Furthermore, the limited number of strains makes it difficult to effectively degrade glyphosate.
A strain of Aureimonas ureilytica L12 was isolated from glyphosate-resistant male Amaranthus fragrans plants. The strain was cultured in a modified enrichment medium and then inoculated into a medium containing glyphosate for microbial degradation, achieving a highly efficient degradation effect of glyphosate.
After being cultured in the dark at 28℃ and 180rpm for 5 days, strain L12 achieved a glyphosate degradation rate of 85.44%, significantly reducing glyphosate residues in the environment and mitigating harm to the ecosystem and human health.
Smart Images
Figure FT_1 
Figure FT_2 
Figure FT_3
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial degradation, specifically, it relates to strains. Aureimonas ureilytica L12 and its applications. Background Technology
[0002] Glyphosate was chemically synthesized in 1959 and began to be used as an agricultural herbicide in 1971. Due to its high efficiency, broad spectrum, and low cost, it is widely used to control weeds in crops. It has been found to eliminate over a hundred species of annual weeds and 60 species of perennial weeds, making it the most widely used and highest-producing pesticide in the world, consistently ranking first in annual sales. However, with the global promotion and use of glyphosate, some negative effects have become increasingly apparent. Large-scale use of glyphosate leaves residues in the environment, which not only impact ecosystems and disrupt ecological balance but may also adversely affect animals, plants, and microorganisms. Glyphosate residues in the food chain can enter the human body through bioaccumulation, causing concentrations in the human body to exceed those in the environment, and even causing systemic poisoning, threatening human health. Therefore, how to eliminate glyphosate residues in soil has become an urgent problem to be solved.
[0003] The degradation methods for pesticide residues include physical degradation, chemical degradation, and biodegradation. Compared to physical and chemical degradation, microbial degradation has advantages such as high degradation efficiency, multiple metabolic pathways, and no secondary pollution, making it the main method for removing pesticide residues from soil and water environments, effectively reducing the harm caused by pesticide residues. Many microorganisms can degrade organophosphorus pesticides, including bacteria, fungi, actinomycetes, and algae. Among them, bacteria play an important role in degrading organophosphorus pesticides due to their diverse physiological and biochemical adaptability and the ease with which mutant strains can be induced. Utilizing microorganisms and their produced degrading enzymes to degrade organophosphorus pesticide residues in the environment has shown good results and is a major research direction in organophosphorus pesticide degradation in recent years.
[0004] Currently, many microbial strains resistant to glyphosate or capable of degrading and utilizing glyphosate have been obtained both domestically and internationally. These strains have played a crucial role in eliminating glyphosate accumulation in nature and preventing ecological damage. However, the effectiveness of microbial degradation of glyphosate varies depending on the strain, and current strains suffer from degradation issues and are relatively few in number, severely limiting research on glyphosate-degrading microorganisms. This study isolated endophytic bacteria from a glyphosate-resistant population of *Amaranthus longicornis* and determined their glyphosate-degrading capacity, aiming to screen for glyphosate-degrading strains to enrich the microbial library. Summary of the Invention
[0005] The purpose of this invention is to provide a bacterial strain capable of degrading glyphosate. Aureimonas ureilytica L12 and its applications.
[0006] To achieve the objectives of this invention, in a first aspect, this invention provides a glyphosate-degrading strain—*Cyclomonella chilli* L12—isolated from fresh leaves of a glyphosate-resistant male *Amaranthus longicornis* plant, and classified as... Aureimonas ureilytica The strain is now deposited at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, accession number CGMCC No. 33169, deposit date December 23, 2024.
[0007] In a second aspect, the present invention provides a microbial preparation comprising one or more of the following: bacterial cells, bacterial powder, bacterial suspension, or bacterial extract selected from the strain L12.
[0008] Thirdly, the present invention provides a method for preparing the microbial preparation, the method comprising the step of culturing the strain L12.
[0009] Furthermore, the conditions for culturing strain L12 were: 28°C, 180 rpm shaker, and in the dark.
[0010] Furthermore, strain L12 was cultured using a modified enrichment medium.
[0011] The modified enrichment medium is formulated as follows: glucose 1 g / L, peptone 5 g / L, potassium nitrate 1 g / L of sodium chloride and 2 g / L of glyphosate (added to a concentration of 100 mg / L); pH value 6.6-7.6.
[0012] Fourthly, the present invention provides the application of the strain L12 or the microbial preparation in the degradation of glyphosate.
[0013] Fifthly, the present invention provides a method for degrading glyphosate, the method comprising: inoculating the strain L12 into a modified enrichment medium containing glyphosate.
[0014] In one specific embodiment of the present invention, the method for degrading glyphosate includes: converting OD... 600 A bacterial suspension with a value of 0.5 (the viable bacteria content in the bacterial suspension is approximately 1.67 × 10⁻⁵). 7 1 mL of glyphosate (CFU / mL) was inoculated into 30 mL of modified enrichment medium containing 100 mg / L glyphosate and cultured at 28°C and 180 rpm in the dark for 5 days.
[0015] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:
[0016] This invention provides a bacterial strain capable of degrading glyphosate. Aureimonas ureilytica L12, OD 600 One mL of a bacterial suspension with a pH of 0.5 was inoculated into 30 mL of a modified enrichment medium containing 100 mg / L glyphosate. After culturing at 28°C and 180 rpm for 5 days, the glyphosate content in the culture supernatant (0.5 mL) was measured. The results showed that the degradation rate of glyphosate by this bacterium could reach 85.44%. The strain L12 provided by this invention enriches the germplasm resource bank of pesticide-degrading bacteria. By using this strain, the environmental residues of glyphosate can be significantly reduced, thereby reducing its harm to ecosystems and human health. Attached Figure Description
[0017] Figure 1 This is a diagram showing the growth and colony morphology distribution of strain L12 on LB solid plates in a preferred embodiment of the present invention.
[0018] Figure 2 This is a phylogenetic tree diagram of the 16S rRNA of strain L12 in a preferred embodiment of the present invention.
[0019] Figure 3 This is a scatter plot showing the relationship between glyphosate and peak area for strain L12 in a preferred embodiment of the present invention.
[0020] Figure 4 This is a bar chart showing the degradation rate of glyphosate by strain L12 in a preferred embodiment of the present invention.
[0021] Figure 5 The colony morphology of strain L12 in a preferred embodiment of the present invention on LB medium with pH values of 5, 5.3, 5.4, 7, 8, 9, and 10 (from left to right) is shown. Detailed Implementation
[0022] This invention addresses the issue of glyphosate residues in soil and their harmful effects on human health. Starting from the field of microbial degradation, it isolates glyphosate-degrading bacterial strains from fresh leaves of glyphosate-resistant male Amaranth spp. The details are as follows:
[0023] Fresh amaranth leaf mill extract was diluted and inoculated onto TSA / R2A / LB / NA solid media via plate spreading. After cultivation, the strains were purified and identified. The strains significantly enriched in the isolated resistant population were inoculated onto LB medium and cultured for 24 h. The cultured bacterial suspension was centrifuged at 3800 rpm, the supernatant was discarded, and the bacterial cells were washed twice with sterile water. The bacterial suspension was then purified using an OD400 concentration. 600 The sample was inoculated into 30 mL of modified enrichment medium containing 100 mg / L glyphosate with a glyphosate concentration of 0.5. After 5 days, the sample was collected and the glyphosate residue was measured, thus obtaining a glyphosate-degrading strain—Cyclomonella coldwaterii L12.
[0024] The 16S rRNA of this bacterium was amplified using bacterial primers (27F and 1492R), and the PCR product fragment was sequenced by a bioengineering sequencing company (SEQ ID NO:1). The assembled sequence was uploaded to the NCBI website for sequence alignment, and phylogenetic analysis was performed using MEGA (version 7.0) software. Based on high homology and high similarity, the strain was ultimately identified as... Aureimonas ureilytica .
[0025] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.
[0026] Example 1: Classification, purification and identification of strain L12
[0027] 1. Sample collection
[0028] Seeds of *Amaranthus longicornis* from Jilin, China were collected, and fresh plant leaves were harvested after planting.
[0029] 2. Isolation and identification of endophytic fungi from the leaves of Amaranth laurentii
[0030] Fresh leaves from resistant *Amaranthus longicornis* plants were collected and rinsed with running water. Approximately 0.05 g of leaves were transferred to 50 mL centrifuge tubes for surface sterilization: rinsed with sterile water for 30 s, treated with 75% alcohol for 1 min, treated with 2% NaClO for 3 min, and finally rinsed with sterile water for 30 s. 10 mL of sterile water was added, and the mixture was ground into a suspension. The suspension was diluted to different concentrations, and a 10-fold dilution was selected for purification. 100 μL of the diluted solution was inoculated onto TSA / R2A / LB / NA solid medium, air-dried, and incubated at 28°C for 2 days (incubation conditions may vary depending on specific circumstances). Single colonies were picked with a sterile pipette tip and streaked for purification. The growth and colony morphology distribution of strain L12 on LB solid plates are shown in [the table below]. Figure 1 Finally, the DNA of the strain was observed and extracted and purified. The 16S rRNA gene of the isolated and cultured bacteria was identified by high-throughput sequencing using two-sided tag PCR amplification, thus determining the taxonomic position of the bacteria.
[0031] The 16S rRNA gene was amplified using bacterial primers (27F and 1492R). The PCR fragments were sequenced by a bioengineering sequencing company. The assembled sequences were uploaded to the NCBI website for sequence alignment, and phylogenetic analysis was performed using MEGA (version 7.0) software. The constructed phylogenetic tree is shown below. Figure 2 As shown. Based on high homology and high similarity, strain L12 was ultimately identified as... Aureimonas ureilytica .
[0032] 3. Colony microbiological characteristics
[0033] The colony morphology of L12 colony after 2 days of incubation on LB agar plates is as follows: Figure 1 As shown, the colonies are round, yellow, with smooth, regular edges, a glossy surface, a raised center, and are opaque. Strain L12 is Gram-negative.
[0034] Example 2 Determination of glyphosate degradation by bacterial strains
[0035] The significantly enriched strains from the isolated resistant populations were inoculated into LB liquid medium and incubated on a shaker at 180 rpm and 28°C. After centrifugation, the bacterial cells were collected, washed twice with sterile water, and then cultured at an OD concentration... 600 A bacterial suspension with a value of 0.5 (the viable bacteria content in the bacterial suspension is approximately 1.67 × 10⁻⁵). 7 1 mL of glyphosate (CFU / mL) was inoculated into 30 mL of modified enrichment medium containing 100 mg / L glyphosate. The samples were incubated at 180 rpm in the dark at 28°C for 5 days. The residual glyphosate in the culture medium was determined using pre-column derivatization-high performance liquid chromatography. Each treatment was repeated in triplicate; samples without the strain served as the control group.
[0036] The modified enrichment medium formula is: glucose 1 g / L, peptone 5 g / L, potassium nitrate 1 g / L sodium chloride, 2 g / L glyphosate (added to a concentration of 100 mg / L); pH 6.6-7.6. The method for determining the remaining glyphosate concentration is as follows:
[0037] Preparation of blank solution: Accurately measure 7 mL of water into a 50 mL volumetric flask, then add 0.5 mL of 50% sulfuric acid solution, 0.1 mL of 250 g / L potassium bromide solution, and 0.5 mL of 14 g / L sodium nitrite solution sequentially. Seal the flask tightly and shake thoroughly. After the entire system has reacted for 20 min (reaction temperature not lower than 15℃), dilute to the mark with purified water and shake well, then let stand for 15 min. Preparation of standard solution: Add 10 mg of glyphosate (95% purity) to 100 mL of modified enrichment liquid culture medium containing glyphosate to prepare a stock solution with a concentration of 100 mg / L. Five 50mL volumetric flasks were prepared and numbered. 0.05mL, 0.25mL, 0.5mL, 1mL, and 2mL of glyphosate stock solution were added to each flask, along with 6.95mL, 6.75mL, 6.5mL, 6mL, and 5mL of water, respectively. Then, 0.5mL of sulfuric acid solution, 0.1mL of potassium bromide solution, and 0.5mL of sodium nitrite solution were added sequentially. The flasks were tightly sealed, thoroughly shaken, and allowed to react for 20 minutes. The solutions were then diluted to the mark with purified water, shaken well, and allowed to stand for 15 minutes to obtain glyphosate standard solutions with concentrations of 0.1mg / L, 0.5mg / L, 1.0mg / L, 2.0mg / L, and 4mg / L. Sample solution preparation: After culturing the strain in modified enrichment medium for 5 days, the supernatant was centrifuged and subjected to nitrification reaction according to the method used for standard solutions. Using a blank solution as a control, the absorbance values were recorded at a wavelength of 242 nm, and a standard curve was fitted.
[0038] Calculate the degradation rate of glyphosate by the bacterial community: Degradation rate (%) = (C1-C2) / C2 × 100%
[0039] In the formula: C1 is the original concentration of glyphosate; C2 is the concentration of glyphosate obtained from the standard curve. The units are both mg / L.
[0040] The strain achieved a glyphosate degradation rate of 85.44% after culturing in a modified enrichment medium of 100 mg / L for 5 days. Figure 3 and Figure 4 ).
[0041] Example 3: Stress resistance experiment of strain L12
[0042] To investigate the acid and alkali resistance of strain L12, since the normal growth condition of strain L12 is 7, single colonies were transferred to LB medium with pH values of 5, 5.3, 5.4, 7, 8, 9, and 10 (pH adjusted using hydrochloric acid and sodium hydroxide solution) and cultured at 28°C for 2 days. The growth of the strain was then observed.
[0043] Figure 5The results showed that as the alkalinity of the culture medium increased, the growth of the strain gradually deteriorated, and the alkalinity tolerance could reach pH 10; while acidity had a greater impact on the strain, and the strain grew very poorly at pH 5. As the pH increased, the strain's growth ability gradually became stronger.
[0044] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. Strains Aureimonas ureilytica L12, with accession number CGMCC No. 33169.
2. A microbial preparation, characterized in that, It contains one or more of the following: bacterial cells, bacterial powder, and bacterial suspension selected from strain L12 of claim 1.
3. The method for preparing the microbial preparation according to claim 2, characterized in that, The preparation method includes the step of culturing the strain L12.
4. The preparation method according to claim 3, characterized in that, The conditions for culturing strain L12 were: 28°C, 180 rpm shaker, and in the dark.
5. The preparation method according to claim 3 or 4, characterized in that, The strain L12 was cultured using a modified enrichment medium. The modified enrichment medium has the following formula: glucose 1 g / L, peptone 5 g / L, potassium nitrate 1 g / L and sodium chloride 2 g / L, with glyphosate added to a concentration of 100 mg / L; pH value 6.6-7.
6.
6. A method for degrading glyphosate, characterized in that, The method includes: inoculating the strain L12 of claim 1 into a modified enrichment medium containing glyphosate; The improved enrichment medium is the same as the medium described in claim 5.
7. The method according to claim 6, characterized in that, OD 600 One mL of bacterial suspension with a concentration of 0.5 was inoculated into 30 mL of modified enrichment medium containing 100 mg / L glyphosate and cultured for 5 days at 28°C and 180 rpm in the dark.