Gordonia sp. dly03 and application thereof
By screening out Rhodococcus Gordonii DLY03, the problem of soil and water remediation for oxadiazon and cadmium pollution has been solved, achieving efficient degradation of oxadiazon and cadmium, thus protecting the ecological environment and human health.
Patent Information
- Application Number
- CN202510212287.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-25
- Publication Date
- 2025-11-11
- Estimated Expiration
- 2045-02-25
AI Technical Summary
Existing technologies are insufficient to effectively address soil and water pollution problems caused by oxadiazon and cadmium pollution, especially in environments with complex pollution, where it is difficult to screen out microbial strains that can both degrade oxadiazon and tolerate cadmium.
A strain of *Rhodococcus Gordonii* DLY03 was screened out. This strain can efficiently degrade oxadiazon and exhibits good cadmium tolerance. A liquid inoculant of *Rhodococcus Gordonii* DLY03 was prepared for the remediation of contaminated soil and water.
Rhodococcus Gordonii DLY03 can significantly degrade oxadiazon in a short time, with a degradation rate of 60.68% to 82.06%, and can maintain growth even at cadmium concentrations as high as 100 mg/L. It effectively manages herbicides and cadmium residues in agricultural production, protecting the ecological environment and human health.
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Figure CN120060020B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a type of Rhodococcus Gordonii DLY03 and its applications, belonging to the field of microbial technology. Background Technology
[0002] Oxadiazon, also known as oxadiazon or oxadiazon, is a selective pre-emergence herbicide developed by Rhône-Poulenc in France in 1969. Its chemical name is 5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazon-2(3H)-one, belonging to the nitrogen-containing heterocyclic class of compounds. Oxadiazon technical grade is a white crystalline powder, readily soluble in organic solvents, with a solubility of 0.7 mg / L in water at room temperature. It has a melting point of approximately 90°C and exhibits good stability. Currently, there are many commercial formulations of oxadiazon registered in my country. Among them, primary formulations include 250 g / L and 380 g / L suspension concentrates; secondary formulations mainly include 60% oxadiazon·butachlor EC, 60% oxadiazon·pretilachlor Emulsifiable Concentrate, 54% oxadiazon·acetochlor EC, 20% / 60% oxadiazon·butachlor EC, etc.; tertiary formulations mainly include 48% pretilachlor·propoxachlor·ciprofloxacin EC, 50% propoxachlor·butachlor·ciprofloxacin EC, 43% butachlor·oxadiazon·ciprofloxacin EC, 42% oxadiazon·paratide·ciprofloxacin EC, 28% propoxachlor·ciprofloxacin·isoxazone EC, 40% propoxachlor·pyrazosulfuron·propoxachlor, etc. Oxalide, a highly effective and broad-spectrum herbicide, has been widely used in global agriculture since its introduction, making significant contributions to sustainable agricultural development. It is commonly used to control annual grasses and broadleaf weeds in fields and orchards of various crops (rice, cotton, soybeans, peanuts, potatoes, celery, sugarcane, onions, tea, citrus, and grapes). Previous studies have shown that the extensive use of oxadiazon formulations inevitably pollutes soil ecosystems. Oxalide has a long residual period in soil (30–120 days), and even after microbial degradation and photodegradation, it can still accumulate in the soil over a long period. Through rainwater runoff and agricultural irrigation drainage, oxadiazon can enter surface water and groundwater, causing water pollution and impacting aquatic organisms. Through the food chain, it accumulates from lower to higher organisms, reaching a harmful threshold within organisms, triggering chronic toxic effects, damaging organ function, and affecting survival and reproduction. Therefore, the soil and water pollution caused by the extensive use of oxadiazon has received great attention, and there is an urgent need to develop practical green remediation technologies to solve the ecological and environmental problems caused by oxadiazon-contaminated soil.
[0003] Cadmium is a heavy metal element that is ubiquitous in the natural environment and toxic to humans, animals, and plants. It is a carcinogen, and cadmium is present in almost all soils, surface water, and plant matter. Ingesting even trace amounts of cadmium not only threatens the physiology and health of individual organisms but also affects population size and species distribution. Studies show that cadmium pollution exists to varying degrees in countries worldwide. Soils in areas with sedimentary rocks, particularly carbonate rocks, generally exhibit geochemical anomalies indicating high background cadmium levels. Globally, carbonate rocks cover approximately 12% of the land area, and the karst carbonate rock region in southwestern China, centered on Guizhou, accounts for one-third of the country's land area, approximately 1.5 × 10⁻⁶. 5 km 2 The background cadmium levels in the soil within this region (0.015–2.977 mg / kg) are higher than the national average, indicating a typical cadmium geochemical anomaly area. In recent years, with the widespread application of cadmium in industry and agriculture and the expansion of human activities, coupled with cadmium's characteristics of being unable to be degraded by soil microorganisms, capable of long-distance migration, and easily absorbed and accumulated by plants, soil cadmium pollution has seriously affected the sustainable development of agricultural production and human health in my country, especially in the southwest region. Therefore, the control and remediation of soil cadmium pollution has been a hot topic of concern in recent years.
[0004] In recent years, microbial remediation technology has attracted much attention due to its advantages of safety, efficiency, environmental friendliness, and sustainability. This technology utilizes the adsorption and metabolic capabilities of microorganisms to transform pesticide residues into harmless substances and passivate heavy metals in the soil, preventing their absorption by plants, thus effectively reducing pesticide and heavy metal pollution of the environment and agricultural products. Based on this, many developed countries and large enterprises have invested heavily in the research and development of bio-agents for the remediation of pesticide residues or cadmium pollution. For example, the US-based Engineering Services Bioremediation Company has successfully commercialized pesticide residue degradation formulations. However, the widespread application of microbial remediation technology faces several challenges. On the one hand, pesticide degradation by microorganisms is highly specific, and existing biodegrading bacterial resource libraries are insufficient to meet the actual needs of bioremediation of different types of pesticide residue pollution. On the other hand, actual bioremediation scenarios are often environments with combined organic matter and heavy metal pollution, making it particularly important to screen microbial strains that are both adapted to heavy metal pollution environments and capable of degrading pesticides. Therefore, screening for highly efficient cadmium-resistant microbial strains that degrade oxadiazon and developing corresponding bio-agents, using artificial addition methods to accelerate the decomposition of pesticides and the passivation of heavy metals in the environment, is a key strategy for eliminating pesticide residues and heavy metal hazards. Summary of the Invention
[0005] The purpose of this invention is to provide a *Rhodococcus Gordonii* strain DLY03 and its applications. This strain exhibits highly efficient degradation of oxadiazon and demonstrates good cadmium tolerance.
[0006] The technical solution of the present invention: A Rhodococcus gordoniae DLY03 was deposited at the China Center for Type Culture Collection on April 25, 2022, at Wuhan University, Wuhan, China, and named Rhodococcus gordoniae DLY03, with accession number CCTCC NO: M 2022479.
[0007] Application of Rhodococcus Gordonii DLY03 in the treatment of oxadiazon and cadmium pollution.
[0008] A liquid bacterial agent containing Rhodococcus Gordonii DLY03.
[0009] A method for preparing a liquid bacterial agent containing Rhodococcus Gordonii DLY03, wherein Rhodococcus Gordonii DLY03 is used as the living component to prepare the liquid bacterial agent.
[0010] The beneficial effects of this invention are as follows: Compared with the prior art, the strain involved in this invention was identified as *Rhodococcus gordoniae* through morphological characteristics, physiological and biochemical properties, and 16S rDNA sequence analysis. The colonies of this strain on beef extract peptone agar plates are opaque orange. Physiological and biochemical characterization revealed that strain DLY03 is Gram-positive, catalase-positive, oxidase-negative, aerobic, and can utilize glucose, xylose, L-arabinose, and mannitol, but does not decompose casein.
[0011] The inventors of this application have screened and obtained a strain of *Rhodococcus gordoniae* DLY03 that combines the ability to degrade oxadiazon and is cadmium-tolerant. This strain can efficiently degrade oxadiazon in a short time, exhibiting significant degradation capacity, with a degradation rate of 60.68% within 3 days and 82.06% within 5 days. In cadmium tolerance tests, this strain showed good cadmium tolerance, with a maximum tolerated concentration of 100 mg / L. This strain can be used to remediate natural environments such as soil and water bodies contaminated by oxadiazon and cadmium, effectively controlling excessive herbicide and cadmium residues in agricultural production and related environmental pollution, thereby protecting the ecological environment and human health. The bacterial agent prepared using *Rhodococcus gordoniae* DLY03 has low production costs, convenient application methods, and significant pollutant removal effects. This bacterial agent is suitable for solving problems such as soil and water pollution caused by oxadiazon and cadmium pollution, and has important theoretical significance and practical application value. Attached Figure Description
[0012] Appendix Figure 1 Chromatogram of oxadiazon standard;
[0013] Appendix Figure 2 The colony morphology of Rhodococcus Gordonii DLY03;
[0014] Appendix Figure 3 Phylogenetic tree of Rhodococcus Gordonii DLY03;
[0015] Appendix Figure 4 The degradation rate of oxadiazon (50 mg / L) in selective inorganic salt culture medium (cadmium 20 mg / L) by the degrading bacterial agent was measured.
[0016] Appendix Figure 5 To assess the cadmium tolerance of Rhodococcus Gordonii DLY03. Detailed Implementation
[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.
[0018] Example 1 of the present invention: Isolation and identification of Rhodococcus Gordonii DLY03:
[0019] 1) Isolation and screening of Rhodococcus Gordonii DLY03
[0020] Weigh 10g of soil that has been contaminated with oxadiazon and cadmium for a long time into a 250mL Erlenmeyer flask, add 100mL of selective inorganic salt medium (cadmium 20mg / L), and add oxadiazon to a concentration of 25mg / L. Place the Erlenmeyer flask in a shaker at 28℃ and 150rpm for 5 days. Take 5mL of the culture solution and inoculate it into a new selective inorganic salt medium (cadmium 20mg / L, oxadiazon 25mg / L). Incubate under the same conditions for 5 days. Repeat this process, increasing the concentration of oxadiazon by 25mg / L, until the final concentration of the herbicide in the enriched culture solution reaches 500mg / L, thus obtaining the enriched degrading bacterial solution.
[0021] During the enrichment and isolation of degrading strains, after every two transfers, the bacterial suspension was prepared into 10⁻¹⁰ saturates using sterile water. -2 ~10 -6 A series of dilutions were inoculated onto solid separation medium using the spread plating method and incubated at 28°C for 2 days. Based on the morphological characteristics of the colonies, single colonies were picked and repeatedly streaked for purification. Strains with the same colony characteristics were combined based on both external and microscopic morphological characteristics, and the purified strains were inoculated onto slant solid separation medium for storage.
[0022] After extensive enrichment culture, a strain capable of growing with oxadiazon as the sole carbon source in a medium containing 20 mg / L cadmium and 500 mg / L oxadiazon was successfully isolated. Its degradation efficiency was verified using ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS). The strain was named degrading bacterium DLY03. Under pure culture conditions, this strain was able to degrade more than 80% of oxadiazon at an initial concentration of 50.0 mg / L within 5 days.
[0023] UPLC-MS / MS determination conditions: Ultra-high performance liquid chromatography-triple quadrupole LC-MS / MS system (Agilent 1290II, 6470); Agilent Eclipse Plus C 18 Column (4.6 × 100 mm, 3.5 μm particle size); sheath gas temperature and flow rate: 250 °C and 11.0 L / min; nozzle voltage: 500 V; capillary voltage: 3500 V; nebulizer gas pressure: 15 psi; mobile phase: aqueous solution (A) containing 0.1% formic acid - acetonitrile (B), isocratic elution with 80% acetonitrile for 15 min. Flow rate: 0.3 mL / min, column temperature: 30 °C; injection volume: 20 μL. Retention time: 11.350 min for oxadiazon; typical chromatogram shown in [reference needed]. Figure 1 .
[0024] The degradation rate is calculated as follows:
[0025]
[0026] 2) Identification of Rhodococcus Gordonii DLY03
[0027] (1) Morphological identification of degrading bacteria DLY03: The single colony state of the Gordon red cocci DLY03 obtained by isolation and purification in step one above, which is in the logarithmic growth phase and has a stable colony size, is described, mainly including the size, color, transparency and surface state of the colony.
[0028] The results showed that the degrading bacteria DLY03, isolated and purified through the above steps, grew rapidly on beef extract peptone agar plates, appearing as round or nearly round, 3-4 mm in diameter, orange in color, and opaque. Figure 2 ).
[0029] (2) Physiological and biochemical characteristics analysis
[0030] The physiological and biochemical characteristics of the degrading bacterium DLY03 were determined with reference to the methods described in "Microbiology Experiments" (Shen Ping, Fan Xiurong, and Li Guangwu. Microbiology Experiments (3rd Edition). Beijing: Higher Education Press, 1999) and "Handbook of Systematic Identification of Common Bacteria" (Dong Xiuzhu and Cai Miaoying. Handbook of Systematic Identification of Common Bacteria. Beijing: Science Press, 2011).
[0031] The test results are shown in Table 1. The degrading bacterium DLY03 is Gram-positive, catalase-positive, oxidase-negative, and aerobic. This bacterium can utilize glucose, xylose, L-arabinose, and mannitol; it does not decompose casein.
[0032] Table 1. Physiological and biochemical characteristics of degrading bacterium DLY03
[0033] Experimental Project result Experimental Project result Gram staining Positive glucose + Cell shape club-shaped Xylose + catalase + L-arabinose + Oxidase - Mannitol + Aerobic growth + Starch hydrolysis + Nitrate reduction + Break down casein - Carbohydrate acid production + Using citrate +
[0034] Note: "+" indicates a positive reaction, and "-" indicates a negative reaction.
[0035] (3) Homology analysis of 16S rDNA of degrading bacteria
[0036] DNA sequencing was performed by the Institute of Microbiology, Chinese Academy of Sciences, and homology analysis was conducted using 16S rDNA sequences from Genbank using the Blast software.
[0037] The 16S rDNA of the obtained degrading bacterium DLY03 is detailed in the sequence listing.
[0038] The 16S rDNA sequence of this strain has been submitted to the GenBank database (GenBank accession number ON329176). It was found that this sequence shares 100% homology with gene sequences of strains such as *Rhodococcus gordoniae* MW221328. (See phylogenetic tree). Figure 3 .
[0039] 3) Growth Characteristics Analysis
[0040] Optimal temperature and pH growth experiments were conducted on the strain. Selective inorganic salt medium was used, and temperatures were set at 20℃, 25℃, 30℃, 35℃, and 40℃, with each treatment replicated three times. The acidity of the medium was adjusted to pH 4, pH 5, pH 6, pH 7, pH 8, and pH 9, with each treatment replicated three times. The optimal temperature and pH for the strain's growth were cultured, observed, and recorded. The results showed that the optimal growth temperature for the degrading bacterium DLY03 was 30–35℃, and the optimal growth pH was 7–8.
[0041] Based on the morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis results of the above-mentioned degrading bacterium DLY03, it was identified as *Rhodococcus gordoniae*. Strain *Rhodococcus gordoniae* DLY03 was deposited on April 25, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2022479.
[0042] Example 2 of the present invention: Preparation of the degrading bacterium Rhodococcus Gordonii DLY03 preparation:
[0043] 1) The Rhodococcus Gordonii DLY03, which has both the ability to degrade oxadiazon and is cadmium resistant, was inoculated into an inorganic salt medium and cultured with shaking at 30°C and 150 rpm until the logarithmic growth phase to obtain the strain;
[0044] 2) Inoculate the bacterial strain into a seed bottle containing culture medium at a volume percentage of 10%, and culture at 30°C and 150 rpm until the logarithmic growth phase to obtain the seed culture.
[0045] 3) Inoculate the obtained seed culture into the fermentation medium at an inoculation rate of 10% by volume, and ferment at 30℃ and 150 rpm for 45–50 h to obtain the fermentation broth (OD). 600 (Greater than 20), the fermentation broth is directly diluted into a liquid inoculum. The OD of the liquid inoculum... 600 The value is 2. In addition, the fermentation broth can be adsorbed using diatomaceous earth or biochar to produce a solid inoculum.
[0046] The culture medium mentioned in steps 2) and 3) is the same, and its composition, by mass percentage, includes 0.5% yeast extract, 1% peptone, 0.5% glucose, 1% NaCl, and the balance being water, with a pH of 7.
[0047] Example 3 of the present invention: Degradation effect of Rhodococcus Gordonii DLY03 on oxadiazon:
[0048] Cadmium and oxadiazon were added to an inorganic salt culture medium to achieve final concentrations of 20 mg / L and 50.0 mg / L, respectively; OD was inoculated at a rate of 10%. 600 =2 bacterial cells were inoculated into an inorganic salt culture medium containing cadmium (20 mg / L) and oxadiazon (50.0 mg / L), with an uninoculated culture medium as a control. All samples were incubated in the dark at 30℃ and 150 rpm for 0–5 days, and samples were taken periodically.
[0049] The residue of oxadiazon was determined and its degradation rate was calculated using UPLC-MS / MS. Figure 4 As shown, the degrading bacterium DLY03 exhibited a significant degradation effect on 50.0 mg / L oxadiazon within a short period of time, with a degradation rate reaching 60.68% within 3 days and 82.06% within 5 days, indicating that this strain possesses highly efficient biodegradation capabilities. This result demonstrates that the degrading bacterium DLY03 provided by this invention can efficiently degrade oxadiazon and has broad application potential in the remediation of oxadiazon and cadmium-contaminated soil and water bodies. Furthermore, this strain can also degrade pollutants such as phthalates and aflatoxins.
[0050] Example 4 of the present invention: Cadmium tolerance test of Rhodococcus Gordonii strain DLY03:
[0051] Prepare inorganic salt culture media containing cadmium concentrations of 5, 20, 50, 100, and 200 mg / L (initial concentration of oxadiazon is 50 mg / L), and inoculate OD at a 10% inoculum. 600 =2 Gordon's Rhodococcus DLY03 bacterial suspension, after being incubated in the dark at 30℃ and 150rpm for 0-5 days, the OD of the bacterial suspension was measured.600 Regular sampling is required.
[0052] The results are as follows Figure 4 As shown, *Rhodococcus Gordonii* DLY03 exhibits good growth at cadmium concentrations of 5–50 mg / L, indicating strong cadmium tolerance. When the cadmium concentration increases to 100 mg / L, the growth curve becomes more stable, and the growth of the strain is inhibited, indicating that a cadmium concentration of 100 mg / L is close to or has reached the critical value for cadmium tolerance. When the cadmium concentration increases to 200 mg / L, the OD... 600 The value decreased slowly, the growth of the strain was significantly inhibited, and when the cadmium concentration exceeded the strain's tolerance, the cell concentration decreased.
[0053] The culture medium used in the above embodiments is as follows:
[0054] Inorganic salt culture medium: KH₂PO₄ 0.4g, K₂HPO₄ 0.4g, NH₄Cl 1g, MgCl₂ 0.1g, Na₂SO₄ 1.425g, FeSO₄·7H₂O 0.025g, trace element solution 10mL, diluted to 1L with distilled water, pH 7.0. The trace element solution composition is as follows: ZnSO₄·7H₂O 1.1g, MgSO₄·H₂O 0.58g, (NH₄)₆Mo₇O₇ 24 ·4H2O 0.18g, CoSO4·7H2O 0.024g, CuSO4·5H2O 0.077g, H3BO3 0.029g, NaNO3·4H2O 0.074g, distilled water 1L.
[0055] Selective inorganic salt medium: Add 0.02g CdCl2 to the inorganic salt medium.
[0056] Isolation medium: Selective inorganic salt medium with oxadiazon as the sole carbon source, with different concentrations added according to the experimental design requirements, pH 7.0 (20g agar added to solid medium).
[0057] Beef extract peptone medium: 3.0g beef extract, 5.0g NaCl, 10.0g peptone, 1000mL distilled water, 20g agar, pH 7.0.
[0058] All the above culture media were sterilized in an autoclave at 121°C for 20–30 minutes.
[0059] Sequence list information:
[0060] DTD Version: V1_3
[0061] Filename: Rhodococcus Gordonii DLY03.xml
[0062] Software Name: WIPOSequence
[0063] Software version: 2.3.0
[0064] Generation Date: 2025-02-21
[0065] Basic Information:
[0066] Current application / applicant file name: 12520000429203011T
[0067] Applicant's Name or Title: Guizhou University
[0068] Applicant's name or title / language:zh
[0069] Applicant's Name or Title / Latin Name: guizhou University Invention Title: A Rhodococcus Gordonii DLY03 and Its Application (zh) Total Sequences: 1
[0070] sequence:
[0071] Serial Number (ID): 1
[0072] Length: 1426
[0073] Molecular type: RNA
[0074] Feature location / qualifier:
[0075] -source,1..1426
[0076] >mol_type, other RNA
[0077] >organism, synthetic construct
[0078] residues:
[0079]
[0080]
[0081]
Claims
1. A type of Rhodococcus Gordonii DLY03 ( Rhodococcus gordoniae DLY03), characterized by: It was deposited on April 25, 2022, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, and named *Rhodococcus Gordonii* DLY03. Rhodococcus gordoniae DLY03), with accession number CCTCC NO:M 2022479.
2. The application of Rhodococcus Gordonii DLY03 as described in claim 1 in the treatment of oxadiazon and cadmium pollution.
3. A liquid bacterial agent containing Rhodococcus Gordonii DLY03 as described in claim 1.
4. A method for preparing a liquid bacterial agent containing *Rhodococcus Gordonii* DLY03 as described in claim 3, characterized in that: A liquid bacterial agent was prepared by using Rhodococcus Gordonii DLY03 as the living component.