Rhodococcus gordonus DLY03 and application thereof

Abstract

The invention discloses Rhodococcus gordoniae DLY03 and application thereof, the Rhodococcus gordoniae DLY03 is preserved in China Center for Type Culture Collection (CCTCC) on April 25, 2022, the address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 2022479. The degradation rate of oxadiazon with the initial concentration of 50.0 mg / L in a selective inorganic salt culture medium (20 mg / L of cadmium) reaches 60.68% within 3 days, and the degradation rate reaches 82.06% within 5 days. When the strain is cultured in inorganic salt culture media with different cadmium concentrations, the maximum tolerance concentration to cadmium can reach 100 mg / L. The rhodococcus gordonus DLY03 not only can efficiently degrade the oxadiazon, but also has good cadmium tolerance, provides powerful technical support for treatment of compound pollution of the oxadiazon and cadmium, and has important significance for solving the problem of complex environmental pollution.

Description

Technical Field

[0001] The present invention relates to Rhodococcus gordonii DLY03 and its application, belonging to the technical field of microorganisms. Background Art

[0002] Oxadiazon, also known as Ronstar, is a selective pre-emergence herbicide developed by Rhône-Poulenc in 1969. Its chemical name is 5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2(3H)-one, belonging to the nitrogen-containing heterocyclic compounds. The technical oxadiazon is a white crystal, soluble in organic solvents, with a solubility of 0.7 mg / L at room temperature in water, a melting point of about 90 °C, and good stability. Currently, there are many commercial formulations of oxadiazon registered in China. Among them, the single-ingredient formulations mainly include 250 g / L and 380 g / L suspension concentrates; the two-ingredient compound formulations mainly include 60% oxadiazon & butachlor EC, 60% oxadiazon & pretilachlor EW, 54% oxadiazon & acetochlor EC, 20% / 60% oxadiazon & butachlor EC, etc.; the three-ingredient compound formulations mainly include 48% pretilachlor & oxadiazon & simetryn EC, 50% oxadiazon & butachlor & simetryn EC, 43% butachlor & oxadiazon & simetryn EC, 42% oxadiazon & anilofos & simetryn EC, 28% oxadiazon & simetryn & clomazone EC, 40% pretilachlor & pyrazolate & oxadiazon, etc. As an efficient and broad-spectrum herbicide, oxadiazon has been widely used in the global agricultural field since its introduction, making important contributions to the sustainable development of agriculture. It is commonly used to control annual gramineous and broad-leaved weeds in various crop (rice, cotton, soybean, peanut, potato, celery, sugarcane, onion, tea tree, citrus, grape) fields / gardens. Previous studies have shown that the extensive use of oxadiazon formulations inevitably pollutes the soil ecosystem. The residue period of oxadiazon in the soil is relatively long (30 - 120 d). Even after processes such as microbial degradation and photolysis, it may still accumulate in the soil for a long time. Under the action of rainwashing, farmland irrigation drainage, etc., oxadiazon will enter surface water and groundwater, causing pollution to water sources and aquatic organisms. With the transfer through the food chain, it accumulates from lower organisms to higher organisms, and can reach the hazard threshold in organisms, triggering chronic toxic effects, damaging the functions of biological organs, and affecting the survival and reproduction of organisms. Therefore, the soil and water pollution caused by the extensive use of oxadiazon has received great attention, and it is urgent to develop practical green remediation technologies to solve the ecological environment problems brought by oxadiazon-polluted soil.

[0003] Cadmium is a heavy metal element that is widespread in the natural environment and is toxic to humans, animals and plants. It is a carcinogen. Cadmium is contained in almost all soils, surface waters and plants. Ingesting trace amounts of cadmium not only threatens the physiology and health of individual organisms, but also affects the population size and species distribution of organisms. Research shows that there are varying degrees of cadmium pollution problems in countries around the world. There is a common phenomenon of high cadmium background with geochemical anomalies in the soils in the areas where sedimentary rocks represented by carbonate rocks are developed. The outcropping area of carbonate rocks in the world accounts for about 12% of the land area. The southwestern karst carbonate rock area in China centered on Guizhou accounts for 1 / 3 of the national land area, about 1.5×10 5 km 2 The background value of soil cadmium (0.015 - 2.977 mg / kg) in the region is higher than the national average, and it is a typical cadmium geochemical anomaly area. In recent years, with the extensive application of cadmium in the industrial and agricultural fields and the extension of the scope of human activities, coupled with the characteristics that cadmium cannot be degraded by soil microorganisms, can migrate over long distances and is easily absorbed and accumulated by plants, soil cadmium pollution has seriously affected the sustainable development of agricultural production and human health in China, especially in the southwestern region. Therefore, the control and treatment of soil cadmium pollution have always been one of the hot issues concerned by people in recent years.

[0004] In recent years, the microbial remediation technology has attracted much attention due to its advantages such as safety, high efficiency, environmental protection and sustainability. Through the adsorption and metabolism ability of microorganisms, this technology converts pesticide residues into harmless substances, passivates soil heavy metals so that they cannot be absorbed by plants, and effectively reduces the pollution of pesticides and heavy metals to the environment and agricultural products. Based on this, many developed countries and large enterprises have invested a large amount of funds in the research and development of biological agents for treating pesticide residues or cadmium pollution. For example, the Engineering Services Bioremediation Company in the United States has successfully realized the productization of pesticide residue degradation preparations. There are many problems in the popularization and application of the microbial remediation technology. On the one hand, the microbial degradation of pesticides has strong specificity, and the existing degradation bacteria resource library is difficult to meet the actual needs of bioremediation of different types of pesticide residue pollution; on the other hand, the actual bioremediation scenarios are mostly complex pollution environments of organic matter and heavy metals, which makes it particularly important to screen out microbial strains that are adapted to heavy metal pollution environments and have the function of degrading pesticides. Therefore, screening cadmium-tolerant microbial strains that can efficiently degrade oxadiazon and developing corresponding biological agents, and using artificial addition means to accelerate the decomposition of pesticides and the passivation of heavy metals in the environment is a key strategy to eliminate the hazards of pesticide residues and heavy metals. Summary of the Invention

[0005] The purpose of the present invention is to provide a Rhodococcus gordonae DLY03 and its application. This strain has the function of efficiently degrading oxadiazon and shows good cadmium tolerance.

[0006] Technical solution of the present invention: A Rhodococcus gordoniae DLY03 was deposited with the China Center for Type Culture Collection on April 25, 2022. The deposit address is Wuhan University, Wuhan, China, and it is named Rhodococcus gordoniae DLY03, with the deposit number CCTCC NO: M 2022479.

[0007] Application of a Rhodococcus gordoniae DLY03 in treating oxyfluorfen and cadmium pollution.

[0008] A liquid bacterial agent containing Rhodococcus gordoniae DLY03.

[0009] A preparation method of a liquid bacterial agent containing Rhodococcus gordoniae DLY03, using Rhodococcus gordoniae DLY03 as a living component to prepare the liquid bacterial agent.

[0010] Beneficial effects of the present invention: Compared with the prior art, the strain involved in the present invention was identified as Rhodococcus gordoniae through morphological characteristics, physiological and biochemical characteristics, and 16S rDNA sequence analysis. The colonies of this strain on the nutrient agar solid plate are opaque orange. Through physiological and biochemical property determination, it was found that strain DLY03 is Gram-positive, catalase-positive, oxidase-negative, aerobic growth, can utilize glucose, xylose, L-arabinose, and mannitol, and does not decompose casein.

[0011] The inventors' team of the present application screened and obtained a Rhodococcus gordoniae DLY03 that has both the ability to degrade oxyfluorfen and cadmium tolerance. This strain can efficiently degrade oxyfluorfen within a short time, showing significant degradation ability. Its degradation rate reaches 60.68% within 3 days and 82.06% within 5 days; this strain shows good cadmium tolerance in the cadmium tolerance determination, and the maximum tolerable concentration of cadmium can reach 100 mg / L. This strain can be used to repair natural environments such as soil and water bodies polluted by oxyfluorfen and cadmium, effectively control the excessive residues of herbicides and cadmium in agricultural product production and related environmental pollution, thereby protecting the ecological environment and human health. The bacterial agent produced and prepared using Rhodococcus gordoniae DLY03 has a low production cost, a convenient usage method, and a significant pollutant removal effect. This bacterial agent is suitable for solving problems such as soil and water environmental pollution caused by oxyfluorfen and cadmium pollution, and has important theoretical significance and practical application value. Description of the drawings

[0012] Appendix Figure 1 Is the chromatogram of the oxyfluorfen standard product;

[0013] Appendix Figure 2 Is the colony morphology of Rhodococcus gordoniae DLY03;

[0014] Attached Figure 3 is the phylogenetic tree of Rhodococcus gordoniae DLY03;

[0015] Attached Figure 4 is the degradation rate of the degradation agent on oxadiazon (50 mg / L) in the selective inorganic salt medium (cadmium 20 mg / L);

[0016] Attached Figure 5 is the cadmium tolerance of Rhodococcus gordoniae DLY03. Specific Embodiments

[0017] The present invention will be further described below in conjunction with the accompanying drawings and embodiments, but it shall not be used as a basis for limiting the present invention.

[0018] Example 1 of the present invention: Isolation and identification of Rhodococcus gordoniae DLY03:

[0019] 1) Isolation and screening of Rhodococcus gordoniae DLY03

[0020] Weigh 10 g of soil contaminated with oxadiazon and cadmium for a long time into a 250 mL conical flask, add 100 mL of selective inorganic salt medium (cadmium 20 mg / L), add oxadiazon to a concentration of 25 mg / L, place the conical flask on a shaker at 28 °C and 150 rpm for 5 d. Take 5 mL of the culture solution and inoculate it into a new selective inorganic salt medium (cadmium 20 mg / L, oxadiazon 25 mg / L), and culture it under the same conditions for 5 d. By analogy, increase the concentration gradient of oxadiazon by 25 mg / L each time until the concentration of the herbicide in the final enriched culture solution reaches 500 mg / L, obtaining an enriched degradation bacterial solution.

[0021] During the enrichment and isolation process of the degradation strain, after every 2 transfers, prepare the bacterial suspension into a 10 -2 ~10 -6 serial dilution solution with sterile water, and use the coating method to inoculate it into the solid separation medium respectively, and culture it at 28 °C for 2 d. According to the morphological characteristics of the colonies, pick single colonies and purify them by repeated streaking. Combine the same strains according to the colony appearance morphological characteristics and microscopic morphological characteristics, and inoculate the purified strains into the slant solid separation medium for storage for later use.

[0022] After a large amount of enrichment culture, a strain that can grow with oxadiazon as the sole carbon source in the separation medium containing 20 mg / L of cadmium and 500 mg / L of oxadiazon was successfully isolated, and its degradation effect was verified by ultra-high performance liquid chromatography (UPLC-MS / MS). The strain was named degradation bacterium DLY03. Under pure culture conditions, the strain could degrade more than 80% of the initial concentration of 50.0 mg / L of oxadiazon within 5 d.

[0023] UPLC-MS / MS determination conditions: Ultra Performance Liquid Chromatography-Triple Quadrupole Liquid Chromatography-Mass Spectrometry (Agilent 1290 II, 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; Nebulizing gas pressure: 15 psi; The mobile phase was an aqueous solution containing 0.1% formic acid (A)-acetonitrile (B), and isocratic elution with 80% acetonitrile for 15 min. The flow rate was 0.3 mL / min, the column temperature was 30 °C; The injection volume was 20 μL. Retention time: Oxadiazon was 11.350 min, and the typical chromatogram is shown in Figure 1 .

[0024] The calculation method of the degradation rate is as follows:

[0025]

[0026] 2) Identification of Rhodococcus gordoniae DLY03

[0027] (1) Morphological identification of the degrading bacterium DLY03: Rhodococcus gordoniae DLY03 isolated and purified in the above step 1 at the logarithmic growth phase with stable colony size was described in the single colony state, mainly including the size, color, transparency, and surface state of the colony.

[0028] It was found that the degrading bacterium DLY03 isolated and purified by the above steps grew rapidly on the nutrient agar solid plate, was round or approximately round, 3-4 mm in diameter, orange, and opaque ( Figure 2 ).

[0029] (2) Analysis of physiological and biochemical characteristics

[0030] Refer to "Microbiology Experiments" (Shen Ping, Fan Xiurong, Li Guangwu. Microbiology Experiments (Third Edition). Beijing: Higher Education Press, 1999.) and "Handbook for the Systematic Identification of Common Bacteria" (Dong Xiuzhu, Cai Miaoying. Handbook for the Systematic Identification of Common Bacteria. Beijing: Science Press, 2011.) to determine the physiological and biochemical characteristics of the degrading bacterium DLY03.

[0031] The determination results are shown in Table 1. The degrading bacterium DLY03 was Gram-positive, catalase-positive, oxidase-negative, and aerobic. This bacterium could utilize glucose, xylose, L-arabinose, and mannitol; it did not decompose casein.

[0032] Table 1 Physiological and biochemical characteristics of the degrading bacterium DLY03

[0033] Test item Result Test item Result Gram stain Positive Glucose + Cell shape Coccobacillus Xylose + Catalase + L-Arabinose + Oxidase - Mannitol + Aerobic growth + Starch hydrolysis + Nitrate reduction + Casein decomposition - Carbohydrate acid production + Utilization of citrate +

[0034] Note: “+” indicates a positive reaction, and “-” indicates a negative reaction

[0035] (3) Homology analysis of the 16S rDNA of the degrading bacterium

[0036] DNA sequencing was completed by the Institute of Microbiology, Chinese Academy of Sciences, and homology analysis was performed using the 16S rDNA sequences in the Genbank with Blast software

[0037] The 16S rDNA of the obtained degrading bacterium DLY03 is shown in the sequence listing information

[0038] The 16S rDNA sequence of this strain has been submitted to the GenBank database (GenBank accession number ON329176), and it is found that the homology of this sequence with the gene sequences of strains such as Rhodococcus gordoniae MW221328 reaches 100%. The phylogenetic tree is shown in Figure 3 .

[0039] (3) Growth characteristic analysis

[0040] Optimal temperature and pH growth experiments of the strain were carried out. Using a selective inorganic salt medium, the temperatures were set at 20°C, 25°C, 30°C, 35°C, and 40°C respectively, with 3 replicates for each treatment. The acidity of the medium was adjusted to pH4, pH5, pH6, pH7, pH8, and pH9 respectively, with 3 replicates for each treatment. Cultivate, observe, and record the most suitable temperature and pH for the growth of the strain. The results show that the optimal growth temperature of the degrading bacterium DLY03 is 30 - 35°C, and the optimal growth pH is 7 - 8

[0041] In view of the morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis results of the above degrading bacterium DLY03, the degrading bacterium DLY03 was identified as Rhodococcus gordoniae. The strain Rhodococcus gordoniae DLY03 was preserved in the China Center for Type Culture Collection (CCTCC) on April 25, 2022, at the address of Wuhan University, Wuhan, China, with the preservation number CCTCC NO: M 2022479

[0042] Example 2 of the present invention: Preparation of the Rhodococcus gordoniae DLY03 preparation

[0043] (1) Inoculate Rhodococcus gordoniae DLY03, which has both the ability to degrade oxadiazon and cadmium tolerance, into an inorganic salt medium, and shake-culture at 30°C and 150 rpm until the logarithmic growth phase to obtain the bacterial strain

[0044] 2) Inoculate the strain into a seed flask containing a medium at an inoculation amount of 10% by volume, and culture it at 30 °C and 150 rpm until the logarithmic growth phase to obtain a seed liquid;

[0045] 3) Inoculate the obtained seed liquid into a fermentation medium at an inoculation amount of 10% by volume, and ferment and culture it at 30 °C and 150 rpm for 45 - 50 h to obtain a fermentation broth (OD 600 greater than 20), directly dilute the fermentation broth into a liquid microbial agent, and the OD of the liquid microbial agent 600 is 2. In addition, the fermentation broth can be adsorbed with diatomaceous earth or biochar to make a solid microbial agent.

[0046] The media mentioned in steps 2) and 3) are the same. Its composition is calculated by mass percentage and contains 0.5% yeast extract, 1% peptone, 0.5% glucose, 1% NaCl, the balance being water, and pH 7.

[0047] Example 3 of the present invention: Degradation effect of Rhodococcus gordoniae DLY03 on oxadiazon:

[0048] Add cadmium and oxadiazon to an inorganic salt medium so that their final concentrations are 20 mg / L and 50.0 mg / L respectively; inoculate the cells with OD 600 = 2 into an inorganic salt medium containing cadmium (20 mg / L) and oxadiazon (50.0 mg / L) at an inoculation amount of 10%, and use the uninoculated medium as a control. All samples are cultured in the dark at 30 °C and 150 rpm for 0 - 5 d, and samples are taken regularly.

[0049] Use the UPLC-MS / MS method to determine the residual amount of oxadiazon and calculate its degradation rate. As Figure 4 shown, the degradation bacterium DLY03 shows a significant degradation effect on 50.0 mg / L of oxadiazon in a short time. Its degradation rate reaches 60.68% within 3 d and 82.06% within 5 d, indicating that this strain has high biodegradation performance. This result shows that the degradation bacterium DLY03 provided by the present invention can efficiently degrade oxadiazon and has broad application potential in repairing soil and water bodies polluted by oxadiazon and cadmium. In addition, this strain can also degrade pollutants such as phthalic acid esters and aflatoxins.

[0050] Example 4 of the present invention: Cadmium tolerance determination of Rhodococcus gordoniae strain DLY03:

[0051] Prepare inorganic salt media containing cadmium concentrations of 5, 20, 50, 100, and 200 mg / L respectively (the initial concentration of oxadiazon is 50 mg / L), inoculate the Rhodococcus gordoniae DLY03 bacterial liquid with OD 600 = 2 at an inoculation amount of 10%, and measure the OD of the bacterial liquid after culturing in the dark at 30 °C and 150 rpm for 0 - 5 d600 , sample regularly.

[0052] The results are as Figure 4 shown. When the cadmium concentration is 5 - 50 mg / L, Rhodococcus Gordonae DLY03 grows well and the strain has strong cadmium tolerance. When the cadmium concentration increases to 100 mg / L, the growth curve is relatively stable and the growth of the strain is inhibited, indicating that the cadmium concentration of 100 mg / L has approached or reached the critical value of the cadmium tolerance of the bacterial cells. When the cadmium concentration increases to 200 mg / L, the OD 600 value decreases slowly, the growth of the strain is significantly inhibited, the cadmium concentration exceeds the tolerance of the strain, and the cell concentration decreases.

[0053] The culture media used in the above examples are as follows:

[0054] Inorganic salt medium: KH 2 PO 4 0.4 g, K 2 HPO 4 0.4 g, NH 4 Cl 1 g, MgCl 2 0.1 g, Na 2 SO 4 1.425 g, FeSO 4 ·7H 2 O 0.025 g, trace element solution 10 mL, made up to 1 L with distilled water, pH 7.0. The composition of the trace element solution is as follows: ZnSO 4 ·7H 2 O 1.1 g, MgSO 4 ·H 2 O 0.58 g, (NH 4 ) 6 Mo 7 O 24 ·4H 2 O 0.18 g, CoSO 4 ·7H 2 O 0.024 g, CuSO 4 ·5H 2 O 0.077 g, H 3 BO 3 0.029 g, NaNO 3 ·4H 2 O 0.074 g, distilled water 1 L.

[0055] Selective inorganic salt medium: Add 0.02 g of CdCl 2 .

[0056] Isolation medium: Oxadiazon is used as the sole carbon source in the selective inorganic salt medium, and different concentrations are added according to the experimental design requirements. The pH is 7.0 (20 g of agar is added to the solid medium).

[0057] Beef extract peptone medium: 3.0 g of beef extract, 5.0 g of NaCl, 10.0 g of peptone, 1000 mL of distilled water, 20 g of agar, pH 7.0.

[0058] The above media are all sterilized in an autoclave at 121 °C for 20 - 30 minutes.

[0059] Sequence list information:

[0060] DTD version: V1_3

[0061] File name: Gordonia rubripertincta 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 name: Guizhou University

[0068] Applicant name / language: zh

[0069] Applicant name / Latin name: guizhouUniversity Invention name: A Gordonia rubripertincta DLY03 and its application (zh) Total number of sequences: 1

[0070] Sequence:

[0071] Sequence 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] Residue:

[0079]

[0080]

[0081]

Claims

1. A Rhodococcus gordonii DLY03, characterized in that: It was deposited in the China Center for Type Culture Collection on April 25, 2022, with the deposit address at Wuhan University, Wuhan, China, and named Rhodococcus gordoniae DLY03 (Rhodococcus gordoniae DLY03), with the deposit number CCTCC NO: M2022479.

2. Use of the Rhodococcus gordonii DLY03 as claimed in claim 1 in the treatment of oxadiazon and cadmium pollution.

3. A liquid bacterial agent containing the Rhodococcus gordonii DLY03 according to claim 1.

4. A method for preparing a liquid bacterial agent containing the Rhodococcus gordonii DLY03 according to claim 3, characterized in that: The liquid bacterial agent was prepared by using Rhodococcus gordonii DLY03 as a living component.

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