Atrazine degrading bacterium and application thereof

Abstract

The invention provides an atrazine degrading bacterium, the atrazine degrading bacterium is arthrobacter ureafaciens H1, the arthrobacter ureafaciens H1 is preserved in China General Microbiological Culture Collection Center on August 19, 2024, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.31684. The invention further provides a preparation method of the atrazine degrading bacterium. Meanwhile, the invention provides an application of the atrazine degrading bacterium in remediation of soil or water body polluted by atrazine. The atrazine degrading bacterium is not repelled by indigenous bacteria when being applied to soil remediation as an engineering bacterium, can well adapt to the natural environment of farmland, and achieves a better degradation effect.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial degradation, and specifically relates to atrazine-degrading bacteria and application thereof. Background Art

[0002] Atrazine is widely used in corn, sugarcane and sorghum fields in more than 80 countries around the world, with annual consumption ranging from 7,000 to 9,000 tons. However, less than 30% of the applied herbicide can effectively penetrate the target weeds, and most of it will spread into the surrounding environment and eventually enter the soil. The stable chemical structure of atrazine enables it to persist in the soil for a long time, posing a threat to the health of agricultural ecosystems. It is worth noting that atrazine has been confirmed to be an endocrine disruptor and may be carcinogenic. Therefore, the development of advanced atrazine soil remediation technology is particularly important to effectively reduce its environmental and health risks, especially when it is not feasible to ban atrazine.

[0003] At present, the methods used for the remediation of atrazine-contaminated soil mainly include physical, chemical and biological methods. Among them, physical and chemical methods have a greater impact on the soil and are costly, and are not suitable for the remediation of non-point source pollution in farmland soil. The biological method is a greener and more environmentally friendly technology that can degrade herbicides or convert them into non-toxic metabolites. Bioremediation mainly includes phytoremediation, microbial remediation and plant-microorganism combined remediation, which are currently the in-situ remediation methods that have attracted much attention in the current farmland soil pollution control. Microbial remediation is the most commonly used bioremediation method.

[0004] However, many degradation strains are currently screened from soil or activated sludge in pesticide factories. They cannot adapt well to soil environments with low pollution concentrations in actual farmland remediation applications. In addition, these strains have high requirements for environmental temperature and pH conditions, especially in an environment of 25-35°C and pH=6-8. They cannot achieve good degradation effects and are not suitable for large-scale promotion. Therefore, it is very necessary to develop degradation strains that are adapted to farmland environments. Summary of the invention

[0005] In view of the defects of the prior art, the present invention provides an atrazine-degrading bacterium and an application thereof. The atrazine-degrading bacterium is applied as an engineered bacterium to soil remediation without being rejected by indigenous bacteria, can adapt well to the natural environment of farmland, and achieves a better degradation effect.

[0006] An atrazine-degrading bacterium, wherein the atrazine-degrading bacterium is Arthrobacter ureagenes ( Paenarthrobacter ureafaciens )H1, the Arthrobacter ureagenes ( Paenarthrobacter ureafaciens )H1 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 19, 2024, with the deposit number CGMCC No. 31684.

[0007] Preferably, the base sequence of 16S rDNA of the atrazine-degrading bacteria is as shown in SEQ ID NO.1.

[0008] The application of the atrazine-degrading bacteria is specifically: using the atrazine-degrading bacteria to repair soil or water contaminated by atrazine.

[0009] Preferably, the application is to use the atrazine-degrading bacteria for the remediation of soil contaminated by atrazine.

[0010] The atrazine-degrading bacteria are used for the restoration of soil contaminated by atrazine, specifically as follows: the atrazine-degrading bacteria are inoculated into LB culture medium, placed in a constant temperature shaker, and cultured at 30-35° C. and 160-180 r / min. After the bacterial solution becomes turbid, it is centrifuged, resuspended with sterile water, and then the bacterial solution is sprayed on the soil to be restored and stirred evenly.

[0011] Preferably, after the bacterial solution is sprayed on the soil to be repaired, the concentration of the atrazine-degrading bacteria reaches 1×10 8 -1×10 9 UFC / g dry soil.

[0012] Preferably, the moisture content of the soil to be repaired is between 20-25%.

[0013] Preferably, the formula of the LB medium is as follows: 5 g / L beef extract, 10 g / L peptone, and 5 g / L NaCl.

[0014] Arthrobacter ureagenes of the present invention ( Paenarthrobacter ureafaciens ) The collection information of H1 is as follows: Deposit date: August 19, 2024; Depository: China General Microbiology Center (CGMCC); Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit number: CGMCC No. 31684; Classification name: Arthrobacter ureagenes ( Paenarthrobacter ureafaciens ).

[0015] Advantages of the present invention: (1) The strain of the present invention has good degradation properties for atrazine and can be used to repair soil and water contaminated by atrazine; (2) The strain of the present invention is screened from farmland soil. When applied as an engineered bacterium to soil remediation, it will not be rejected by native bacteria and can reproduce and grow rapidly to achieve the purpose of removing atrazine from the soil; (3) The strain of the present invention can rapidly degrade atrazine at a temperature of 25-35°C and a pH of 6-8, and can adapt well to the natural conditions of farmland; (4) The strain of the present invention can completely degrade 50 mg / L of atrazine into cyanuric acid within 16 hours, and has great potential for engineering applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Arthrobacter ureagenes ( Paenarthrobacter ureafaciens ) H1 Phylogenetic tree based on 16S rDNA sequence and NCBI database; Figure 2 Arthrobacter ureagenes ( Paenarthrobacter ureafaciens )The curve of H1 degradation of atrazine and cyanuric acid content; Figure 3 Arthrobacter ureagenes ( Paenarthrobacter ureafaciens )H1 atrazine degradation diagram at different temperatures; Figure 4 Arthrobacter ureagenes ( Paenarthrobacter ureafaciens ) H1 atrazine degradation diagram at different pH; Figure 5 Arthrobacter ureagenes ( Paenarthrobacter ureafaciens ) The remediation results of H1 on soils contaminated with different atrazines. DETAILED DESCRIPTION

[0017] The inoculation amount % described in the present invention refers to the volume percentage.

[0018] Example 1 Acquisition of strains 1. Soil collection The soil samples were collected from the farmland where atrazine was applied for a long time in Fuxin Mongolian Autonomous County, Fuxin City, in the Black Soil Protection Project Experimental Demonstration Area of ​​the Chinese Academy of Sciences. The soil was brown soil.

[0019] 2. Culture medium Basic culture medium (MSM): K2HPO4 1.79 g, KH2PO4 0.45 g, MgSO4·7H2O 0.2 g, NaCl 0.4 g, glucose 3 g, FeSO4·7H2O 0.01 g, 3% CaCl2 1 mL, trace element solution 1 mL, dilute to 1 L with distilled water; The trace element liquid components: CoCl2·6H2O 0.1g, MnCl·4H2O 0.425g, ZnCl2 0.05g, NiCi2·6H2O 0.01g, CuSO4·5H2O 0.015g, Na2MoO4·2H2O 0.01g, H3BO4 0.1g, dilute to 1L with distilled water; LB medium: 5 g beef extract, 10 g peptone, 5 g NaCl, dilute to 1 L with distilled water.

[0020] 3. Strain enrichment screening (1) Atrazine was added to 100 mL of basal medium at a concentration of 100 mg / L, and then 10 g of soil sample was added. Atrazine was used as the sole nitrogen source and the medium was placed on a constant temperature shaker at 30 °C and 160 r / min for acclimatization. (2) Every 7 days, 5 mL (5% inoculation volume) of culture medium was inoculated into 100 mL of new basal medium. At the same time, the atrazine content in the new basal medium was increased by 100 mg / L compared with the previous time. The enrichment process ended after 35 consecutive days (i.e., with atrazine as the only nitrogen source, starting from the first 7 days, the atrazine content was 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L, respectively); (3) diluting the culture medium obtained in step (2) and spreading it on LB culture medium containing 100 mg / L atrazine, and then placing it in a constant temperature incubator at 30°C; (4) After 2-3 days of culture, screen the single colonies on the plate for the next step of plate streaking. Repeat streaking 2-3 times until all single bacteria are selected; (5) Store the obtained plate in a refrigerator at 4°C.

[0021] Example 2 Identification of strain 16S rDNA 1. Use the Sangon DNA extraction kit to extract DNA; 2. Use the extracted genomic DNA of the strain as a template and then perform PCR amplification: primers are 27F (5-AGAGTTTGATCCTGGCTCAG-3) and 1492R (5-GGTTACCTTGTTACGACTT-3); The PCR reaction system was: genomic DNA (20 ng / μL) 1 μL, 10× PCR Buffer (Mg 2+ plus) 5 μL, Taq polymerase (5 U / μL) 1 μL, dNTP (1×10 4 uM) 1 μL, 27F primer (10 uM) 1.5 μL, 1492R primer (10uM) 1.5 μL, ddH2O 39 μL, the total volume is 50 μL; the 10×PCR Buffer (Mg 2+ plus) purchased from Takara brand; The PCR amplification conditions were pre-denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 58°C for 30 s, extension at 72°C for 1.5 min, and extension at 72°C for 7 min; 3. The PCR product was recovered using the AxyPrep DNA gel recovery kit and sent to Nanjing Paisono Biotechnology Co., Ltd. for sequencing. The strain was identified by 16SrDNA and the results were compared with the NCBI Blast program for homology. Paenarthrobacter ureafaciens sp. strain The homology of the strain is 100%. The base sequence of the 16SrDNA of the strain is shown in SEQ ID No. 1. The strain Arthrobacter ureagenes ( Paenarthrobacter ureafaciens )H1 Phylogenetic tree based on 16S rDNA sequence and NCBI database is shown in Figure 1 shown.

[0022] Example 3 Determination of degradation curve and cyanuric acid content curve Arthrobacter ureagenes Paenarthrobacter ureafaciens ) H1 was inoculated into LB medium and cultured in a constant temperature shaker at 30°C and 160 r / min. When the bacterial solution became turbid, it was centrifuged at 8000 rpm / min for 5 min and then resuspended with sterile water. The OD600 of the bacterial solution was adjusted to 1.0; According to the inoculation amount of 7%, the bacterial solution with OD600=1.0 was inoculated into the basic culture medium. At this time, atrazine was added at a concentration of 50 mg / L (i.e., the substrate concentration was 50 mg / L). Under the conditions of pH 6.9 and temperature 36.7°C, the culture medium was placed in a constant temperature shaker at a speed of 160 r / min for cultivation. Samples were taken every 2 hours, and the contents of atrazine and cyanuric acid in the bacterial solution were determined by high performance liquid chromatography (HPLC). The ability of the strain to degrade atrazine was determined. Three parallel samples were set up in the experiment. The results are as follows: Figure 2 ; Among them, the detection conditions of atrazine are as follows: the HPLC conditions are as follows: the chromatographic column is Ultimate XB-C18 column (150 mm×4.6 mm, 5 μm), the mobile phase is methanol-water (80:20 v / v), the flow rate is 1.0 mL / min, the column temperature is 25°C, the wavelength is 254 nm, and the injection volume is 5 μL; Detection conditions of cyanuric acid: The chromatographic column was Ultimate XB-C18 column (150 mm×4.6 mm, 5 μm), the mobile phase was a mixture of 0.002 mol / L potassium dihydrogen phosphate and 0.005 mol / L potassium hydrogen phosphate-methanol (95:5 V / V), the flow rate was 1.0 mL / min, the column temperature was 35°C, the wavelength was 213 nm, and the injection volume was 10 μL; Depend on Figure 2 It can be seen that the strain Arthrobacter ureagenes ( Paenarthrobacter ureafaciens ) H1 can completely degrade 50 mg / L atrazine within 16 h. As the concentration of atrazine decreases, the concentration of cyanuric acid gradually increases, which indicates that atrazine is gradually converted into cyanuric acid.

[0023] Example 4 Effect of different temperatures on degradation According to the inoculation amount of 7%, the bacterial solution with OD600=1.0 was inoculated into the basic culture medium. At this time, atrazine was added at a concentration of 100 mg / L (i.e., the substrate concentration was 100 mg / L). The culture was carried out at a constant temperature shaker at pH 6.9 and different temperatures (20, 25, 30, 35, 40°C) at a speed of 160 r / min for 24 hours. The degradation rate of atrazine at different temperatures was investigated. The detection method was the same as in Example 3. The results are shown in Table 3. Figure 3 ; Depend on Figure 3 It can be seen that the strain Arthrobacter ureagenes ( Paenarthrobacter ureafaciens ) H1 has a good degradation effect on atrazine within the temperature range of 20-40℃, which can reach more than 70%; especially the degradation effect is best within the temperature range of 25-35℃, which can reach more than 80%.

[0024] Example 5 Effect of different pH on degradation According to the inoculation amount of 7%, the bacterial solution with OD600=1.0 was inoculated into the basic culture medium. At this time, atrazine was added at a concentration of 100 mg / L (i.e., the substrate concentration was 100 mg / L). Different pH conditions (5, 6, 7, 8, 9) were set. The culture was placed in a constant temperature shaker at 30°C and a speed of 160 r / min for 24 hours. The degradation rate of atrazine at different pH was investigated. The detection method was the same as in Example 3. The results are shown in FIG. Figure 4 ; Depend on Figure 4 It can be seen that the strain Arthrobacter ureagenes ( Paenarthrobacter ureafaciens ) In the pH range of 6-8, the degradation rate of atrazine by H1 was above 80%.

[0025] Example 6 Remediation of soil contaminated by atrazine by bacterial strains Arthrobacter ureagenes Paenarthrobacter ureafaciens) H1 was inoculated into LB medium and placed in a constant temperature shaker at 30°C and 160 r / min. When the bacterial solution became turbid, it was centrifuged at 8000 rpm / min for 5 min and then resuspended in sterile water. The bacterial solution was evenly sprayed on the atrazine-contaminated soil and stirred evenly to make the bacterial concentration reach 1×10 8 UFC / g dry soil. In the experiment, each pot contained 500 g of soil. In order to approach the natural conditions of farmland, the light duration was set to light: dark = 16:8, the temperature was 25-30°C, and the soil moisture content was 24% of the dry soil weight. Samples were collected on the 1st, 3rd, 5th and 7th days to detect the residual amount of atrazine in the soil; all samples were repeated three times, and a blank culture medium was set as a control; The atrazine contents in the two atrazine-contaminated soils were 5 mg / kg and 20 mg / kg, respectively, and the pH was 6.18. The degradation effects were shown in Table 1. Figure 5 Figure (a) and Figure (b) in Figure 5 It can be seen that compared with CK, in the 5mg / kg contaminated soil sample, the strain Arthrobacter ureagenes ( Paenarthrobacter ureafaciens ) H1 promoted the removal of atrazine by 92.30%, 83.72%, 67.90% and 40.93% in the soil at 1, 3, 5 and 7 days, respectively; in the soil contaminated with 20 mg / kg atrazine, the strain Arthrobacter ureagenes ( Paenarthrobacter ureafaciens )H1 promoted the removal of 64.98%, 67.81%, 67.51% and 28.38% of atrazine in the soil, respectively, indicating that Arthrobacter ureagenes ( Paenarthrobacter ureafaciens )H1 can significantly promote the removal of atrazine in soil.

Claims

1. An atrazine-degrading bacterium, characterized in that: The atrazine-degrading bacteria is Arthrobacter ureagenes ( Paenarthrobacter ureafaciens )H1, the ureagenic Arthrobacter ( Paenarthrobacter ureafaciens )H1 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 19, 2024, with the deposit number CGMCC No. 31684.

2. The atrazine-degrading bacteria according to claim 1, characterized in that: The base sequence of 16S rDNA of the atrazine-degrading bacteria is shown in SEQ ID NO.

1.

3. The use of atrazine-degrading bacteria according to claim 1 or 2, characterized in that: The atrazine-degrading bacteria are used for repairing soil or water bodies contaminated by atrazine.

4. The use according to claim 3, characterized in that: The atrazine-degrading bacteria are used for remediation of soil contaminated by atrazine.

5. The use according to claim 4, characterized in that: The atrazine-degrading bacteria are inoculated into LB culture medium, placed in a constant temperature shaker, and cultured at 30-35°C and 160-180 r / min. After the bacterial solution becomes turbid, it is centrifuged and resuspended with sterile water. Then, the bacterial solution is sprayed on the soil to be repaired and stirred evenly.

6. The use according to claim 5, characterized in that: After the bacterial solution is sprayed on the soil to be repaired, the concentration of the atrazine-degrading bacteria reaches 1×10 8 -1×10 9 UFC / g dry soil.

7. The use according to claim 5, characterized in that: The moisture content of the soil to be repaired is between 20-25%.

8. The use according to claim 5, characterized in that: The formula of the LB medium is as follows: 5 g / L beef extract, 10 g / L peptone, and 5 g / L NaCl.

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