Arthrobacter AD8 capable of efficiently degrading atrazine and application of arthrobacter AD8

Abstract

The invention discloses arthrobacter AD8 capable of efficiently degrading atrazine and application of the arthrobacter AD8, the strain can efficiently degrade atrazine and is effectively used for repairing pollution caused by atrazine to soil and water, the classification name is Paenarthrobacter sp., and the arthrobacter AD8 is preserved in China General Microbiological Culture Collection Center on March 18, 2025, and has the preservation number of CGMCC NO. The address is Institute of Microbiology, Chinese Academy of Sciences, No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.33865. The triazine herbicide has the advantages of high efficiency in degradation of atrazine in soil and water and remediation of atrazine-polluted water and soil, good effect and high degradation efficiency, is a great innovation in remediation of triazine herbicide-polluted water and soil, and has significant economic and social benefits.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to Arthrobacter paraffineus AD8 with high efficiency in degrading atrazine and its application. Background Art

[0002] Atrazine, trade name Aatrex, is a triazine herbicide widely used worldwide and is widely used for controlling broad-leaved weeds in corn, sugarcane, and sorghum fields. Due to its relatively long half-life in the soil, it is likely to cause phytotoxicity to some sensitive succeeding crops. Research shows that atrazine is an endocrine disruptor. It can interfere with the regulatory function of hormones, cause reproductive defects in humans and amphibians, induce tumors and cancers, and its ecological toxicological risks cannot be ignored. This herbicide is stable in nature and is very widely distributed in the environment, causing serious pollution and damage to the ecological environment, threatening the safety of food and drinking water, and the environmental pollution problems it causes have attracted worldwide attention.

[0003] Using the method of biodegradation to control the environmental pollution caused by triazine herbicides is an effective means. Microorganisms that can degrade pollutants include bacteria, fungi, actinomycetes, etc. Among them, bacteria have more species and stronger adaptability and play an important role. Due to differences in climate, soil quality, and the characteristics of the degrading bacteria themselves in different regions, there are differences between the actual application effects of some isolated degrading bacteria and the expectations. Therefore, it is very necessary to isolate more degrading strains adapted to different environments. Summary of the Invention

[0004] In view of the above situation, in order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide Arthrobacter paraffineus AD8 with high efficiency in degrading atrazine and its application. This strain can efficiently degrade atrazine and is effectively used for efficiently repairing the pollution of soil and water caused by atrazine.

[0005] The technical solution solved by the present invention is an Arthrobacter paraffineus AD8 with high efficiency in degrading atrazine, classified and named as Paenarthrobacter sp., deposited in the China General Microbiological Culture Collection Center on March 18, 2025, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 33865.

[0006] The application of the described Arthrobacter paraffineus AD8 with high efficiency in degrading atrazine in repairing atrazine-polluted water bodies and soil.

[0007] The present invention relates to a Paenarthrobacter sp. AD8 screened from atrazine-contaminated soil, which has the ability to efficiently degrade atrazine in soil and water bodies, repair atrazine-contaminated water bodies and soil, with good effects and high degradation efficiency. It is a major innovation in the remediation of water bodies and soil contaminated by triazine herbicides, and has significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 It is a Gram staining diagram of Paenarthrobacter sp. AD8 of the present invention;

[0009] Figure 2 It is a phylogenetic tree diagram of Paenarthrobacter sp. AD8 of the present invention;

[0010] Figure 3 It is a diagram showing the effect of externally added carbon and nitrogen sources on the degradation of atrazine by Paenarthrobacter sp. AD8 of the present invention;

[0011] Figure 4 It is a diagram showing the effect of sucrose addition amount on the degradation of atrazine by Paenarthrobacter sp. AD8 of the present invention;

[0012] Figure 5 It is a diagram showing the effect of pH on the degradation of atrazine by Paenarthrobacter sp. AD8 of the present invention;

[0013] Figure 6 It is a diagram showing the effect of temperature on the degradation of atrazine by Paenarthrobacter sp. AD8 of the present invention;

[0014] Figure 7 It is a diagram showing the degradation effect of Paenarthrobacter sp. AD8 of the present invention under different initial atrazine levels;

[0015] Figure 8 It is a diagram showing the field degradation effect of Paenarthrobacter sp. AD8 of the present invention on atrazine. DETAILED DESCRIPTION OF THE INVENTION

[0016] The following will describe in detail the specific implementation manners of the present invention in combination with examples and specific situations.

[0017] The specific implementation of the present invention is given by the following examples.

[0018] Obtaining of Paenarthrobacter sp. AD8 with high efficiency in degrading atrazine of the present invention:

[0019] First, prepare an inorganic salt medium: K2HPO4 1.5 g / L, KH2PO4 0.5 g / L, MgSO4 0.5 g / L, NaCl 1 g / L. During use, 1 g / L ammonium nitrate can be added as the sole nitrogen source or 3 g / L glucose can be added as the sole carbon source.

[0020] LB medium: Sodium chloride 10 g / L, yeast powder 5 g / L, tryptone 10 g / L, pH 7.0 - 7.2.

[0021] Weigh 5 g of soil samples from a cornfield in Zhumadian area, Henan Province, where atrazine herbicide has been applied for many years, and place them in a carbon source-containing inorganic salt medium liquid containing 100 mg / L of atrazine. Incubate at 180 r / min and 30 °C for 7 d. Then take out 5 mL and add it to the inorganic salt culture solution, with the atrazine concentration being 200 mg / L; and so on, gradually increase the atrazine concentration to 500 mg / L. After gradient dilution of the obtained bacterial solution, take 200 μL and spread it on a solid plate. Place the plate in an incubator at 30 °C and culture for 48 h. Select single colonies for isolation and purification to obtain purified strains.

[0022] Inoculate the purified strains into a carbon source-containing inorganic salt culture solution containing 100 mg / L of atrazine, and culture at 30 °C and 180 r / min for 7 d. Use samples without inoculation as a control. Use high performance liquid chromatography to measure the residual concentration of atrazine in each sample, and calculate the degradation rate to screen out highly efficient atrazine-degrading bacteria. The liquid chromatography detection conditions for atrazine are as follows: Chromatographic column: 4.6 mm * 150 mm, Eclipse XDB-C18, mobile phase: methanol∶water = 55∶45 (V∶V), column temperature 30 °C, flow rate 1.0 mL / min, detection wavelength 222 nm.

[0023] Degradation rate (%) = (control atrazine concentration - treated atrazine concentration) / control atrazine concentration.

[0024] After enrichment culture, 8 bacterial strains that can grow using atrazine as the sole nitrogen source were screened out. Use high performance liquid chromatography to measure the degradation rate of the 8 strains of bacteria. The results are shown in Table 1. It can be seen from Table 1 that among the 8 strains of bacteria, AD8 has the best degradation effect, and the degradation rate reaches 43.09%.

[0025] Table 1 Degradation effects of different strains on atrazine

[0026]

[0027]

[0028] 1. Identification of atrazine-degrading strain AD8

[0029] Refer to the "Handbook for the Systematic Identification of Common Bacteria" to detect the morphological characteristics and physiological and biochemical characteristics of strain AD8: The morphological characteristics of strain AD8 are rod-shaped, and the Gram stain is positive. See Figure 1; The physiological and biochemical characteristics of strain AD8 are as follows: indole test positive, methyl red test negative, VP test positive, citrate utilization positive, starch hydrolysis detection positive, catalase test positive. The genomic DNA of strain AD8 was extracted using a bacterial genomic DNA extraction kit (Solarbio). The 16S rDNA of the strain was sequenced using the bacterial universal primers 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R: 5′-TACCTTGTTACGACTT-3′, and the sequence is SEQ ID No.1; homologous sequence alignment was performed in the GenBank database of NCBI, and multiple sequences with high similarity were found and a phylogenetic tree was constructed, as shown in Figure 2 ;

[0030] Combined with the morphological characteristics, physiological and biochemical characteristics, and molecular biology identification results of strain AD8, strain AD8 was identified as Paenarthrobacter AD8, classified and named as Paenarthrobacter sp., and deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on March 18, 2025. Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 33865;

[0031] 2. Experiment on the effect of adding external carbon and nitrogen sources on the degradation of atrazine by AD8

[0032] Based on the inorganic salt medium containing 100 mg / L atrazine, in one group, glucose, sucrose, starch, or sodium citrate with a final concentration of 3 g / L was added as an external carbon source respectively, and in another group, ammonium nitrate, sodium nitrate, or potassium nitrate with a concentration of 1 g / L was added as an external nitrogen source respectively; in another group, sucrose and sodium nitrate were added simultaneously; the AD8 bacterial suspension was inoculated into the above culture media respectively, and cultured at 30 °C and 180 r / min for 7 d. Each treatment was repeated 3 times, and the non-inoculated sample was used as a control. The residual concentration of atrazine in each sample was measured using high performance liquid chromatography, and the degradation rate was calculated;

[0033] The results are shown in Figure 3 , and it is shown by Figure 3 that adding different external nitrogen sources will all inhibit the degradation effect of AD8 on atrazine, indicating that this strain can use atrazine as the sole nitrogen source for growth and does not require additional nitrogen source addition; the effects of adding external carbon sources on the degradation of atrazine by AD8 vary greatly. Glucose and sucrose show a promoting effect on the degradation effect of AD8, and among them, the promoting effect of sucrose is the best. Subsequently, the effect of different sucrose addition amounts on the degradation effect of AD8 was tested, and the results are shown in Figure 4 as shown, and it can be seen from Figure 4 that when the concentration of sucrose in the culture medium is 5 g / L, the degradation rate of atrazine by AD8 is the highest.

[0034] 3. Experiment on the Influence of Environmental Factors on the Degradation Effect of Atrazine by Strain AD8

[0035] To experiment on the influence of different environmental factors such as pH and temperature on the degradation of atrazine by strain AD8, the present invention respectively set treatment groups with different initial medium pH values (4, 5, 6, 7, 8, 9, 10, 11) and different culture temperatures (4, 10, 20, 30, 35, 40, 45 °C). AD8 was inoculated into an inorganic salt medium containing 100 mg / L atrazine as the nitrogen source and 5 g / L sucrose as the carbon source. After all samples were treated according to the above different pH values and different temperatures, they were cultured with shaking. At the same time, samples without inoculating the strain were set as the control. Each treatment group had three replicates. After 7 days, the concentration of residual atrazine in the medium was detected by high performance liquid chromatography, and the degradation rate was calculated. On the basis of the optimal pH and temperature, the influence of different initial atrazine concentrations (10, 50, 100, 200 mg / L) on the degradation effect of atrazine by strain AD8 was studied.

[0036] The results are shown in Figure 5 、 Figure 6 , Figure 5 、 Figure 6 It shows that when the pH is 7 and the culture temperature is 35 °C, the degradation effect of AD8 on atrazine is the best, and the degradation rate of 100 mg / L atrazine can reach 100%; from Figure 7 it can be seen that under the condition of the same culture time, with the increase of atrazine concentration, the degradation effect of strain AD8 gradually decreases. When the atrazine concentration is less than or equal to 50 mg / L, strain AD8 can completely degrade it on the 3rd day of culture. When the atrazine concentration is 200 mg / L, strain AD8 cannot completely degrade it within 7 days, and the degradation rate on the 7th day is only 64.93%; in summary, it can be known that strain AD8 has the best degradation ability for atrazine with a concentration of less than 50 mg / L.

[0037] 4. Experiment on the Field Degradation Effect of Atrazine by Strain AD8

[0038] A corn field where atrazine herbicide has been applied continuously for several years (with extremely high atrazine residue concentration) was selected to conduct the field degradation effect of atrazine by strain AD8. The experiment had a total of four treatments: Treatment 1, control group (without inoculating AD8 bacterial agent); Treatment 2, inoculating 10 mL / m 2 group; Treatment 3, inoculating 20 mL / m 2 group; Treatment 4, inoculating 30 mL / m 2 group; Each group had 3 replicates, and the plots were randomly arranged in blocks. The area of each plot was 30 m 2, on the 7th day, 14th day, and 21st day after applying the microbial agent, the residual situation of atrazine in the field soil of each treatment was analyzed by high performance liquid chromatography, and the results are shown in Figure 8 ;

[0039] It can be seen from Figure 8 that in the control group without inoculating the microbial agent, the change in the atrazine content in the soil was very small, and there was no significant change in the atrazine concentration during the detection period from the 7th day to the 21st day. While in the treatment group inoculated with the AD8 microbial agent, there was an obvious downward trend in the residual concentration of atrazine during the detection period. Especially for treatment 4 inoculated with a high dose of the AD8 microbial agent, the residual concentration of atrazine was only 2.85 mg / kg at the 21st day, showing a high degradation effect.

[0040] The experiment shows that the Arthrobacter sp. AD8 of the present invention grows with atrazine as the sole nitrogen source, and the addition of sucrose has a significant promoting effect on the degradation effect of AD8. When the concentration of sucrose in the culture solution is 5 g / L, the promoting effect on the degradation rate of atrazine by AD8 is the highest;

[0041] When the pH is 7 and the culture temperature is 35 °C, the degradation effect of AD8 on atrazine is the best, and the degradation rate of 100 mg / L atrazine can reach 100%; the strain AD8 has the best degradation ability for atrazine with a concentration of less than 50 mg / L and can completely degrade it within 3 days.

[0042] To sum up, under the conditions of pH value of 7 and 35 °C for 3 - 7 days of cultivation, the degradation rate of atrazine with a concentration of 50 - 100 mg / L reaches 100%, truly realizing the high-efficiency degradation of atrazine. The microbial agent of the present invention has rich raw materials, a simple preparation method, and low production costs. It is an Arthrobacter sp. AD8 screened from atrazine-contaminated soil, classified and named as Paenarthrobacter sp., which has the ability to efficiently degrade atrazine in soil and water bodies, repair atrazine-contaminated water bodies and soil, with good effects and high degradation efficiency, and realizes the application in repairing atrazine-contaminated water bodies and soil. It is a major innovation in the repair of triazine herbicides, especially atrazine-contaminated water bodies and soil, and has significant economic and social benefits.

Claims

1. An Arthrobacter sp. AD8 with high efficiency in degrading atrazine, classified and named as Paenarthrobacter sp., was deposited in the China General Microbiological Culture Collection Center on March 18, 2025. Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 33865.

2. Use of Arthrobacter AD8 capable of efficiently degrading atrazine according to claim 1 in repairing atrazine-polluted water bodies and soil.

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