Strain A-6 and application thereof

Abstract

The invention discloses a strain A-6, which is classified and named as Paenarthrobacter sp., is preserved in China General Microbiological Culture Collection Center (CGMCC) on October 9, 2023, and has the preservation number of CGMCC NO.28594. The invention also discloses a preparation method of the strain A-6. The strain A-6 is named as Paenarthrobacter sp., and the strain A-6 is preserved in China General Microbiological Culture Collection Center (CGMCC). The invention also discloses an application of the bacillus subtilis in degradation of atrazine. The invention also discloses a microbial inoculum prepared from the strain and application of the microbial inoculum in degradation of atrazine. The screened strain A-6 is applied to the field of bioremediation of residual atrazine in the soil environment, the strain not only has efficient atrazine degradation capacity, but also has high environmental adaptability, and the prepared solid microbial inoculum can be stored for a long time and has a good application prospect in the field of bioremediation of residual atrazine in the soil environment. An efficient degradation strain resource is provided for biological reduction, resistance and control of residual atrazine in a soil environment, and important theoretical and application values are achieved.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology, specifically to a strain A-6 and its applications. Background Technology

[0002] Atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) is a synthetic triazine herbicide. Since its introduction into production in 1959, it has been rapidly and widely used globally due to its low production cost and effective weed control, primarily as a specialized chemical herbicide for crops such as corn, sorghum, and sugarcane. It is reported that approximately 80,000 tons of atrazine are consumed globally each year. However, due to its long half-life, high migration rate, and good solubility, sprayed atrazine easily migrates into groundwater and deep soil, posing a threat to the ecological environment and human drinking water sources. Furthermore, high residue levels and poor degradation rates of atrazine can accumulate in soil and crops. In northern my country, where atrazine is widely used, there have been numerous incidents of industrial wastewater exceeding atrazine concentration standards being illegally discharged into the environment, resulting in crop poisoning. Atrazine residues not only directly affect crop quality but also threaten human health through the food chain.

[0003] Currently, atrazine degradation mainly includes chemical, physical, and biological methods. Chemical methods include photolysis and oxidation, while physical methods include ion exchange and activated carbon fiber adsorption. However, physical and chemical methods often suffer from drawbacks such as high cost, low efficiency, and susceptibility to secondary pollution. In contrast, biological methods, which eliminate pollutants through microbial metabolism, offer advantages such as high efficiency, economy, safety, and no secondary pollution. Therefore, screening for efficient, stable, and environmentally adaptable atrazine-degrading strains will provide microbial strain resources and technical support for the remediation of atrazine pollution in the environment. Summary of the Invention

[0004] The purpose of this invention is to provide a strain A-6 and its applications to address the shortcomings of existing technologies.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides a strain A-6, which is classified and named Arthroblastus (…). Paenarthrobacter sp . The sample was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 9, 2023, with accession number CGMCCNO.28594.

[0006] A second aspect of the present invention provides the application of the above-mentioned strain A-6 in the degradation of atrazine.

[0007] Furthermore, the degradation of atrazine refers to the degradation of atrazine in soil.

[0008] A third aspect of the present invention provides the use of the above-mentioned strain A-6 in the preparation of an agent for degrading atrazine.

[0009] Furthermore, the degradation of atrazine refers to the degradation of atrazine in soil.

[0010] A fourth aspect of the present invention provides a bacterial agent prepared from the above-mentioned strain A-6, the bacterial agent comprising a solid bacterial agent.

[0011] Furthermore, the solid microbial agent is prepared by the following steps: 1) Inoculate strain A-6 into a liquid culture medium for liquid fermentation, wherein the liquid culture medium includes LB liquid medium, and the liquid fermentation conditions are: temperature 28-30℃, rotation speed 160-180 rpm, and liquid fermentation to OD. 600 The concentration was 0.8-1.2, and the fermentation broth of strain A-6 was obtained; 2) Inoculate the fermentation broth of strain A-6 into a sterile carrier at an inoculation ratio of 8v / m%-12v / m%, mix thoroughly, and culture at 28-30℃ for 3-4 days to obtain the solid bacterial agent.

[0012] Furthermore, in step 2), the sterile carrier includes sterilized earthworm castings, pig manure, sheep manure, or chicken manure.

[0013] The fifth aspect of this invention provides the application of the above-mentioned bacterial agent in the degradation of atrazine.

[0014] Furthermore, the degradation of atrazine refers to the degradation of atrazine in soil.

[0015] The beneficial effects of this invention are: 1. This invention isolates and screens a strain A-6 from soil that can efficiently degrade atrazine. At 30°C, it completely degrades 50 mg / L of atrazine within 24 hours. Strain A-6 can also efficiently degrade atrazine at low temperatures (15°C), completely degrading 50 mg / L of atrazine within 80 hours at 15°C. This strain A-6 provides an important degrading strain resource for the biological reduction and control of atrazine residues in the soil environment, exhibiting excellent degradation ability and promising application prospects.

[0016] 2. The strain A-6 screened in this invention can degrade atrazine under conditions of 15℃-37℃ and pH 6.0-9.0, indicating that strain A-6 has strong environmental adaptability and good application potential.

[0017] 3. The strain A-6 screened in this invention was successfully used to prepare a solid bacterial agent, which is convenient for long-term storage and application, and is of great significance for the bio-enhanced remediation of atrazine in the soil environment.

[0018] In summary, the strain A-6 screened in this invention can be applied to the field of bioremediation of atrazine residues in the soil environment. This strain not only has a high efficiency in atrazine degradation but also has a strong environmental adaptability. The prepared solid bacterial agent can be stored for a long time, providing a highly efficient degradation strain resource for the bioreduction and control of atrazine residues in the soil environment, which has important theoretical and applied value. Attached Figure Description

[0019] Figure 1 Photograph (A) and scanning electron microscope image (B) of the colony morphology of strain A-6 on LB solid medium.

[0020] Figure 2 A phylogenetic tree for strain A-6 based on the 16S rRNA gene sequence.

[0021] Figure 3 The growth characteristics of strain A-6 under different temperature conditions.

[0022] Figure 4 The growth characteristics of strain A-6 under different pH conditions are shown.

[0023] Figure 5 The degradation and growth of atrazine by strain A-6.

[0024] Figure 6 A-6 solid bacterial agent prepared for different carriers.

[0025] Figure 7 The degradation effects of A-6 solid bacterial agents prepared for different carriers on atrazine in soil. Information on the preservation of biological materials

[0026] A-6, classified as Arthroblasts (… Paenarthrobacter sp . The sample is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is October 9, 2023, and the accession number is CGMCC NO.28594. Detailed Implementation

[0027] The present invention will be further explained below with reference to embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0028] The culture medium formulations involved in the following examples are as follows: Inorganic salt liquid medium (MSM, g / L): NaCl 1.0 g, NH4Cl 1.0 g, K2HPO4 1.5 g, KH2PO4 0.5 g, MgSO4•7H2O 0.2 g, diluted to 1 L with deionized water, pH 7.0. Inorganic salt solid medium is prepared by adding 1.6 g agar powder per L to the inorganic salt liquid medium.

[0029] LB liquid medium (g / L): 10.0 g peptone, 5.0 g yeast extract, 5.0 g NaCl, and deionized water to a final volume of 1 L, pH 7.0. LB solid medium is prepared by adding 1.6 g agar powder per L to the LB liquid medium.

[0030] The atrazine stock solution involved in the following examples: 1g of atrazine was dissolved in methanol and diluted to 100mL using a volumetric flask to prepare an atrazine stock solution with a concentration of 10000mg / L. The solution was then ultrasonicated at 40KHz for 30min in an ultrasonic cleaner and finally stored in a brown glass bottle at room temperature for later use.

[0031] Example 1: Isolation of strain A-6 Soil samples were collected from soils in Gongzhuling City, Jilin Province, where atrazine had been applied for a long period. The samples were divided into multiple portions, each containing 50g, for the isolation and screening of atrazine-degrading bacteria. Each 50g soil sample was placed in a 2L Erlenmeyer flask containing 1000mL of inorganic salt medium and placed in a shaker at 30℃ and 180rpm for 4 hours to activate the soil and obtain soil liquid. Prepare a sterilized 250mL Erlenmeyer flask in advance, and add 50μL of atrazine stock solution to the laminar flow hood. After the organic solvent (methanol) dissolving atrazine has evaporated to dryness in a fume hood, add 100mL of inorganic salt liquid culture medium. The atrazine concentration in this system is 5mg / L (subsequent inorganic salt liquid or solid culture medium containing atrazine is prepared using the same steps, adjusting the volume of atrazine stock solution added according to the atrazine concentration). This ensures that the system contains only atrazine as a carbon source, and this single carbon source is used to screen atrazine-degrading bacteria. Then, use a pipette to take 10mL of soil liquid and place it into the aforementioned 250mL Erlenmeyer flask containing 100mL of inorganic salt liquid culture medium containing 5mg / L atrazine, as the first-generation enrichment solution. Incubate the first-generation enrichment solution in a low-temperature shaker at 15℃ and 180rpm. Every 5 days thereafter, 5 mL of the enriched solution was added to 95 mL of inorganic salt liquid medium containing atrazine for subculturing, i.e., subculturing at a concentration of 5 v / v%. The concentration of atrazine in the inorganic salt liquid medium was gradually increased in a gradient of 5 mg / L (first-generation enriched solution), 10 mg / L (second-generation enriched solution), 20 mg / L (third-generation enriched solution), and 50 mg / L (fourth-generation enriched solution). 3 mL of the fourth-generation enriched solution after 5 days of culture was transferred to a 10 mL centrifuge tube, and an equal volume of dichloromethane was added for extraction. The mixture was vortexed for 10 min, centrifuged at 8000 rpm for 10 min, and the supernatant aqueous phase was removed. Anhydrous sodium sulfate was added to remove the remaining aqueous phase. Finally, the concentration of atrazine in the organic phase was detected using high-performance liquid chromatography (HPLC). The HPLC detection conditions were as follows: 250 mm × 4.6 mm C18 reverse-phase column; mobile phase: methanol / water (80:20, V:V); flow rate: 0.8 mL / min; column temperature: 30 °C; injection volume: 20 μL; detection wavelength: 226 nm. The same method was used for the HPLC detection of atrazine concentration.

[0032] The fourth-generation enrichment solution with atrazine degradation effect was serially diluted, with a dilution factor of 10. -5 10 -6 10 -7The fourth-generation enrichment solution was spread onto inorganic salt solid medium plates containing 300 mg / L atrazine and incubated at 15°C for 5 days. After 5 days, single colonies with a clear clear zone were picked from the plates and transferred to 5 mL LB liquid medium tubes for further culture: incubated at 30°C and 180 rpm for 3 days. After further culture, 2 mL of the bacterial culture was transferred to a centrifuge tube. The centrifuge tube was centrifuged at 6000 rpm for 5 min, the liquid was then aspirated, and the bacteria at the bottom of the centrifuge tube were mixed by pipetting with 10 mL of inorganic salt liquid medium. The tube was then centrifuged again at 6000 rpm for 5 min. This process was repeated three times to completely wash away the LB liquid medium, resulting in the bacterial culture in inorganic salt liquid medium. Take 5 mL of bacterial culture in inorganic salt liquid culture medium and inoculate it into 95 mL of inorganic salt liquid culture medium containing 50 mg / L atrazine. Incubate at 15 °C and 180 rpm with shaking. Take samples at regular intervals and use HPLC to detect the concentration of atrazine in the samples.

[0033] The atrazine-degrading bacterial suspension in inorganic salt liquid medium was serially diluted again and plated onto inorganic salt solid medium plates containing 300 mg / L atrazine. Purification was carried out at 15°C. Single colonies with a clear clear zone were picked and cultured again in LB liquid medium tubes (under the same conditions). Then, 1 mL of the bacterial suspension was transferred to a 2 mL preservation tube, and an equal volume of 40 v / v% glycerol was added and mixed thoroughly (i.e., glycerol tube preservation). The tubes were then frozen at -80°C for later use. Through enrichment culture and dilution plating isolation, an atrazine-degrading strain was successfully screened and named A-6.

[0034] Example 2 Identification and biological characteristics of strain A-6 Strain A-6 was cultured on LB solid medium at 30℃ for 3-4 days. On LB solid medium plates, strain A-6 appeared as round, white, opaque, and moist specimens. Figure 1 A). The main physiological and biochemical characteristics of strain A-6 are that it is a Gram-positive bacterium. A-6 cells are approximately 4.0 μm long and 1.0 μm wide, rod-shaped, with capsules, and peritrichous flagella (…). Figure 1 B). Using fresh bacterial culture of strain A-6 as a template, PCR amplification was performed using universal primers 27F (as shown in SEQ ID NO.1) and 1492R (as shown in SEQ ID NO.2) for the 16S rRNA gene sequence. The PCR product was collected by gel extraction, and the 16S rRNA gene was then combined with a vector, which was then transformed into competent cells. E. coliIn DH5α, clones were selected for testing. The obtained 16S rRNA gene sequences (as shown in SEQ ID NO.3) were uploaded to NCBI for BLAST alignment. Sequences with high similarity and representative characteristics were selected, their FASTA format files were downloaded, and the files were imported into MAGA 5.0 for sequence alignment. A phylogenetic tree was then constructed. Figure 2 The results showed that strain A-6 and strain Paenarthrobacter nitroguajacolicus Q5 showed the highest similarity, reaching 99.93%. Based on the colony morphology, physiological and biochemical characteristics, and 16S rRNA gene phylogenetic tree of strain A-6, it was identified as Arthroblastus (…). Paenarthrobacter sp . It has been deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.28594.

[0035] Example 3: Growth and Degradation Test of Strain A-6 3.1 Preparation of fermentation broth for strain A-6 After thawing strain A-6, preserved in glycerol tubes at -80℃, on ice, the strain was streaked in three zones on an LB agar plate using an inoculation loop. The LB agar plates were then incubated at 30℃ for 3 days. Single colonies were then picked and transferred to LB liquid medium and incubated at 30℃ and 180 rpm until OD500 reached. 600 =1.0, which is the fermentation broth of strain A-6.

[0036] 3.2 Growth of strain A-6 under different temperature conditions Centrifuge the fermentation broth of strain A-6 at 6000 rpm for 5 min, discard the supernatant, add an equal volume of inorganic salt liquid culture medium to the supernatant, mix well by pipetting, and centrifuge again at 6000 rpm for 5 min; repeat this process three times, wash away the residual LB liquid culture medium to obtain the fermentation broth of strain A-6 in inorganic salt liquid culture medium.

[0037] The fermentation broth of strain A-6 in inorganic salt liquid culture medium was added at a ratio of 1% (v of fermentation broth of strain A-6 in inorganic salt liquid culture medium / v of inorganic salt liquid culture medium containing 50 mg / L atrazine) to 100 mL of inorganic salt liquid culture medium containing 50 mg / L atrazine. The medium was then placed in shakers at 15℃, 23℃, 28℃, 30℃, and 37℃, and cultured at 180 rpm. Samples were taken periodically, and the atrazine concentration was determined by HPLC. The experiment was repeated three times. The results are as follows: Figure 3As shown, strain A-6 can degrade atrazine within the temperature range of 15℃-37℃, and the degradation rate of atrazine varies significantly under different temperature conditions. The optimal temperature for atrazine degradation by strain A-6 is 30℃. Under this temperature condition, after 12 hours of cultivation in 100 mL of inorganic salt liquid medium, 50% of 50 mg / L atrazine can be degraded, 91.4% can be degraded after 20 hours, and 50 mg / L atrazine can be completely degraded within 24 hours.

[0038] 3.3 Growth of strain A-6 under different pH conditions Inorganic salt liquid culture media with pH values ​​of 5.0, 6.0, 7.0, 8.0, and 9.0 were prepared (pH adjusted with 1 mol NaOH and 1 mol HCl). After sterilization at 121℃ for 20 min and cooling, atrazine was added to achieve a final concentration of 50 mg / L. Then, 1% (v of fermentation broth of strain A-6 in inorganic salt liquid culture media at different pH values ​​ / v of inorganic salt liquid culture media containing 50 mg / L atrazine) of strain A-6 fermentation broth (prepared as in 3.2) was inoculated into these media at 30℃ in a shaker at 180 rpm. Samples were taken periodically, and the atrazine concentration was determined by HPLC. The experiment was repeated three times. The results are shown below. Figure 4 As shown, strain A-6 can degrade atrazine between pH 6.0 and 9.0, and the optimal pH range for strain A-6 to degrade atrazine is 7.0-8.0.

[0039] 3.4 Degradation and growth of atrazine by strain A-6 The fermentation broth of strain A-6 in inorganic salt liquid culture medium (prepared as in 3.2) was inoculated into inorganic salt liquid culture medium containing 50 mg / L atrazine at a ratio of 1% (inorganic salt liquid culture medium strain A-6 fermentation broth v / inorganic salt liquid culture medium containing 50 mg / L atrazine v). The medium was incubated at 15℃ and 180 rpm, with samples taken periodically. The atrazine concentration was detected by HPLC, and the OD was measured by UV-Vis spectrophotometer. 600 The experiment was set up with three replicates. The results are as follows: Figure 5 As shown, strain A-6 can completely degrade 50 mg / L atrazine within 80 h at 15 °C, and OD 600 It will increase from 0 to approximately 0.05. At 15℃, in the first 20 hours, the growth and atrazine degradation of strain A-6 were extremely slow, with OD... 600 The growth of strain A-6 increased only from 0 to 0.006, with the majority of growth concentrated in the last 20 hours, specifically between hours 60 and 80. At this point, the OD...600 It will increase from 0.018 to 0.05, and reduce the concentration of atrazine from around 22 mg / L to 0 mg / L.

[0040] Example 4: Preparation of A-6 solid inoculant and its degradation effect on atrazine in soil After thawing strain A-6, preserved in glycerol tubes at -80℃, on ice, the strain was streaked in three zones on an LB agar plate using an inoculation loop. The LB agar plates were then incubated at 30℃ for 3 days. Single colonies were then picked and transferred to LB liquid medium and incubated at 30℃ and 180 rpm until OD500 reached. 600 =1.0, which is the fermentation broth of strain A-6. The degradation performance of strain A-6 on atrazine was determined according to the above method to ensure the function of strain A-6. Seven carriers—biochar (corn stalk biochar), corn stalk powder, vermiculite, earthworm castings, pig manure, sheep manure, and chicken manure—were all air-dried naturally for 3 days and then passed through a 40-mesh sieve. The sieved carriers were then placed in sterilization bags and subjected to two autoclave sterilization processes (121℃, 30 min) for later use. For each of the above different carriers, 200g of carrier was accurately weighed into a sterilization bag under aseptic conditions, and strain A-6 fermentation broth was added at a ratio of 10% (strain A-6 fermentation broth v (mL) / carrier m (g)), and mixed thoroughly. After sealing the sterilization bag, five holes were randomly punched in the bag as ventilation holes. Then, another sterilization bag was placed over the bag, with five holes randomly punched in the same way. After incubating at 30℃ for 3 days, the A-6 solid bacterial agent was obtained. The experiment was conducted in triplicate. The physical morphology of each A-6 solid bacterial agent is shown below. Figure 6 As shown, the A-6 solid microbial agent prepared from biochar (corn stalk biochar), corn stalk powder, earthworm castings, pig manure, sheep manure and chicken manure biomass carriers is in a loose granular form, which can provide a stable microenvironment for functional strains.

[0041] Each of the prepared A-6 solid microbial agents was placed in a cool, dry place. Every 30 days over 120 days, 2g of each A-6 solid microbial agent was added to a 500mL Erlenmeyer flask containing 180mL of sterile water, and glass beads were added. The flasks were then shaken at 200rpm for 30min. The bacterial suspensions were serially diluted, and the diluted solutions were spread onto LB agar plates and incubated at 30℃ for 3 days. The viable cell counts in each A-6 solid microbial agent were then counted using the plate count method. The experiment was conducted in triplicate. After 120 days of storage in a cool, dry place, the viable cell counts of the four A-6 solid microbial agents prepared using earthworm castings, pig manure, sheep manure, and chicken manure as carriers all met the national quality standards for agricultural microbial agents (≥2×10⁻⁶). 8(CFU / g). Therefore, A-6 solid microbial agents prepared from four carriers—earthworm castings, sheep manure, pig manure, and chicken manure—were selected for subsequent experiments on the degradation of atrazine in soil.

[0042] Atrazine stock solution was added to the test soil (black soil from farmland in Gongzhuling City, Jilin Province, naturally air-dried for 3 days, and passed through a 30-mesh sieve) to achieve a final concentration of 5 mg / kg dry soil. The mixture was thoroughly stirred and allowed to stand for 24 hours to allow the methanol in the added atrazine stock solution to completely evaporate. The soil moisture content was then adjusted to 25 wt% with deionized water to obtain the soil sample. 180 g of the soil sample was accurately weighed, and 20 g of A-6 solid microbial agent (prepared from earthworm castings, sheep manure, pig manure, and chicken manure, respectively, using four carriers) that met the national agricultural microbial agent industry quality standards and had been stored for 120 days was added. The mixture was thoroughly mixed and placed in a black plastic cup (10 cm in diameter and 10 cm in height, without a lid), filling the cup to 3 / 4 full. The cup was then incubated in a light incubator. The incubation conditions were: 12 hours of white light at 20℃; 12 hours of darkness at 15℃. Soil moisture content was maintained at approximately 25 wt% by daily weighing, and the soil in the container was shaken thoroughly. Samples were taken periodically over 14 days, and the atrazine concentration in the soil was determined by HPLC after dichloromethane extraction. The experiment was conducted in triplicate, with a control set up without the addition of A-6 solid inoculant. Results are as follows: Figure 7 As shown, in the treatment groups treated with A-6 solid microbial agents, the degradation efficiency of atrazine varied depending on the carrier used. The A-6 solid microbial agent prepared using earthworm castings as a carrier exhibited the fastest degradation rate of atrazine. After being mixed with soil for 14 days, the degradation efficiencies of atrazine for each of the A-6 solid microbial agents prepared using earthworm castings, sheep manure, pig manure, and chicken manure as carriers were 99.56%, 91.73%, 97.89%, and 90.63%, respectively.

Claims

1. A strain A-6, characterized in that, Its classification name is Arthroblastus ( Paenarthrobacter sp . The sample was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 9, 2023, with accession number CGMCCNO.28594.

2. The use of strain A-6 according to claim 1 in the degradation of atrazine.

3. The application according to claim 2, characterized in that, The degradation of atrazine refers to the degradation of atrazine in soil.

4. The use of strain A-6 according to claim 1 in the preparation of an atrazine-degrading bacterial agent.

5. The application according to claim 4, characterized in that, The degradation of atrazine refers to the degradation of atrazine in soil.

6. The bacterial agent prepared from strain A-6 according to claim 1, characterized in that, The bacterial agent includes solid bacterial agents.

7. The microbial agent according to claim 6, characterized in that, The solid microbial agent is prepared by the following steps: 1) Inoculate strain A-6 into a liquid culture medium for liquid fermentation, wherein the liquid culture medium includes LB liquid medium, and the liquid fermentation conditions are: temperature 28-30℃, rotation speed 160-180 rpm, and liquid fermentation to OD. 600 The concentration was 0.8-1.2, and the fermentation broth of strain A-6 was obtained; 2) Inoculate the fermentation broth of strain A-6 into a sterile carrier at an inoculation ratio of 8v / m%-12v / m%, mix thoroughly, and culture at 28-30℃ for 3-4 days to obtain the solid bacterial agent.

8. The microbial agent according to claim 7, characterized in that, In step 2), the sterile carrier includes sterilized earthworm castings, pig manure, sheep manure, or chicken manure.

9. The use of the microbial agent according to any one of claims 6-8 in the degradation of atrazine.

10. The application according to claim 9, characterized in that, The degradation of atrazine refers to the degradation of atrazine in soil.

Images