Vampiric bacteria, bacterial agent and application thereof and method for degrading atrazine

Abstract

The present application relates to the field of microorganism, disclose the method for degrading Atrazine and the application of voracious bacteriophage and microbial inoculants, wherein the preservation number of the voracious bacteriophage is CGMCC No.25130.The voracious bacteriophage can survive and grow in the pollutants containing Atrazine, and can degrade Atrazine, and the degradation rate can reach more than 90%.The present application provides a useful strain for biodegradation of Atrazine, and has the advantages of high efficiency, economy, simple operation, less interference and less secondary pollution, and has important significance in the remediation of farmland contaminated soil and contaminated water.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and more specifically to a strain of glutathione (Variovorax sp.), a bacterial agent, and their application in the degradation of atrazine, as well as a method for degrading atrazine. Background Technology

[0002] Atrazine was first introduced in the 20th century and is often used alone or in combination with other herbicides in agricultural soils. It has been produced for over 60 years and widely used worldwide. Atrazine was once considered to have high environmental safety, but with in-depth toxicological research, its negative effects have become increasingly apparent. Firstly, atrazine has a long half-life in the environment, high migration rate, and wide application, resulting in significant residues not only in the soil where it is applied but also in other areas. Furthermore, due to its cumulative effect, its concentration in the environment increases over time. Atrazine also has a low adsorption coefficient, leading to high mobility. It easily migrates from the application site to surrounding lakes and rivers, and can also be carried by rainwater, sediment, and surface volatilization to other areas, causing pollution to surrounding soil and water bodies, endangering environmental safety and the health of organisms. Therefore, finding a biodegradable technology that does not produce secondary pollution is of paramount importance for controlling atrazine pollution, improving the environment, and protecting human health.

[0003] Existing methods for treating atrazine include adsorption and redox methods. Adsorption utilizes porous materials such as activated carbon and nanomaterials to adsorb atrazine, while redox methods use strong oxidants such as ozone and hydroxylamine to oxidize atrazine. However, both adsorption and redox methods require the preparation of large quantities of chemicals to be effective, resulting in excessive costs. Furthermore, the uncontrollable reaction process can potentially cause secondary pollution. Finally, the inability to properly recover oxidants and adsorbents from the treated environment can lead to further environmental damage.

[0004] Microbial degradation refers to the method of using environmental microorganisms to transform organic pollutants such as atrazine into simple, non-toxic inorganic substances. Microbial removal largely occurs through microbial metabolism. Environmental microorganisms have advantages such as wide availability, diverse species, and strong adaptability; therefore, since the last century, extensive efforts have been made to find highly efficient atrazine-degrading microorganisms. However, currently reported pesticide-degrading microorganisms are few in number, and those that have been reported have low degradation efficiency and are not very thorough in degrading pesticides. Therefore, finding a microorganism to improve the atrazine degradation rate is an important solution. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of limited types of microorganisms and low degradation efficiency in existing technologies, and to provide a microbial strain that can tolerate high concentrations of atrazine and can efficiently degrade atrazine, thereby achieving the goal of rapid and efficient degradation of atrazine.

[0006] To achieve the above objectives, the first aspect of the present invention provides a strain of *Variovorax* sp., which has the accession number CGMCC No. 25130.

[0007] A second aspect of the present invention provides a bacterial agent containing the voracious bacteria (Variovorax sp.) as described above.

[0008] A third aspect of the present invention provides the use of the gluttonous bacteria and / or the bacterial agents described above in the degradation of atrazine.

[0009] A fourth aspect of the present invention provides a method for degrading atrazine, the method comprising: contacting the gluttonous bacteria and / or the bacterial agent as described above with an environment containing atrazine.

[0010] The glutathione bacteria described in this invention can survive and grow in pollutants containing atrazine, and can degrade atrazine with a degradation rate of over 90%.

[0011] The use of the glutathione bacteria provided by this invention for atrazine degradation has advantages such as high efficiency, economy, simple operation, minimal interference, and less secondary pollution, making it significant for the remediation of contaminated farmland soil and polluted water bodies. By degrading atrazine in situ and ex-situ through microorganisms, the use of chemicals can be reduced, thus lowering costs during the remediation process.

[0012] Biological Preservation

[0013] The strain provided by this invention is classified and named Variovorax sp., and was deposited on June 23, 2022 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNo.25130, located at No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0014] Figure 1 This is a microscope image of the colony morphology of the gluttonous bacteria described in this invention;

[0015] Figure 2 This is a scanning electron microscope image of the gluttonous bacteria described in this invention;

[0016] Figure 3 This is the phylogenetic tree of the gluttonous bacteria described in this invention;

[0017] Figure 4 It is the standard curve for the detection of atrazine by liquid chromatography;

[0018] Figure 5 These are the growth curves of the glutathione and the degradation curves of atrazine described in this invention. Detailed Implementation

[0019] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0020] The first aspect of this invention provides a strain of *Variovorax* sp., which has the accession number CGMCC No. 25130.

[0021] The glutathione strains of this invention were screened from the topsoil of farmland where atrazine had been applied for many years. By comparing the degradation rates of atrazine, strains with high degradation rates were selected and identified.

[0022] The strain described in this invention is *Variovorax* sp., which appears as pale yellow colonies on agar plates with a smooth, opaque surface (see...). Figure 1 Strain DA was observed to be arranged singly under a scanning electron microscope, with a long rod-like morphology (see...). Figure 2 Using the strain's genomic DNA as a template, PCR amplification was performed using universal primers for bacterial 16S rDNA, yielding an amplified product of 1435 bp. BLAST alignment showed that the strain shared 99.51% homology with Variovorax sp. S15 (GenBanK accession number HE662647.1). A phylogenetic tree was constructed to determine the strain's evolutionary level (see...). Figure 3 ).

[0023] The strain provided by this invention is classified and named Variovorax sp., and was deposited on June 23, 2022 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNo.25130, located at No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0024] A second aspect of the present invention provides a bacterial agent containing the voracious bacteria (Variovorax sp.) as described above.

[0025] The preparation method of the microbial agent can be a conventional method in the art, such as culturing the strain to obtain a material containing microorganisms (such as fermentation broth or microbial sludge), and then optionally adding auxiliary materials as needed to prepare different types of products.

[0026] The strain can be cultured in a conventional manner in the art, such as at 22-32℃ (preferably 25-30℃), pH 6.5-7 and under aerobic conditions. There is no particular limitation on the culture time, such as 12-48h.

[0027] The culture medium for culturing the strain can be a conventional culture medium in the art (such as LB medium), or it can be a medium containing inorganic salts containing atrazine, for example, a medium with atrazine as the sole carbon source or the sole nitrogen source.

[0028] In this invention, the culture medium using atrazine as the sole carbon source preferably contains 0.5-1.5 g / L K₂HPO₄, 0.5-1.5 g / L KH₂PO₄, 0.05-0.4 g / L MgSO₄·7H₂O, 0.5-1.5 g / L NaCl, 0.01-0.05 g / L CaCl₂, 0.5-1.5 g / L NH₄NO₃, and 10-100 mg / L atrazine. It should be understood that if the culture medium is a solid medium, agar may also be added, with the amount of agar added being 15-20 g / L.

[0029] In this invention, the culture medium using atrazine as the sole nitrogen source preferably contains 0.5-1.5 g / L K₂HPO₄, 0.5-1.5 g / L KH₂PO₄, 0.05-0.4 g / L MgSO₄·7H₂O, 0.5-1.5 g / L NaCl, 0.01-0.05 g / L CaCl₂, 0.5-1.5 g / L sucrose, and 10-100 mg / L atrazine. It should be understood that if the culture medium is a solid medium, agar may also be added, with the amount of agar added being 15-20 g / L.

[0030] Depending on the intended use, the microbial agent provided by this invention can be prepared in different dosage forms, such as liquid or solid microbial agents. The microbial agent may also contain other components, such as protectants and diluents; the specific selection of these components is well known to those skilled in the art, and will not be described in detail here.

[0031] The concentration of the bacterial strain in the bacterial agent is not particularly limited and can be selected according to specific circumstances. Preferably, the viable count in the bacterial agent is 1 × 10⁻⁶. 9 CFU / g or higher.

[0032] A third aspect of the present invention provides the use of the gluttonous bacteria and / or the bacterial agents described above in the degradation of atrazine.

[0033] A fourth aspect of the present invention provides a method for degrading atrazine, the method comprising: contacting the gluttonous bacteria and / or the bacterial agent as described above with an environment containing atrazine.

[0034] In this invention, the form of bacteria added to the atrazine-containing environment is not particularly limited, as long as the strain can function in the atrazine-containing environment and effectively degrade the atrazine after addition. The form of the added gluttonous bacteria can be, for example, activated bacterial cells (bacterial suspension or bacterial precipitate) cultured to the logarithmic phase, freeze-dried bacterial powder, or a bacterial agent obtained by compounding.

[0035] Preferably, the environment containing atrazine is soil and / or water contaminated with atrazine.

[0036] Preferably, the concentration of atrazine in the environment containing atrazine is not higher than 100 mg / L, more preferably not higher than 50 mg / L, and even more preferably not higher than 10 mg / L.

[0037] There are no particular restrictions on the conditions under which the strain comes into contact with the environment. However, in order to improve the degradation efficiency of atrazine, the contact conditions preferably include: a temperature of 15-35°C and a pH of 5.5-8.5; more preferably, a temperature of 22-32°C and a pH of 6.5-7.

[0038] The present invention does not impose any particular limitation on the number of glutathione bacteria added. This can be determined based on the atrazine content in the atrazine-containing environment and the survival ability of the strain in the environment. For example, when the atrazine content in the environment is high or the environment is unfavorable to the survival of the glutathione bacteria, the amount of glutathione bacteria added can be increased; when the atrazine content in the environment is low or the environment has little impact on the survival of the glutathione bacteria, the amount of glutathione bacteria added can be reduced.

[0039] Preferably, the amount of the bacterial strain or the bacterial agent used is such that the viable count in the atrazine-containing environment is 10. 3 -10 9 cfu / mL, more preferably 10 5 -10 9 cfu / mL. It should be understood that the inoculation amount can also be determined according to the inoculation volume, for example, 1-10% of the volume.

[0040] In this invention, the atrazine content can be determined by HPLC.

[0041] The present invention will be described in detail below through embodiments.

[0042] LB liquid medium: 10 g / L peptone -1 5g / L yeast powder -1 10 g·L of sodium chloride -1 The pH value is 6.8.

[0043] Inorganic salt culture medium with atrazine as the sole nitrogen source: K2HPO4 1.0g, KH2PO4 1.0g, MgSO4·7H2O 0.2g, NaCl 1.0g, CaCl2 0.02g, sucrose 1.0g, atrazine 50mg, add distilled water to a final volume of 1L. When preparing solid culture medium, add 15g agar powder, autoclave and use.

[0044] Inorganic salt culture medium with atrazine as the sole carbon source: K2HPO4 1.0g, KH2PO4 1.0g, MgSO4·7H2O 0.2g, NaCl 1.0g, CaCl2 0.02g, NH4NO3 1.0g, atrazine 50mg, add distilled water to a final volume of 1L. When preparing solid culture medium, add 15g of agar powder, autoclave and use.

[0045] HPLC determination of atrazine: 10 ml of the sample containing atrazine was pipetted into a 50 ml centrifuge tube, and 10 ml of dichloromethane was added. The mixture was shaken at 220 rpm and 25°C for 30 min. The liquid was then transferred to a separatory funnel for splitting, and 5 g of NaCl was added until clear separation of the liquid layers was observed. The lower organic layer was collected, filtered through a 0.22 μm disposable syringe filter, and injected into a brown bottle for HPLC determination. The method for determining atrazine using HPLC was as follows: mobile phase composition: methanol:water = 70:30; flow rate: 10 μL / min; column temperature: 25°C; detection wavelength: 225 nm. The standard curve of atrazine was obtained by determining the sample. Figure 4 .

[0046] In the following examples, unless otherwise specified, the reagents and materials used are conventional reagents and materials in the art, and the methods used are conventional methods in the art.

[0047] Example 1

[0048] This embodiment is used to illustrate the screening and identification of the gluttonous bacteria described in this invention.

[0049] Isolation and purification of gluttonous bacteria:

[0050] Soil samples from the top 0-20 cm layer of a maize experimental field in Da'an City, Jilin Province (45°14'25"N, 123°22'46"E) were collected using a five-point sampling method and placed in plastic bags. 5 g of soil was added to 45 mL of sterile physiological saline and shaken at 25°C and 180 rpm for 30 min on a shaker, then allowed to stand at room temperature for 1 h. 1 mL of the supernatant was added to 19 mL of sterile inorganic medium with atrazine as the sole nitrogen source (preliminary experiments showed that the degradation rate of atrazine was higher in the medium with atrazine as the sole nitrogen source than in the medium with atrazine as the sole carbon source). The medium was shaken and cultured at 25°C, 180 rpm, and in the dark, with the medium changed every 3 days. After 10 passages, the culture was plated onto solid medium and purified twice using streak plating.

[0051] Strain identification:

[0052] The bacterial colonies on the culture medium plates are pale yellow with a smooth and opaque surface. Figure 1 The strains were observed to be arranged singly under a scanning electron microscope, and their morphology was long rod-shaped. Figure 2 ).

[0053] Using the strain's genomic DNA as a template, PCR amplification was performed using universal primers for bacterial 16S rDNA, yielding an amplified product of 1435 bp. BLAST alignment showed that the strain shared 99.51% homology with Variovorax sp. S15 (GenBanK accession number HE662647.1). A phylogenetic tree was constructed to determine the evolutionary level of this strain. Figure 3 ).

[0054] Example 2

[0055] This embodiment is used to illustrate the atrazine degradation characteristics of the gluttonous bacteria described in this invention.

[0056] Methods: Single colonies of the strain were picked and pre-cultured in 5 mL of inorganic salt medium with atrazine as the sole nitrogen source for 12 h. After centrifuging the resulting bacterial suspension at 4000 r / min for 5 min, the supernatant was discarded. The bacterial cells were washed with 5 mL of 0.9% sterile physiological saline and resuspended twice. The resuspended suspension was then used as a seed suspension in 5 mL of inorganic salt medium with atrazine as the sole nitrogen source.

[0057] To determine the degradation effect of atrazine, 1 mL of the above seed suspension was inoculated into a 50 mL Erlenmeyer flask containing 19 mL of sterile basal salt medium. The flask was incubated at 25°C and pH 6.8 with shaking at 180 rpm for 3 days. Samples were taken every 12 hours and analyzed using HPLC (the standard curve for atrazine is shown in [reference needed]). Figure 4The residue of atrazine herbicide was determined, and atrazine degradation curves and bacterial growth curves were plotted. Bacterial growth was characterized by the absorbance value (OD600) at 600 nm. Three replicates were performed for each treatment, with an uninoculated strain serving as a control.

[0058] Result: As Figure 5 As shown, the *Gnaphalium affine* of this invention can rapidly degrade atrazine, which can be used as a growth medium, and the degradation of atrazine is positively correlated with the growth of the strain. In a basal salt culture medium containing atrazine, the strain does not have a significant growth retardation phase and rapidly enters the logarithmic growth phase. By day 3, the degradation rate of atrazine reaches 86.2%, while in the control group without inoculation, the atrazine content is not significantly reduced.

[0059] Example 3

[0060] This embodiment illustrates the effect of culture conditions on the atrazine degradation ability of the gluttonous bacteria described in this invention.

[0061] The study investigated the factors influencing the degradation efficiency of atrazine herbicide by *Agrobacterium davidii*. These factors included: different temperatures (20℃, 25℃, 30℃, 35℃); different pH values ​​(4.5, 5.5, 6.5, 7.5, 8.5, 9.5); and different strain dosages (1%, 5%, 10%, 20%, 50%).

[0062] 1. The degradation ability of gluttonous bacteria of atrazine at different temperatures

[0063] The seed suspension of *Gnaphalium affine* prepared according to the method described in Example 2 was inoculated at a rate of 5% by volume into 19 ml of a culture medium with atrazine as the sole nitrogen source, so that the final atrazine concentration was 50 mg / L.

[0064] After culturing at pH 6.8, 180 r / min, and temperatures of 20℃, 25℃, 30℃, and 35℃ for 3 days in a shaker, the degradation rate of atrazine was compared, and it was found that 25–30℃ was the optimal degradation temperature for atrazine.

[0065] The degradation rate decreases when the temperature is below 25℃ or above 30℃. Moreover, the degradation rate of herbicides is significantly lower in high-temperature environments (35℃) than in low-temperature environments (20℃). This may be related to the growth of the phytophagous bacteria and the optimal temperature for the enzymes that degrade herbicides.

[0066] 2. The degradation capacity of atrazine by gluttonous bacteria at different pH levels

[0067] The seed suspension of *Gnaphalium affine* prepared according to the method described in Example 2 was inoculated at a rate of 5% by volume into 19 ml of a culture medium with atrazine as the sole nitrogen source, so that the final atrazine concentration was 50 mg / L.

[0068] The strain was cultured in a shaker for 4 days at 20℃, 180 r / min, and pH values ​​of 4.5, 6.5, 7.0, and 8.5. The results showed that the degradation rate was higher between pH 6.5 and 7.0, indicating a better adaptation to a slightly acidic environment. However, the degradation rate was lower in other pH ranges. At pH 4.5, the degradation rate was approximately 40%, and at pH 8.5, it dropped to below 65%. This is likely because excessive acidity or alkalinity inhibited enzyme activity, thus affecting the microbial degradation of atrazine.

[0069] 3. The degradation capacity of atrazine by gluttonous bacteria at different inoculum sizes

[0070] The atrazine bacteria were inoculated into LB medium at inoculum rates of 1%, 5%, 10%, 20%, and 50% (v / v), ultimately achieving an atrazine concentration of 100 mg / L. After culturing for 3 days at pH 6.8, 25°C, and 180 rpm, the degradation rate remained relatively stable at around 92% for all inoculum rates, showing no significant difference. This may be because after two days of culturing, the number of bacteria in the medium tends to be uniform, resulting in similar degradation rates.

[0071] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A strain of gluttonous bacteria ( Variovorax sp.), characterized in that, The accession number of this gluttonous bacterium is CGMCC No.25130.

2. A microbial agent, characterized in that, This bacterial agent contains the gluttonous bacteria described in claim 1. Variovorax sp.).

3. The microbial agent according to claim 2, wherein, The bacterial agent can be a liquid bacterial agent or a solid bacterial agent.

4. The microbial agent according to claim 2 or 3, wherein, The viable bacteria count in the bacterial agent is 1×10⁻⁶. 9 CFU / g or higher.

5. The use of the glutathione of claim 1 and / or the bacterial agent of any one of claims 2-4 in the degradation of atrazine.

6. A method for degrading atrazine, characterized in that, The method includes contacting the gluttonous bacteria of claim 1 and / or the bacterial agent of any one of claims 2-4 with an environment containing atrazine.

7. The method according to claim 6, wherein, The environment containing atrazine refers to soil and / or water bodies contaminated with atrazine.

8. The method according to claim 6 or 7, wherein, In environments containing atrazine, the concentration of atrazine should not exceed 100 mg / L.

9. The method according to claim 6 or 7, wherein, The contact conditions include a temperature of 15-35°C and a pH of 5.5-8.

5.

10. The method according to claim 8, wherein, The contact conditions include a temperature of 15-35°C and a pH of 5.5-8.

5.

11. The method according to any one of claims 6-7 and 10, wherein, The amount of strain or the bacterial agent used results in a viable count of 10 in the atrazine-containing environment. 3 -10 9 cfu / g.

12. The method according to claim 8, wherein, The amount of strain or the bacterial agent used results in a viable count of 10 in the atrazine-containing environment. 3 -10 9 cfu / g.

13. The method according to claim 9, wherein, The amount of strain or the bacterial agent used results in a viable count of 10 in the atrazine-containing environment. 3 -10 9 cfu / g.

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