A microbial inoculum for degrading pesticide residues, its preparation method and application

Abstract

The present invention provides a microbial agent for degrading pesticide residues, a preparation method thereof and an application thereof, belonging to the technical field of microbiology. The microbial agent can simultaneously degrade various different pesticide residues and is composed of methylotrophic Bacillus sp. L7, Paenibacillus polymyxa DPM03 and an auxiliary agent. The microbial agent can simultaneously degrade one or more of fluazinam, BHC, quintozene, chlorpyrifos, DDT, atrazine and deltamethrin, overcoming the problem in the prior art that the microbial agent can only degrade one or a kind of pesticide residues with similar structures, and can achieve a greater degradation effect and a better soil remediation effect with a smaller application amount. Moreover, methylotrophic Bacillus sp. L7 and Paenibacillus polymyxa DPM03 can promote each other and have a synergistic effect, and the compound microbial agent of the two has a significantly better degradation effect on pesticide residues than the single use of methylotrophic Bacillus sp. L7 and Paenibacillus polymyxa DPM03.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and relates to a microbial agent for degrading pesticide residues, a preparation method thereof, and an application thereof. Background Art

[0002] Chemical pesticides play a huge role in preventing and controlling crop diseases, pests and weeds, eliminating sanitary pests and diseases, etc. due to their advantages such as quick effect, wide control spectrum, stable properties, convenient storage and transportation, and low price. However, the large-scale use of pesticides has also brought side effects such as environmental pollution, killing of natural enemies, and the generation of pesticide resistance in pests, diseases and weeds, resulting in adverse consequences such as increased pesticide residues, reduced soil fertility, and decline in the quality of agricultural products. In particular, pesticides with high toxicity and stable properties have a long residual period and are not easily degraded, posing a serious threat to human health.

[0003] In the remediation of polluted soil, bioremediation has received attention due to its low treatment cost and no secondary pollution. In recent years, the application of microorganisms in agriculture has also been relatively extensive, and it also plays an important role in the degradation of pesticide residues. The degradation effect of microorganisms is the most important and thorough purification of pesticides in the soil. Microorganisms with the ability to degrade pesticides include bacteria, fungi, etc. The bioremediation method of using organisms themselves or biological products to degrade pollutants has the advantages of being non-toxic and residue-free, and is a relatively cheap and effective method for eliminating pesticide residues or reducing the toxicity of residual pesticides. When microbial agents are applied as fertilizers, they show varying degrees of inhibitory effects on crop diseases and pests.

[0004] Due to the wide variety of microorganisms and their extremely rich metabolic types, they can completely decompose pollutants into harmless substances to the environment and the human body such as carbon dioxide, water, and inorganic compounds. In addition, microbial degradation of pesticides has substrate specificity and can only degrade one or a class of pesticides with similar structures, which will increase the repair cost for complex pesticide pollution. On the other hand, further improvement is needed in aspects such as the extension of the shelf life of degradation agent products and the stability of field degradation and repair effects. Summary of the Invention

[0005] According to the technical defects existing in the prior art, the technical problem to be solved by the present invention is: to provide a microbial agent for soil pollution remediation that can simultaneously degrade various different pesticide residues, solve the problem of the need to apply multiple microbial agents due to different pesticide residues, reduce the application amount of microbial agents, reduce the damage to the self-ecological balance of the soil, alleviate the soil pollution situation, and protect the safety of the ecological environment.

[0006] In order to achieve the technical object of the present invention, the present invention provides a microbial agent for degrading pesticide residues, and the microbial agent is composed of methylotrophic Bacillus sp. ( Bacillus methylotrophicus ), L7 and Paenibacillus polymyxa ( Paenibacillus polymyxa)(It consists of) Methylotrophic Bacillus L7 and an adjuvant. The Methylotrophic Bacillus L7 has been deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC No. 1.12059; the Paenibacillus polymyxa DPM03 has been deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC No. 7250.

[0007] Furthermore, in the microbial inoculum for degrading pesticide residues, the effective viable count of the microbial bacteria ≥ 1×10 7 / g.

[0008] Furthermore, in the microbial inoculum for degrading pesticide residues, the composition components and their mass parts of the microbial inoculum are as follows: 1 - 2 parts of Methylotrophic Bacillus L7, 5 - 10 parts of Paenibacillus polymyxa DPM03, and 5 - 8 parts of the adjuvant.

[0009] Furthermore, in the microbial inoculum for degrading pesticide residues, the composition components and their mass parts of the microbial inoculum are as follows: 2 parts of Methylotrophic Bacillus L7, 5 parts of Paenibacillus polymyxa DPM03, and 8 parts of the adjuvant.

[0010] Furthermore, in the microbial inoculum for degrading pesticide residues, the adjuvant is diatomite, humic acid, and ammonium sulfate.

[0011] Furthermore, for the microbial inoculum for degrading pesticide residues, it is characterized in that: in the adjuvant, the mass part ratio of diatomite, humic acid, and ammonium sulfate is 7:2:1.

[0012] The present invention also provides a preparation method for the microbial inoculum for degrading pesticide residues, which includes the following steps:

[0013] (1) Strain activation: Inoculate the Methylotrophic Bacillus L7 and the Paenibacillus polymyxa DPM03 strains preserved on the slant onto the beef extract peptone medium plates respectively, and culture them at 30 - 32°C for 24 - 36 h.

[0014] (2) Seed liquid culture: Pick the activated strains respectively and inoculate them into the liquid beef extract peptone medium, and culture them in a shaking flask at 30 - 32°C for 24 - 36 h.

[0015] (3) Scale-up culture: Inoculate the seed liquid into the liquid fermentation medium according to an inoculation amount of 1 - 10%, culture it at 30 - 32°C for 12 - 24 h, with the rotation speed of 180 - 260 rpm and the ventilation volume of 0.6 - 1.2 V / V·min.

[0016] (4)Preparation of the microbial agent: The cells of the methylotrophic Bacillus sp. L7 and the Paenibacillus polymyxa DPM03 are collected separately, and the methylotrophic Bacillus sp. L7, the Paenibacillus polymyxa DPM03 and the auxiliary agent are uniformly mixed according to the mass ratio of (1-2):(5-10):(5-8).

[0017] Further, step (4) also includes the step of detecting the viable cell count in the microbial agent.

[0018] The present invention also provides the application of the microbial agent for degrading pesticide residues or the microbial agent prepared by the foregoing preparation method in the remediation of soil pollution by pesticide residues, and the pesticide is one or more of fluazinam, BHC, quintozene, chlorpyrifos, DDT, atrazine, deltamethrin.

[0019] The present invention also provides a method for remediating soil pollution by pesticide residues, and the microbial agent for degrading pesticide residues is applied to the soil with pesticide residues.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] The microbial agent of the present invention can simultaneously degrade one or more of fluazinam, BHC, quintozene, chlorpyrifos, DDT, atrazine, deltamethrin, overcomes the problem that the microbial agent in the prior art can only degrade one or a class of pesticide residues with similar structures, and can achieve greater degradation effect and better soil remediation effect with less application amount. Moreover, it is unexpectedly found that the methylotrophic Bacillus sp. L7 and the Paenibacillus polymyxa DPM03 can promote each other and have a synergistic effect, and the degradation effect of the compound microbial agent of the two on pesticide residues is significantly better than that of using the methylotrophic Bacillus sp. L7 and the Paenibacillus polymyxa DPM03 alone. Specific embodiments

[0022] The strains used in the present invention:

[0023] Paenibacillus polymyxa DPM03 has been deposited in the China General Microbiological Culture Collection Center, and the deposit number is CGMCC No.7250. It has been disclosed in the patent CN108034614A and is purchased from the China General Microbiological Culture Collection Center.

[0024] Methylotrophic Bacillus sp. L7 has been deposited in the China General Microbiological Culture Collection Center, and the deposit number is CGMCC No.1.12059. It is purchased from the China General Microbiological Culture Collection Center.

[0025] Bacillus methylotrophicus 962, with the preservation number of CGMCC No. 1.806, was purchased from the General Microbiology Center of the China Committee for Culture Collection of Microorganisms.

[0026] To further elaborate on the beneficial effects of the present invention, the following specific examples were conducted. It should be particularly noted that the following specific examples are intended to better understand the present invention and are by no means limited to the scope of the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all conventional biochemical preparations purchased from stores unless otherwise specified. In the following examples, all quantitative tests were set up with three repeated tests, and the results were averaged.

[0027] Example 1: Preparation of microbial inoculum

[0028] (1) Strain activation: The Bacillus methylotrophicus L7 and Paenibacillus polymyxa DPM03 strains preserved on the slant were respectively inoculated on the beef extract peptone medium plates and cultured at 30 °C for 36 h.

[0029] (2) Seed liquid culture: The activated strains were respectively picked and inoculated into the liquid beef extract peptone medium, and cultured in a shaking flask at 30 °C for 36 h to obtain the Bacillus methylotrophicus L7 seed liquid and the Paenibacillus polymyxa DPM03 strain seed liquid respectively.

[0030] (3) Scale-up culture: The seed liquids obtained in step (2) were respectively inoculated into the liquid fermentation medium at an inoculation amount of 5%, and cultured at 32 °C for 24 h, with a rotation speed of 180 rpm and an aeration rate of 0.8 V / V·min.

[0031] (4) Preparation of inoculum: The cells of Bacillus methylotrophicus L7 and Paenibacillus polymyxa DPM03 in the fermentation broth were respectively collected, and Bacillus methylotrophicus L7, Paenibacillus polymyxa DPM03 and the auxiliary agent were mixed evenly according to a mass ratio of 1:7:5 to obtain the microbial inoculum A. Among them, calculated by mass fraction, diatomaceous earth in the auxiliary agent accounted for 70%, humic acid accounted for 20%, and ammonium sulfate accounted for 10%. The viable bacteria count in the mixed microbial preparation was detected to ensure that the effective viable bacteria count ≥ 1×10 7 / g.

[0032] Example 2: Preparation of microbial inoculum

[0033] (1) Strain activation: The Bacillus methylotrophicus L7 and Paenibacillus polymyxa DPM03 strains preserved on the slant were respectively inoculated on the beef extract peptone medium plates and cultured at 30 °C for 36 h.

[0034] (2) Seed culture: Pick and inoculate the activated strains into the liquid beef extract peptone medium respectively, and culture them in a shaking flask at 30 °C for 24 h to obtain the seed solutions of Bacillus methylotrophicus L7 and Bacillus polymyxa DPM03 respectively.

[0035] (3) Scale-up culture: Inoculate the seed solutions obtained in step (2) into the liquid fermentation medium respectively according to the inoculation amount of 5%, culture at 30 °C for 24 h, the rotation speed is 180 rpm, and the aeration volume is 0.8 V / V·min.

[0036] (4) Preparation of microbial inoculum: Collect the cells of Bacillus methylotrophicus L7 and Bacillus polymyxa DPM03 in the fermentation broth respectively, and mix Bacillus methylotrophicus L7, Bacillus polymyxa DPM03 and the auxiliary agent evenly according to the mass ratio of 2:5:8 to obtain the microbial inoculum B. Among them, calculated by mass fraction, diatomite in the auxiliary agent accounts for 70%, humic acid accounts for 20%, and ammonium sulfate accounts for 10%. Detect the viable count in the mixed microbial preparation to ensure that the effective viable count ≥ 1×10 7 / g.

[0037] Example 3: Preparation of Bacillus methylotrophicus L7 inoculum

[0038] (1) Strain activation: Inoculate the Bacillus methylotrophicus L7 preserved on the slant into the beef extract peptone medium plate and culture at 30 °C for 36 h.

[0039] (2) Seed culture: Pick and inoculate the activated strain into the liquid beef extract peptone medium, and culture it in a shaking flask at 30 °C for 24 h to obtain the seed solution of Bacillus methylotrophicus L7.

[0040] (3) Scale-up culture: Inoculate the seed solution obtained in step (2) into the liquid fermentation medium according to the inoculation amount of 5%, culture at 30 °C for 24 h, the rotation speed is 180 rpm, and the aeration volume is 0.8 V / V·min.

[0041] (4) Preparation of microbial inoculum: Collect the cells of Bacillus methylotrophicus L7 in the fermentation broth, and mix Bacillus methylotrophicus L7 and the auxiliary agent evenly according to the mass ratio of 2:8 to obtain the microbial inoculum C. Among them, calculated by mass fraction, diatomite in the auxiliary agent accounts for 70%, humic acid accounts for 20%, and ammonium sulfate accounts for 10%. Detect the viable count in the mixed microbial preparation to ensure that the effective viable count ≥ 1×10 7 / g.

[0042] Example 4: Preparation of Bacillus polymyxa DPM03 inoculum

[0043] (1) Strain activation: Inoculate the Paenibacillus polymyxa strain DPM03 preserved on the slant onto a nutrient agar medium plate and culture it at 30 °C for 36 h.

[0044] (2) Seed liquid culture: Pick the activated strain and inoculate it into a liquid nutrient agar medium, and culture it in a shaking flask at 30 °C for 24 h to obtain the seed liquid of the Paenibacillus polymyxa strain DPM03 respectively.

[0045] (3) Subculture: Inoculate the seed liquid obtained in step (2) into the liquid fermentation medium at an inoculation amount of 5%, culture it at 30 °C for 24 h, with a rotation speed of 180 rpm and an aeration rate of 0.8 V / V·min.

[0046] (4) Preparation of microbial agent: Collect the cells of Paenibacillus polymyxa strain DPM03 in the fermentation broth, and mix Paenibacillus polymyxa strain DPM03 and the auxiliary agent evenly according to a mass ratio of 5:8 to obtain the microbial agent D. Among them, calculated by mass fraction, diatomite in the auxiliary agent accounts for 70%, humic acid accounts for 20%, and ammonium sulfate accounts for 10%. Detect the viable count in the mixed microbial preparation to ensure that the effective viable count ≥ 1×10 7 / g.

[0047] Example 5: Preparation of other microbial agents

[0048] (1) Strain activation: Inoculate the methylotrophic Bacillus sp. 962 and Paenibacillus polymyxa strain DPM03 preserved on the slant onto a nutrient agar medium plate respectively, and culture them at 30 °C for 36 h.

[0049] (2) Seed liquid culture: Pick the activated strains and inoculate them into a liquid nutrient agar medium respectively, and culture them in a shaking flask at 30 °C for 24 h to obtain the seed liquid of methylotrophic Bacillus sp. 962 and the seed liquid of Paenibacillus polymyxa strain DPM03 respectively.

[0050] (3) Subculture: Inoculate the seed liquid obtained in step (2) into the liquid fermentation medium at an inoculation amount of 5% respectively, culture it at 30 °C for 24 h, with a rotation speed of 180 rpm and an aeration rate of 0.8 V / V·min.

[0051] (4) Preparation of microbial agent: Collect the cells of methylotrophic Bacillus sp. 962 and Paenibacillus polymyxa strain DPM03 in the fermentation broth respectively, and mix methylotrophic Bacillus sp. 962, Paenibacillus polymyxa strain DPM03 and the auxiliary agent evenly according to a mass ratio of 2:5:8 to obtain the microbial agent E. Among them, calculated by mass fraction, diatomite in the auxiliary agent accounts for 70%, humic acid accounts for 20%, and ammonium sulfate accounts for 10%. Detect the viable count in the mixed microbial preparation to ensure that the effective viable count ≥ 1×10 7 / g.

[0052] Example 6: Degradation of Pesticide Residues by Microbial Inoculants

[0053] Add 1 mL of the standard substances of the above pesticides at 100 mg / mL to 2 kg of soil samples that have been detected to contain no fluazinam, hexachlorocyclohexane, quintozene, chlorpyrifos, DDT, atrazine, and deltamethrin. Set up control and experimental groups. Control group 1 does not apply any substances, control group 2 applies a mixture containing only adjuvants, and the experimental groups apply the microbial inoculants prepared in Examples 1-5, with an application rate of 20 g / kg of soil. Detect the residual amounts of pesticide substances in the soil samples after 15 days and 30 days of treatment respectively. The determination of the content of various pesticides in the samples refers to the determination method in Patent CN108034614A.

[0054] After detection, the degradation effects of each microbial inoculant on pesticide residues in the soil are shown in Table 1. The results show that the microbial inoculant C containing only Bacillus methylotrophicus L7 has a good degradation effect on atrazine and deltamethrin, but has almost no degradation effect on fluazinam, hexachlorocyclohexane, quintozene, chlorpyrifos, and DDT; the microbial inoculant D containing only Paenibacillus polymyxa DPM03 has a certain degradation effect on fluazinam, hexachlorocyclohexane, quintozene, chlorpyrifos, and DDT, but has almost no degradation effect on atrazine and deltamethrin. The microbial inoculants A and B containing both Bacillus methylotrophicus L7 and Paenibacillus polymyxa DPM03 not only have good degradation effects on the above 7 pesticides. The microbial inoculant A has the best degradation effect on deltamethrin, and the degradation rate reaches 93.42% after 30 days. The 30-day degradation rates of the 7 pesticides by the microbial inoculant B are all higher than 50%, and the 30-day degradation rates of atrazine and deltamethrin reach 90.52% and 90.66% respectively, and the degradation effects are better than those of the microbial inoculants C and D. When Bacillus methylotrophicus L7 is replaced by Bacillus methylotrophicus 962 to obtain the microbial inoculant E, the degradation effects of the microbial inoculant E on the above seven pesticides are all reduced. Among them, the degradation effects of atrazine and deltamethrin decrease the most, and the 30-day degradation rates are only 9.58% and 7.56%, indicating that the combination of Bacillus methylotrophicus L7 and Paenibacillus polymyxa DPM03 has a synergistic effect in pesticide degradation and cannot be replaced randomly.

[0055] Table 1: Degradation Rates of Microbial Inoculants on Pesticide Residues in Soil

[0056]

[0057] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Any simple modifications, equivalent changes and modifications made to the above examples based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A microbial inoculum for degrading pesticide residues, characterized in that: The microbial bacteria consists of methylotrophic Bacillus sp. ( Bacillus methylotrophicus ), L7, and Paenibacillus polymyxa ( Paenibacillus polymyxa ), DPM03, and an adjuvant. The methylotrophic Bacillus sp. L7 has been deposited in the China General Microbiological Culture Collection Center (CGMCC) with the deposit number CGMCC No. 1.12059. The Paenibacillus polymyxa DPM03 has been deposited in the China General Microbiological Culture Collection Center (CGMCC) with the deposit number CGMCC No. 7250.

2. The microbial inoculum for degrading pesticide residues according to claim 1, wherein: The effective viable count of the microbial bacteria ≥ 1×10 7 / g.

3. The microbial inoculum for degrading pesticide residues according to any one of claims 1 or 2, characterized in that: The composition components of the microbial inoculant and their parts by mass are as follows: 1 - 2 parts of methylotrophic Bacillus sp. L7, 5 - 10 parts of Paenibacillus polymyxa DPM03, and 5 - 8 parts of adjuvant.

4. The microbial inoculum for degrading pesticide residues according to claim 3, characterized in that: The composition components of the microbial inoculant and their parts by mass are as follows: 2 parts of methylotrophic Bacillus sp. L7, 5 parts of Paenibacillus polymyxa DPM03, and 8 parts of adjuvant.

5. The microbial inoculum for degrading pesticide residues according to claim 1, characterized in that: The adjuvant is diatomite, humic acid, and ammonium sulfate.

6. The microbial inoculum for degrading pesticide residues according to claim 5, characterized in that: Among the adjuvants, the mass ratio of diatomite, humic acid, and ammonium sulfate is 7:2:

1.

7. The preparation method of the microbial agent for degrading pesticide residues according to claim 1, characterized in that: It includes the following steps: (1) Strain activation: Inoculate the methylotrophic Bacillus sp. L7 and the Paenibacillus polymyxa DPM03 strains preserved on the slant onto the beef extract peptone medium plates respectively, and culture them at 30 - 32 °C for 24 - 36 h; (2) Seed liquid culture: Selectively pick the activated strains and inoculate them into the liquid beef extract peptone medium, and culture them in a shaking flask at 30 - 32 °C for 24 - 36 h; (3) Scale - up culture: Inoculate the seed liquid into the liquid fermentation medium at an inoculation amount of 1 - 10%, culture it at 30 - 32 °C for 12 - 24 h, the rotation speed is 180 - 260 rpm, and the ventilation rate is 0.6 - 1.2 V / V·min; (4) Preparation of the inoculant: Collect the cells of the methylotrophic Bacillus sp. L7 and the Paenibacillus polymyxa DPM03 respectively, and mix the methylotrophic Bacillus sp. L7, Paenibacillus polymyxa DPM03, and the adjuvant evenly according to the mass ratio of (1 - 2):(5 - 10):(5 - 8).

8. The preparation method of the microbial agent for degrading pesticide residues according to claim 7, characterized in that: Step (4) also includes the step of detecting the viable count in the inoculant.

9. Use of the microbial agent for degrading pesticide residues according to any one of claims 1-6 or the microbial agent prepared by the preparation method according to any one of claims 7-8 in the remediation of soil pollution by pesticide residues, characterized in that: The pesticide is one or more of fluazinam, BHC, quintozene, chlorpyrifos, DDT, atrazine, deltamethrin.

10. A method for repairing soil pollution caused by pesticide residues, characterized in that: Apply the microbial inoculant for degrading pesticide residues as described in any one of claims 1 - 6 to the soil polluted by pesticide residues.

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