Microbial complex microbial inoculant for degrading herbicide and preparation method of microbial complex microbial inoculant

Abstract

The invention belongs to the field of microbial agents, and particularly relates to a microbial composite agent for degrading herbicides and a preparation method of the microbial composite agent. The preparation method of the microbial complex microbial inoculant comprises the following preparation steps: mixing bran hydrolysate, peat soil and diatomite, sterilizing, adding a protective agent and a complex flora suspension in a sterile environment, uniformly mixing, kneading and forming, preparing into slices, naturally air-drying, and crushing to obtain the microbial inoculant, and uniformly mixing the microbial agent with the adsorbent, the surfactant and the nutrient substances to obtain the microbial complex microbial inoculant. According to the invention, domesticated microorganisms form a composite flora to prepare the microbial agent, and the composite flora exerts unique degradation capability and generates synergistic effect, so that the herbicide can be degraded more comprehensively and efficiently, and the proportion of each microorganism, an adsorbent, a surfactant and other components in the microbial agent can be adjusted according to different herbicide types and polluted environments; and efficient degradation of specific herbicides is realized.

Description

Technical Field

[0001] The present invention belongs to the field of preparation of microbial agents, and particularly relates to a microbial composite agent for degrading herbicides and a preparation method thereof. Background Art

[0002] With the development of agricultural modernization, herbicides are widely used in agricultural production to control weed growth and increase crop yields. However, the extensive use of herbicides has brought a series of environmental and ecological problems. Some herbicides are prone to residue in the soil, resulting in serious problems such as soil quality decline, crop growth inhibition, and groundwater pollution. Physical remediation and chemical remediation both belong to traditional remediation methods, but they both have certain limitations. Physical remediation methods such as soil washing and soil replacement can remove herbicides in the soil to a certain extent, but they are costly and may cause greater damage to soil structure and ecological environment. Chemical remediation is prone to secondary pollution. Chemical remediation methods usually use chemical oxidants or reductants to react with herbicides to degrade or convert them into harmless substances. However, these chemical reagents themselves may cause secondary pollution to the environment, and the reaction conditions are relatively harsh and difficult to control.

[0003] In contrast, microbial remediation has certain advantages. Degrading herbicides with microbial agents is a bioremediation method. Microorganisms use their own metabolic activities to convert herbicides into harmless carbon dioxide, water, and other small-molecule substances during the degradation process, without generating secondary pollution, which conforms to the concept of sustainable development. The microbial remediation process usually does not require complex equipment and a large amount of chemical reagents. As long as suitable environmental conditions are provided, microorganisms can grow and reproduce on their own and play a degradation role, with relatively low costs. At the same time, microbial remediation can be carried out in-situ, reducing costs such as soil transportation. Moreover, different microorganisms have different degradation abilities for specific herbicides. Through screening and domestication, microbial strains or composite microbial communities with high degradation abilities for target herbicides can be obtained to specifically solve the problem of herbicide pollution. During the process of microorganisms degrading herbicides, their metabolic activities can also improve the ecological environment of the soil, promote the growth and reproduction of beneficial microorganisms in the soil, restore the balance of the soil microbial community, and thus improve the self-purification ability and ecological function of the soil.

[0004] Patent CN 110358717A discloses a microbial agent for degrading fomesafen, a preparation method thereof, and an application. The bacteria used in this invention are a mixed bacteria of Bacillus velezensis FB11 and Stenotrophomonas maltophilia FB14. After mixing the two strains of bacteria, auxiliary materials are added to prepare the microbial agent. It is used to degrade the herbicide fomesafen and has a significant degradation effect on the herbicide fomesafen in the soil.

[0005] However, the degradation effect of the existing microbial inoculants on various complex herbicides still needs to be improved, and it is of great significance to continue researching highly efficient herbicide component-degrading inoculants. Summary of the Invention

[0006] The present invention discloses a microbial composite inoculant for degrading herbicides and a preparation method thereof. The present invention forms a composite microbial community from domesticated microorganisms to prepare a microbial inoculant. They each play a unique degradation ability and produce a synergistic effect, enabling more comprehensive and efficient degradation of herbicides. Compared with single microorganisms, they have a wider degradation spectrum and higher degradation efficiency. A protective agent, an adsorbent, a surfactant, and nutrients are compounded, and the proportions of various microorganisms in the composite microbial community, as well as the dosages of components such as the adsorbent and the surfactant, can be adjusted according to different herbicide types and polluted environments to achieve efficient degradation of specific herbicides.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides a preparation method of a microbial composite inoculant for degrading herbicides, including the following preparation steps:

[0009] (1) Mix wheat bran hydrolysate, peat soil, and diatomaceous earth, sterilize, and in a sterile environment, add a protective agent and a composite microbial community suspension, mix evenly, knead into a shape, make into thin slices, and naturally air-dry for 40 - 80 h, then crush to obtain a microbial inoculant;

[0010] (2) Mix the microbial inoculant obtained in step (1) evenly with an adsorbent, a surfactant, and nutrients to obtain a microbial composite inoculant.

[0011] In some embodiments, the mass ratio of the wheat bran hydrolysate, peat soil, and diatomaceous earth is 3:(3 - 6):(2 - 5).

[0012] Preferably, the mass ratio of the wheat bran hydrolysate, peat soil, and diatomaceous earth is 3:5:3.5.

[0013] In some embodiments, the preparation method of the wheat bran hydrolysate is as follows:

[0014] Add water to the crushed wheat bran, boil and cool, then add a composite enzyme, enzymatically hydrolyze for 2 - 4 h, inactivate the enzyme, and cool to obtain the wheat bran hydrolysate.

[0015] In some embodiments, the dosage of the wheat bran is 20 - 35 wt% of the water.

[0016] In some embodiments, the composite enzyme includes cellulase, glucoamylase, and phytase; the usage amount of the composite enzyme is 0.5 - 3 wt% of the wheat bran.

[0017] Preferably, the usage amounts of the cellulase, glucoamylase, and phytase are respectively 0.5 - 2 wt%, 0.1 - 0.5 wt%, and 0.05 - 0.2 wt% of the wheat bran.

[0018] In some embodiments, the protective agent comprises cyclodextrin, trehalose, sodium carboxymethyl cellulose, and glycerol in a mass ratio of (2 - 5):(3 - 6):2:(2 - 4); the usage amount of the protective agent is 5 - 15 wt% of the complex bacterial community suspension.

[0019] Preferably, the protective agent comprises cyclodextrin, trehalose, sodium carboxymethyl cellulose, and glycerol in a mass ratio of 4:5:2:3.

[0020] In some embodiments, the complex bacterial community suspension comprises Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Streptomyces, and Aspergillus niger in a quantity ratio of (2 - 3):(1.5 - 2.5):(1 - 2):(2 - 3):2.

[0021] Preferably, the complex bacterial community suspension comprises Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Streptomyces, and Aspergillus niger in a quantity ratio of 2.5:2:1.5:2.5:2.

[0022] Further preferably, Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Streptomyces, and Aspergillus niger are all domesticated;

[0023] The operating steps of the domestication are as follows:

[0024] S1: Respectively inoculate the activated Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Streptomyces, and Aspergillus niger into a medium containing 10 - 50 mg / L of herbicide, and culture until the growth stationary phase; among them, Bacillus subtilis, Pseudomonas aeruginosa, and Acinetobacter baumannii are cultured with shaking at 30 - 37 °C, pH 6.5 - 7.5, and 150 - 200 r / min; Streptomyces is cultured with shaking at 28 - 30 °C, pH 7.0 - 7.5, and 100 - 150 r / min; Aspergillus niger is statically cultured under the conditions of 25 - 30 °C and pH 5.0 - 6.0;

[0025] S2: Transfer the stationary - phase Bacillus subtilis obtained in step S1 to a medium containing a higher concentration of herbicide for continuous culture, gradually increase the concentration of the herbicide in a gradient of 10 - 20 mg / L, and perform sub - culture 3 - 7 times to respectively obtain the domesticated Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Streptomyces, and Aspergillus niger.

[0026] Preferably, the domesticated Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Streptomyces, and Aspergillus niger are enriched, cultured, diluted to obtain a bacterial suspension, and then the bacterial suspensions are mixed in proportion to obtain a composite bacterial suspension.

[0027] In some embodiments, the concentration of the strains in the composite bacterial suspension in the thin slice is (1-10)×10 9- 10 CFU / g.

[0028] In some embodiments, the adsorbent is selected from activated carbon and / or bentonite.

[0029] In some embodiments, the surfactant is selected from one or more of Tween, Span, alkylbenzene sulfonate, and alkyl sulfate.

[0030] The second aspect of the present invention provides a microbial composite agent prepared by the preparation method described in the above scheme.

[0031] The present invention relates to a composite microbial community composed of domesticated Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Streptomyces, and Aspergillus niger in a certain proportion, which has a specific degradation ability for herbicides. Among them, Bacillus subtilis can, on the one hand, produce various enzymes such as protease, amylase, lipase, etc. These enzymes can preliminarily decompose the chemical structure of herbicides, break some chemical bonds in the herbicide molecules, making their structures simpler and more conducive to subsequent degradation. On the other hand, during its growth, it can produce a large amount of extracellular polysaccharides and other substances, which help to improve the aggregate structure of the soil, increase the air permeability and water retention of the soil, and indirectly promote the degradation of herbicides. Pseudomonas aeruginosa can metabolize herbicides as substitutes for carbon or nitrogen sources, and gradually decompose herbicides through its own metabolic activities. Moreover, this bacterium has strong adaptability to the environment. During the process of degrading herbicides, even if the environmental conditions fluctuate, Pseudomonas aeruginosa can still maintain a certain activity and continue to play a degradation role, increasing the stability of the microbial complex agent in different environments. Acinetobacter baumannii has a certain tolerance to harsh environments and can maintain a relatively stable growth state when the growth of other microorganisms is inhibited. When degrading herbicides, it can participate in the degradation process of herbicides through its own metabolic system. Although its degradation ability may be relatively weak compared to some strains, it can supplement the degradation pathway and cooperate with other strains to jointly complete the degradation of herbicides. Streptomyces can produce rich secondary metabolites, some of which have the activity of degrading herbicides. The mycelium of Streptomyces can interact with soil particles to improve the soil structure. Aspergillus niger can secrete various enzymes such as cellulase, amylase, protease, and some special oxidases. These enzymes can enzymatically decompose herbicides into small molecule substances. In addition, Aspergillus niger secretes organic acids during its growth, regulating the pH value of the soil and making the soil environment more conducive to the growth of other microorganisms and the exertion of degradation effects. During the degradation process, the metabolites of each strain may provide nutrients for other strains or create more favorable metabolic conditions. After Aspergillus niger regulates the soil pH value, it may enhance the degradation enzyme activities of Bacillus subtilis and Pseudomonas aeruginosa, thereby improving the overall degradation efficiency. At the same time, the various enzymes secreted by different strains can carry out continuous and multi-step degradation of herbicides, which is more efficient than the action of a single enzyme. Moreover, different strains have different adaptabilities to environmental conditions, and their synergistic effects can enable the microbial complex agent to play a role under a wider range of environmental conditions. The tolerance of Acinetobacter baumannii combined with the environmental adaptability of Pseudomonas aeruginosa enables the composite microbial agent to still maintain a certain degradation ability when environmental factors such as temperature, pH value, and salinity change, ensuring the effective degradation of herbicides in soils with different geographical locations and different pollution levels.

[0032] The present invention also adds a protective agent composed of cyclodextrin, trehalose, sodium carboxymethyl cellulose, and glycerol in a specific proportion to the microbial inoculum, which can improve the stability of microorganisms during storage and use. Cyclodextrin can encapsulate harmful substances and protect microorganisms from adverse external factors; trehalose can form a protective film on the surface of microbial cells to maintain the structure of cell membranes and proteins; sodium carboxymethyl cellulose increases the viscosity of the system and makes the microorganisms disperse evenly; glycerol can regulate the cell osmotic pressure and prevent microorganisms from losing water. In addition, nutrients such as carbon sources, nitrogen sources, and trace elements in the bran hydrolysate are added to the raw materials of the microbial complex inoculum of the present invention, providing energy and material basis for the growth and reproduction of microorganisms. Under sufficient nutritional conditions, microorganisms can multiply in large numbers and maintain high activity, thereby enhancing the degradation ability of herbicides. In addition, adsorbents such as activated carbon and bentonite have a large specific surface area and adsorption capacity, can adsorb herbicides in soil or water, enrich herbicides around microorganisms, increase the contact opportunities between microorganisms and herbicides, and thus improve the degradation efficiency. Surfactants are also added to the raw materials of the microbial complex inoculum. Surfactants such as Tween, Span, alkylbenzene sulfonates, and alkyl sulfates can reduce the surface tension between herbicides and soil particles or water, make herbicides more easily dispersed, increase their contact area with microorganisms, and contribute to the adsorption and degradation of herbicides by microorganisms.

[0033] The microbial complex inoculum of the present invention comprises a complex microbial community composed of domesticated microorganisms, each playing a unique degradation ability, and their synergistic effect can degrade herbicides more comprehensively and efficiently. It has a wider degradation spectrum and higher degradation efficiency compared with single microorganisms; low-cost raw materials such as bran hydrolysate are used in the raw materials, and the preparation process is relatively simple, without the need for complex equipment and expensive chemical reagents, reducing the production cost; the use of the protective agent improves the stability of the microbial inoculum under storage and different environmental conditions, extends the product shelf life, and enhances its adaptability in practical applications. The microbial inoculum of the present invention can also adjust the proportions of various microorganisms in the complex microbial community, as well as components such as adsorbents and surfactants according to different herbicide types and polluted environments, to achieve the efficient degradation of specific herbicides.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] 1. The present invention makes a microbial inoculum by forming a complex microbial community composed of domesticated microorganisms. They each play a unique degradation ability and produce a synergistic effect, capable of degrading herbicides more comprehensively and efficiently. It has a wider degradation spectrum and higher degradation efficiency compared with single microorganisms, and can also adjust the proportions of various microorganisms in the complex microbial community, as well as components such as adsorbents and surfactants according to different herbicide types and polluted environments, to achieve the efficient degradation of specific herbicides.

[0036] 2. In the raw materials of the microbial complex bactericide of the present invention, low-cost raw materials such as wheat bran hydrolysate are used, and the preparation process is relatively simple, without the need for complex equipment and expensive chemical reagents, reducing the production cost.

[0037] 3. The use of a protective agent in the raw materials of the microbial complex bactericide of the present invention improves the stability of the microbial bactericide under storage and different environmental conditions, extends the product shelf life, and enhances its adaptability in practical applications. Detailed implementation manners

[0038] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention. It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0039] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present application are only exemplary.

[0040] Regarding the use of "comprising", "including", "having", or "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0041] The parts by mass described in the following examples can be grams, kilograms, tons, or other mass units.

[0042] The compound monomers and related reagents used in the following examples are all commercially available. Among them, the enzyme activities of the cellulase, glucoamylase, and phytase used in the present invention are all 100,000 U / g; the cyclodextrin used is β-cyclodextrin; the number-average molecular weight of the carboxymethyl cellulose sodium used is 100,000; the activated carbon used is 100-mesh coconut shell activated carbon; the bentonite used is 250-mesh bentonite;

[0043] The formulation of trace elements used in the examples is as follows: 0.25 g of ferrous sulfate, 0.05 g / L of manganese sulfate, 0.05 g of zinc sulfate, 0.005 g of copper sulfate, 0.005 g of sodium molybdate, 0.001 g of cobalt chloride, and 1 L of deionized water.

[0044] Preparation Example 1

[0045] The preparation method of the wheat bran hydrolysate is as follows:

[0046] Add water with a weight three times that of the wheat bran after crushing to 40 mesh, boil for 10 min, cool to 50 °C, then add 2 wt% of the composite enzyme, enzymatically hydrolyze for 3 h, inactivate the enzyme at 100 °C for 10 min, and cool to room temperature to obtain the wheat bran hydrolysate.

[0047] The composite enzyme contains cellulase, glucoamylase, and phytase, and their usage amounts are 1.5 wt%, 0.4 wt%, and 0.1 wt% of the wheat bran respectively.

[0048] Preparation Example 2

[0049] The preparation method of the wheat bran hydrolysate is as follows:

[0050] Add water with a weight twice that of the wheat bran after crushing to 20 mesh, boil for 15 min, cool to 40 °C, then add 2.7 wt% of the composite enzyme, enzymatically hydrolyze for 2 h, inactivate the enzyme at 100 °C for 15 min, and cool to room temperature to obtain the wheat bran hydrolysate.

[0051] The composite enzyme contains cellulase, glucoamylase, and phytase, and their usage amounts are 2 wt%, 0.5 wt%, and 0.2 wt% of the wheat bran respectively.

[0052] Preparation Example 3

[0053] The preparation method of the wheat bran hydrolysate is as follows:

[0054] Add water with a weight four times that of the wheat bran after crushing to 60 mesh, boil for 20 min, cool to 45 °C, then add 0.65 wt% of the composite enzyme, enzymatically hydrolyze for 4 h, inactivate the enzyme at 100 °C for 20 min, and cool to room temperature to obtain the wheat bran hydrolysate.

[0055] The composite enzyme contains cellulase, glucoamylase, and phytase, and their usage amounts are 0.5 wt%, 0.1 wt%, and 0.05 wt% of the wheat bran respectively.

[0056] Preparation Example 4

[0057] The preparation method of the wheat bran hydrolysate is substantially the same as that of Preparation Example 1, except that phytase is not added to the composite enzyme.

[0058] Preparation Example 5

[0059] The domestication steps of Bacillus subtilis are as follows:

[0060] S1: Inoculate the activated Bacillus subtilis into an LB medium containing 30 mg / L imazamox, and culture it by shaking at 37 °C, pH 7, and 150 r / min for 5 days to obtain Bacillus subtilis in the stationary phase;

[0061] S2: Transfer the Bacillus subtilis in the stationary phase obtained in step S1 to an LB medium containing 40 mg / L imazamox and continue culturing under the same conditions. Passage culture is carried out 4 times in sequence, and the imazamox concentration for each passage culture is increased by 10 mg / L to obtain domesticated Bacillus subtilis.

[0062] Preparation Example 6

[0063] The domestication steps of Pseudomonas aeruginosa are as follows:

[0064] S1: Inoculate the activated Pseudomonas aeruginosa into King's B medium containing 30 mg / L imazamox, and culture it by shaking at 37 °C, pH 7, and 150 r / min for 5 days to obtain Pseudomonas aeruginosa in the stationary phase;

[0065] S2: Transfer the Pseudomonas aeruginosa in the stationary phase obtained in step S1 to King's B medium containing 40 mg / L imazamox and continue culturing under the same conditions. Passage culture is carried out 4 times in sequence, and the imazamox concentration for each passage culture is increased by 10 mg / L to obtain domesticated Pseudomonas aeruginosa.

[0066] Preparation Example 7

[0067] The domestication steps of Acinetobacter baumannii are as follows:

[0068] S1: Inoculate the activated Acinetobacter baumannii into MacConkey medium containing 30 mg / L imazamox, and culture it by shaking at 37 °C, pH 7, and 150 r / min for 5 days to obtain Acinetobacter baumannii in the stationary phase;

[0069] S2: Transfer the Acinetobacter baumannii in the stationary phase obtained in step S1 to MacConkey medium containing 40 mg / L imazamox and continue culturing under the same conditions. Passage culture is carried out 4 times in sequence, and the imazamox concentration for each passage culture is increased by 10 mg / L to obtain domesticated Acinetobacter baumannii.

[0070] Preparation Example 8

[0071] The domestication steps of Streptomyces are as follows:

[0072] S1: Inoculate the activated Streptomyces into Gause's No. 1 medium containing 20 mg / L imazamox, and culture it by shaking at 30 °C, pH 7, and 150 r / min for 7 days to obtain stationary-phase Streptomyces;

[0073] S2: Transfer the stationary-phase Streptomyces obtained in step S1 to Gause's No. 1 medium containing 30 mg / L imazamox and continue culturing under the same conditions. Subculture it 5 times in sequence, with the imazamox concentration increased by 10 mg / L each time during subculture, to obtain domesticated Streptomyces.

[0074] Preparation Example 9

[0075] The domestication steps of Aspergillus niger are as follows:

[0076] S1: Inoculate the activated Aspergillus niger into PDA medium containing 20 mg / L imazamox, and culture it statically at 28 °C and pH 6 for 7 days to obtain stationary-phase Aspergillus niger;

[0077] S2: Transfer the stationary-phase Aspergillus niger obtained in step S1 to PDA medium containing 30 mg / L imazamox and continue culturing under the same conditions. Subculture it 5 times in sequence, with the imazamox concentration increased by 10 mg / L each time, to obtain domesticated Aspergillus niger.

[0078] Preparation Example 10

[0079] Centrifugally separate the domesticated Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Streptomyces, and Aspergillus niger obtained in Preparation Examples 5 - 9 respectively, wash and dilute the obtained thalli with sterile water to obtain Bacillus subtilis suspension, Pseudomonas aeruginosa suspension, Acinetobacter baumannii suspension, Streptomyces suspension, and Aspergillus niger suspension with a concentration of 1×10 10 CFU / mL.

[0080] Example 1

[0081] A preparation method of a microbial complex bactericide for degrading herbicides includes the following preparation steps:

[0082] (1) By mass, mix 3 parts of bran hydrolysate, 5 parts of peat soil, and 4 parts of diatomaceous earth, sterilize at 120 °C for 20 min, and in a sterile environment, add a complex microbial community suspension and a 10 wt% protective agent, mix evenly, knead into a shape, and make it into thin slices (the concentration of the complex microbial community suspension in the thin slices is 5×10 10 CFU / g), dry at low temperature, and crush to 0.5 mm to obtain a microbial bactericide;

[0083] (2) Mix 100 parts by mass of the microbial inoculant from step (1) with 30 parts of bentonite, 1 part of Tween 80, and 3.5 parts of nutrients (including 2 parts of glucose, 1 part of urea, and 0.1 part of trace elements) evenly to obtain a microbial compound inoculant.

[0084] The bran hydrolysate used is obtained from Preparation Example 1.

[0085] The protective agent used is cyclodextrin, trehalose, sodium carboxymethyl cellulose, and glycerol with a mass ratio of 4:5:2:3.

[0086] The compound microbial suspension used contains domesticated Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Streptomyces, and Aspergillus niger with a quantity ratio of 2.5:2:1.5:2.5:2.

[0087] Example 2

[0088] A preparation method of a microbial compound inoculant for degrading herbicides, comprising the following preparation steps:

[0089] (1) Mix 3 parts of bran hydrolysate, 3 parts of peat soil, and 2 parts of diatomaceous earth by mass, sterilize at 120 °C for 20 min, add the compound microbial suspension and 5 wt% of the protective agent in a sterile environment, mix evenly, knead and form into a thin sheet (the concentration of the compound microbial suspension in the thin sheet is 1×10 9 CFU / g), dry at low temperature, and crush to 0.1 mm to obtain a microbial inoculant;

[0090] (2) Mix 100 parts by mass of the microbial inoculant from step (1) with 20 parts of bentonite, 0.5 part of Span 60, and 2.2 parts of nutrients (including 1.5 parts of glucose, 0.5 part of urea, and 0.2 part of trace elements) evenly to obtain a microbial compound inoculant.

[0091] The bran hydrolysate used is obtained from Preparation Example 2.

[0092] The protective agent used is cyclodextrin, trehalose, sodium carboxymethyl cellulose, and glycerol with a mass ratio of 2:3:2:2.

[0093] The compound microbial suspension used contains domesticated Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Streptomyces, and Aspergillus niger with a quantity ratio of 2:1.5:1:2:2.

[0094] Example 3

[0095] A preparation method of a microbial compound inoculant for degrading herbicides, comprising the following preparation steps:

[0096] (1) Mix 3 parts of wheat bran hydrolysate, 6 parts of peat soil, and 5 parts of diatomaceous earth by mass. Sterilize at 120 °C for 20 min. In a sterile environment, add a complex microbial community suspension and 15 wt% of a protective agent, mix evenly, knead into a shape, and make into thin slices (the concentration of the complex microbial community suspension in the thin slices is 1×10 11 CFU / g). Dry at low temperature and crush to 1 mm to obtain a microbial inoculant;

[0097] (2) Mix 100 parts of the microbial inoculant from step (1) with 40 parts of activated carbon, 2 parts of sodium dodecylbenzenesulfonate, and 4.3 parts of nutrients (including 2.5 parts of glucose, 1.5 parts of urea, and 0.3 parts of trace elements) by mass to obtain a composite microbial inoculant.

[0098] The wheat bran hydrolysate used is obtained from Preparation Example 3.

[0099] The protective agent used is cyclodextrin, trehalose, sodium carboxymethylcellulose, and glycerol with a mass ratio of 5:6:2:4.

[0100] The complex microbial community suspension used contains domesticated Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Streptomyces, and Aspergillus niger with a quantitative ratio of 3:2.5:2:3:2.

[0101] Example 4

[0102] A preparation method of a composite microbial inoculant for degrading herbicides, the specific implementation method is substantially the same as that of Example 1, the difference is that the wheat bran hydrolysate used is obtained from Preparation Example 4.

[0103] Example 5

[0104] A preparation method of a composite microbial inoculant for degrading herbicides, the specific implementation method is substantially the same as that of Example 1, the difference is that trehalose is not added to the protective agent used.

[0105] Example 6

[0106] A preparation method of a composite microbial inoculant for degrading herbicides, the specific implementation method is substantially the same as that of Example 1, the difference is that glycerol is not added to the protective agent used.

[0107] Example 7

[0108] A preparation method of a composite microbial inoculant for degrading herbicides, the specific implementation method is substantially the same as that of Example 1, the difference is that Acinetobacter baumannii is not added to the complex microbial community suspension used.

[0109] Example 8

[0110] A preparation method of a microbial complex agent for degrading herbicides, the specific implementation manner is substantially the same as that of Example 1, except that Aspergillus niger is not added to the complex microbial suspension used.

[0111] Example 9

[0112] A preparation method of a microbial complex agent for degrading herbicides, the specific implementation manner is substantially the same as that of Example 1, except that Streptomyces is not added to the complex microbial suspension used.

[0113] Control Example 1

[0114] A preparation method of a microbial complex agent for degrading herbicides, the specific implementation manner is substantially the same as that of Example 1, except that a protective agent is not added in step (1).

[0115] Control Example 2

[0116] A preparation method of a microbial complex agent for degrading herbicides, the specific implementation manner is substantially the same as that of Example 1, except that Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Streptomyces, and Aspergillus niger in the complex microbial suspension used are not domesticated.

[0117] Control Example 3

[0118] A preparation method of a microbial complex agent for degrading herbicides, the specific implementation manner is substantially the same as that of Example 1, except that bentonite is not added in step (2).

[0119] Performance Test

[0120] 1. Water body experiment

[0121] The microbial complex agents obtained in each example and control example were added to 20 mL of an inorganic salt medium with a final concentration of 100 mg / L of imazamox at an addition amount of 2 wt%, and cultured at 30 °C and 150 r / min for 7 d. A blank control group and a control group were set up (the complex microbial suspension used in Example 1 was inoculated into 20 mL of an inorganic salt medium with a final concentration of 100 mg / L of imazamox at an inoculation amount of 2 wt%, and then 10 wt% of the protective agent used in Example 1 was added), and both were cultured at 30 °C and 150 r / min for 7 d. The concentration of imazamox was measured by HPLC, and the degradation rate of imazamox in each group was calculated. Three parallel groups were set up for each group, and the average value was taken. The specific test data are shown in Table 1.

[0122] Table 1

[0123]

[0124]

[0125] As can be seen from Table 1, in the blank control group, the degradation rate of imazamox is very low; in contrast, the microbial complex agents prepared in Examples 1-3 have excellent degradation effects on imazamox. Compared with Example 1, the preparation method of the wheat bran hydrolysate in Example 4 is changed, which may reduce the nutrients provided for the strains, resulting in a decrease in the degradation rate of imazamox by the strains; in Examples 5 and 6, the components of the protective agent used are changed, the protective effect on the strains is weakened, and the environmental harm is increased, resulting in a decrease in the degradation rate of imazamox by the strains; in Examples 7, 8, and 9, the components of the complex bacteria used are changed, resulting in a decrease in the synergistic effect between the strains and a decrease in the degradation rate of imazamox; in Comparative Examples 1 and 3, the protective agent and bentonite are missing respectively, and in Comparative Example 2, the strains used are not domesticated, all of which result in a significant decrease in the degradation rate of imazamox by the microbial community.

[0126] 2. Soil experiment

[0127] Imazamox was added to the test soil to make its content 100 mg / kg. After thorough mixing, it was left standing at room temperature for 1 day. The soil water content was adjusted to 25%, and 5 wt% of the microbial complex agent of Example 1 was added. After thorough mixing, the experiment was carried out (alternately at 30 °C, in a light environment for 12 h and at 20 °C, in a dark environment for 12 h). The residual amount of imazamox in the soil was tested by HPLC on the 7th day. At the same time, a blank control group without the microbial complex agent was set up, and 3 parallel groups were set for each treatment group, and the average value was taken. The specific test data are shown in Table 2.

[0128] Table 2

[0129]

[0130] As can be seen from Table 2, the microbial complex agent prepared in Example 1 also has excellent degradation effects on imazamox in the soil degradation experiment.

[0131] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on this application. Although this application is disclosed as above with preferred embodiments, it is not intended to limit this application. Any person skilled in the art, without departing from the scope of the technical solution of this application, making some changes or modifications using the technical content disclosed above is equivalent to equivalent implementation cases. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution.

Claims

1. A preparation method of a microbial complex agent for degrading herbicides, characterized in that, It includes the following preparation steps: (1) Mix wheat bran hydrolysate, peat soil, and diatomaceous earth, sterilize, and in a sterile environment, add a protective agent and a complex microbial community suspension, mix evenly, knead into a shape, make into thin slices, air-dry naturally, and crush to obtain a microbial inoculant; (2) Mix the microbial inoculant in step (1) evenly with an adsorbent, a surfactant, and nutrients to obtain a composite microbial inoculant.

2. The preparation method of the microbial complex bacterial agent according to claim 1, characterized in that, The mass ratio of the wheat bran hydrolysate, peat soil, and diatomaceous earth is 3:(3 - 6):(2 - 5).

3. The preparation method of the microbial complex bacterial agent according to claim 1, wherein The preparation method of the wheat bran hydrolysate is as follows: Add water to the crushed wheat bran, boil and cool, then add a complex enzyme, enzymolyze for 2 - 4 h, inactivate the enzyme, and cool to obtain the wheat bran hydrolysate.

4. The preparation method of the microbial complex bacterial agent according to claim 1, wherein, The complex enzyme includes cellulase, glucoamylase, and phytase; the usage amount of the complex enzyme is 0.5 - 3 wt% of the wheat bran.

5. The preparation method of the microbial complex bacterium agent according to claim 1, characterized in that The protective agent includes cyclodextrin, trehalose, sodium carboxymethylcellulose, and glycerol with a mass ratio of (2 - 5):(3 - 6):2:(2 - 4); the usage amount of the protective agent is 5 - 15 wt% of the complex microbial community suspension.

6. The preparation method of the microbial complex bacterial agent according to claim 1, characterized in that, The complex microbial community suspension includes Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Streptomyces, and Aspergillus niger with a quantity ratio of (2 - 3):(1.5 - 2.5):(1 - 2):(2 - 3):

2.

7. The preparation method of the microbial complex bacterial agent according to claim 6, wherein The concentration of the strains in the composite microbial suspension in the thin slice is (1-10)×10 9-10 CFU / g.

8. The preparation method of the microbial complex bacterium agent according to claim 1, wherein The adsorbent is selected from activated carbon and / or bentonite.

9. The preparation method of the microbial complex bacterial agent according to claim 1, wherein, The surfactant is selected from one or more of Tween, Span, alkylbenzene sulfonate, and alkyl sulfate.

10. A composite microbial inoculant prepared by the preparation method of the composite microbial inoculant according to any one of claims 1 - 9.

Images