CN114027322A - Biochar-based bacillus thuringiensis sustained-release microspheres as well as preparation method and application thereof - Google Patents
Biochar-based bacillus thuringiensis sustained-release microspheres as well as preparation method and application thereof Download PDFInfo
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- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/26—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests in coated particulate form
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Abstract
The invention discloses a biochar-based bacillus thuringiensis sustained-release microsphere as well as a preparation method and application thereof, wherein the preparation method comprises the following steps: adding biochar and bacillus thuringiensis raw powder into distilled water, shaking, standing, centrifuging, performing normal-temperature vacuum drying and crushing on the precipitate to obtain biochar powder adsorbed and fixed with bacillus thuringiensis, uniformly mixing the biochar powder with a sodium alginate solution, dripping the obtained medicine mixed suspension into a cross-linking agent solution, performing cross-linking solidification, filtering and separating to obtain biochar-based bacillus thuringiensis/sodium alginate microspheres; and transferring the obtained microspheres into a chitosan acetic acid solution for film covering, washing and drying in vacuum to obtain the biochar-based bacillus thuringiensis slow-release microspheres. The microsphere can remarkably improve the embedding rate of the bacillus thuringiensis microsphere, the survival rate of the bacillus thuringiensis, the stability of the pesticide effect and the environmental adaptability, thereby effectively prolonging the pesticide effect duration and meeting the requirement of green agricultural development.
Description
Technical Field
The invention relates to the field of biopesticides, and in particular relates to a biochar-based bacillus thuringiensis slow-release microsphere as well as a preparation method and application thereof.
Background
Bacillus thuringiensis (Bt) is a microbial pesticide which is most researched and widely applied in the world, is safe to people and livestock and does not destroy ecological balance, and in the process of forming spores, parasporal crystals (Parasporil crystals) with insecticidal activity can be formed, and the parasporal crystals consist of one or more crystal proteins with high specific insecticidal activity, namely Insecticidal Crystal Proteins (ICPs) or delta-endotoxins.
Although Bt has obvious advantages as a biological insecticide, Bt has the defects of short lasting period, easy control effect by external climate and ecological conditions such as sunlight, temperature, rainwater and the like due to the fact that the main insecticidal component of Bt is insecticidal crystal protein and the capacities of resisting ultraviolet rays and adverse environmental conditions are poor, the field application lasting period is a bottleneck for restricting the development of the insecticide, and how to improve the insecticidal effect is a common problem faced by related research institutions and production enterprises.
At present, the main dosage forms of Bt comprise powder, wettable powder, suspending agent, oil emulsion, granules, tablets and the like, but the dosage forms do not completely solve the problems. Although many studies have been made on Bt microcapsules at home and abroad, the Bt microcapsules are mainly produced by using natural materials such as gelatin, starch, cellulose, gum arabic, poly gamma-glutamic acid (gamma-PGA) and the like as wall materials, but still have the problems of stability and environmental suitability of biological preparations. Therefore, the existing Bt dosage forms cannot meet the requirements of field application on prolonging the lasting period, stable control effect and high tolerance to external conditions such as ultraviolet and the like, and the improvement of the existing dosage forms is urgently needed.
Biochar is a product obtained by pyrolyzing organic substances under the condition of low temperature and oxygen deficiency, mainly comprises aromatic hydrocarbon and simple substance carbon or carbon with a graphite structure, contains more than 60 percent of carbon element, is widely concerned by scholars at home and abroad in recent years in the aspects of slow release carrier of fertilizer, soil conditioner, pollutant remediation, carbon emission reduction and the like, has developed porosity and large specific surface area, and can be used as an excellent functional microbial carrier. At present, the method for realizing the continuous degradation of environmental pollutants by using biochar to fix degradation bacteria has been researched. The animal-derived biochar is prepared from animal bone meal serving as a main raw material, not only has a typical plant-derived biochar component, but also contains a large amount of nitrogen-containing groups and inorganic mineral component hydroxyapatite, and after the animal-derived biochar is prepared into nano biochar, the dispersing capacity, the adsorption capacity and the fixing capacity of the nano biochar are greatly improved.
Aiming at the defects of the existing Bt bacterial preparation, the invention prepares the biochar-based Bacillus thuringiensis microspheres by introducing animal-derived nano biochar to adsorb and fix Bacillus thuringiensis and further embedding and laminating by utilizing natural polymer materials such as sodium alginate, chitosan and the like, improves the ultraviolet resistance of the Bt biological pesticide preparation, improves the prevention and treatment effect and lays a foundation for the application and popularization of the Bt bacterial preparation.
Disclosure of Invention
Aiming at the problems of unstable drug effect and poor environmental adaptability of the traditional bacillus thuringiensis preparation at present, the invention provides a biochar-based bacillus thuringiensis microsphere as well as a preparation method and application thereof.
In order to solve the above problems, the scheme provided by the present application is specifically as follows:
in a first aspect, the present application further provides a preparation method of the biochar-based bacillus thuringiensis sustained-release microspheres, which comprises the following steps:
(1) adding biochar and bacillus thuringiensis raw powder into sterilized distilled water, shaking for 2-3 hours by a shaking table, standing, performing centrifugal separation, performing normal-temperature vacuum drying on the precipitate obtained by centrifugation, and crushing to obtain biochar powder adsorbed and fixed with bacillus thuringiensis; wherein the total mass concentration of the biochar and the bacillus thuringiensis is 30-100 g/L;
(2) uniformly mixing the charcoal powder adsorbed and fixed with the bacillus thuringiensis with a sodium alginate solution with the concentration of 10-30 g/L to obtain a medicine mixed suspension;
(3) dripping the obtained medicine mixed suspension into a calcium chloride solution with the concentration of 10-40 g/L, performing crosslinking and curing for 2-3h, filtering and separating to obtain a biochar-based bacillus thuringiensis/sodium alginate microsphere;
(4) and transferring the obtained bacillus thuringiensis/sodium alginate microspheres into a chitosan acetic acid solution with the concentration of 1-10 g/L for film covering, filtering, separating, washing, and drying at normal temperature in vacuum to obtain the biochar-based bacillus thuringiensis microspheres. Preferably, the film covering time is 0.5-1 h.
Preferably, in the step (2), the mass ratio of the biochar combined with the bacillus thuringiensis to the sodium alginate in the medicine mixed suspension is 1 (3-7.5).
Preferably, the cross-linking agent is calcium chloride; chitosan is used as a film covering agent; further preferably, the coating agent is a chitosan acetic acid solution.
Alternatively, the Bacillus thuringiensis strain B-Y7-1, other Bacillus thuringiensis may be used in other embodiments.
Preferably, in the biochar-based bacillus thuringiensis slow-release microspheres, the bacillus thuringiensis raw powder comprises spores and insecticidal crystal protein.
Preferably, in the biochar-based bacillus thuringiensis slow-release microspheres, the adsorption fixer biochar is nano biochar prepared by taking animal bone meal as a raw material. More preferably, the adsorption fixer biochar is obtained by carbonizing animal bone powder serving as a raw material at 500 ℃ for 2 hours under an anoxic condition and then grinding the carbonized animal bone powder for 12 hours by using a ball mill.
Optionally, in the biochar-based bacillus thuringiensis slow-release microspheres, the mass ratio of the biochar to the bacillus thuringiensis raw powder is 1: 1-3: 1. Preferably, the mass ratio of the biochar to the bacillus thuringiensis raw powder is 2:1, and the biochar-based bacillus thuringiensis microspheres prepared under the condition are excellent in performance in all aspects.
In a second aspect, the invention provides a biochar-based bacillus thuringiensis microsphere which is mainly prepared from the following components: the bacillus thuringiensis powder is prepared from bacillus thuringiensis raw powder, biochar, sodium alginate, calcium chloride and chitosan by the preparation method.
In the microspheres, the bacillus thuringiensis raw powder is used as an active ingredient, the charcoal is used as an adsorption fixing agent, the sodium alginate and calcium chloride are crosslinked and calcified to be used as a carrier, and the bacillus thuringiensis biological agent prepared by coating chitosan can obviously improve the embedding rate of the bacillus thuringiensis microspheres, the survival rate of bacillus thuringiensis, the pharmacodynamic stability and the environmental adaptability, thereby effectively prolonging the pharmacodynamic lasting period.
Through detection, the embedding rate of the prepared biochar-based bacillus thuringiensis slow-release microspheres is over 85 percent, the particle size is 1.0-1.5 mm, and the uvioresistant performance of the prepared biological agent is improved.
In a third aspect, the application also provides the application of the biochar-based bacillus thuringiensis slow-release microspheres as a biological insecticide.
The invention has the following beneficial effects:
the biochar-based bacillus thuringiensis slow-release microspheres have the advantages of few raw materials in the formula, simple preparation method, stable drug effect and long lasting period, and are easy to industrially produce, popularize and apply. The novel environment-friendly bacillus thuringiensis is used as a model drug, animal-derived nano biochar is used as an adsorption fixing agent, sodium alginate and calcium chloride are used as carriers for cross-linking and calcification, and the biological agent prepared by chitosan film covering can obviously improve the drug effect stability and environmental adaptability of the biochar-based bacillus thuringiensis microsphere and prolong the drug effect duration of the biochar-based bacillus thuringiensis microsphere.
Drawings
FIG. 1 is an external view of a Bacillus thuringiensis microsphere (FIG. B) prepared with biochar of animal origin (FIG. A) and without biochar;
FIG. 2 is an electron micrograph of Bacillus thuringiensis microspheres prepared with biochar of animal origin (FIG. A) and without biochar (FIG. B).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In the present invention, the equipment and materials used are commercially available or commonly used in the art, if not specified. The methods in the following examples are conventional in the art unless otherwise specified.
Experimental materials:
animal-derived nano biochar: using commercially available animal bone powder as raw material, carbonizing at 500 deg.C for 2 hr in a carbonization furnace, and further grinding with ball mill for 12 hr. The grain diameter is nano-scale.
Bacillus thuringiensis raw powder: comprises spores and insecticidal crystal protein, and has a bacterial content of 3 × 1010cfu/g, the bacillus thuringiensis is a bacillus thuringiensis strain B-Y7-1 provided by plant medical college of Qingdao agricultural university, and the preservation number is as follows: CCTCC No. m2011267, has been disclosed in patent application No. 201711256177.8.
Bacillus thuringiensis wettable powder (8000 IU/mg) was purchased from Qingdao Taiyuan scientific and technological development Co., Ltd.
Example 1
The embodiment provides a biochar-based bacillus thuringiensis slow-release microsphere, and the preparation method comprises the following steps:
(1) adding animal-derived nano biochar and bacillus thuringiensis raw powder into sterilized distilled water according to the mass ratio of 1:1, keeping the total mass concentration of the animal-derived nano biochar and the bacillus thuringiensis raw powder to be 50g/L, fully mixing, shaking for 2 hours at 160r/min by a shaking table, standing, centrifugally separating a biochar layer, and carrying out vacuum drying and crushing to obtain the charcoal powder for adsorbing and fixing bacillus thuringiensis and insecticidal crystal protein.
(2) Adding 0.4g of charcoal powder adsorbing and fixing the bacillus thuringiensis in the step (1) into 100mL of prepared sodium alginate solution of 30g/L, and uniformly shearing and mixing at low speed by a shearing machine to prepare a suspension of the charcoal and the sodium alginate.
(3) And (3) dripping the suspension obtained in the step (2) into 100mL of prepared 10g/L calcium chloride solution at a constant speed by adopting an orifice method, crosslinking and solidifying for 2h, and separating the bacillus thuringiensis/sodium alginate microspheres by precipitation and filtration.
(4) And (3) transferring the bacillus thuringiensis/sodium alginate microspheres obtained in the step (3) into 100mL of prepared 1g/L chitosan acetic acid solution, slightly stirring, coating for 0.5h, filtering, separating and washing the microspheres, and drying in vacuum to obtain a biochar-based bacillus thuringiensis microsphere product.
Example 2
The embodiment provides a biochar-based bacillus thuringiensis slow-release microsphere, and the preparation method comprises the following steps:
(1) adding animal-derived nano biochar and bacillus thuringiensis raw powder into sterilized distilled water according to the mass ratio of 2:1, keeping the mass concentration of the animal-derived nano biochar and the bacillus thuringiensis raw powder at 50g/L, fully mixing, shaking for 2 hours at 160r/min by a shaking table, standing, centrifugally separating to obtain a biochar layer, and carrying out vacuum drying and crushing to obtain the biochar powder for adsorbing and fixing bacillus thuringiensis and insecticidal crystal protein.
(2) Adding 0.6g of charcoal powder adsorbing and fixing the bacillus thuringiensis in the step (1) into 100mL of prepared 20g/L sodium alginate solution, and uniformly shearing and mixing at low speed by a shearing machine to prepare a suspension of the charcoal and the sodium alginate.
(3) And (3) dripping the suspension obtained in the step (2) into 100mL of prepared 20g/L calcium chloride solution at a constant speed by adopting an orifice method, crosslinking and solidifying for 2h, and separating the bacillus thuringiensis/sodium alginate microspheres by precipitation and filtration.
(4) And (3) transferring the bacillus thuringiensis/sodium alginate microspheres obtained in the step (3) into 100mL of prepared 1g/L chitosan acetic acid solution, slightly stirring, coating for 0.5h, filtering, separating and washing the microspheres, and drying in vacuum to obtain a biochar-based bacillus thuringiensis microsphere product.
Example 3
The embodiment provides a biochar-based bacillus thuringiensis slow-release microsphere, the mass ratio of the animal-derived nano biochar to the bacillus thuringiensis is 3:1, and the preparation method is the same as that in embodiment 1.
Comparative example 1
Preparing the plant source biochar: selecting walnut shells as raw materials, crushing, putting the walnut shells into a crucible for compaction, heating the walnut shells to 500 ℃ in a muffle furnace at the heating rate of 10 ℃/min, keeping the temperature at a constant temperature for 2 hours, cooling, taking out the walnut shells, washing the walnut shells by deionized water to be neutral, thus obtaining the plant-source biochar, and further preparing the plant-source biochar-based bacillus thuringiensis microspheres according to test example 1.
Comparative example 2
Compared with example 1, the raw material of the bacillus thuringiensis microsphere prepared by the comparative example omits the component of biochar, other components are the same, and the preparation method is also referred to the content of example 1 for preparation.
Example 4 characterization of biochar-based Bacillus thuringiensis microspheres and determination of stability
1. Experimental methods and procedures
(1) The method for measuring the embedding rate (EE%) of the microspheres comprises the following steps: taking 1mL of original bacterial liquid, diluting, counting and culturing in PCA culture medium, and recording as N0. Taking a microsphere sample prepared by the same bacterial quantity, adding a proper amount of sterilized sodium citrate solution with the mass fraction of 3.8%,performing ultrasonic treatment for 10min to break and dissolve microsphere, taking clear liquid to PCA culture medium for counting culture, and recording as N1. The embedding rate was calculated according to equation 1.
(2) The method for measuring the ultraviolet resistance comprises the following steps:
bt microsphere products with the same bacterial content and untreated Bt bacterial suspension are respectively paved in a sterile culture dish and irradiated under an ultraviolet lamp (30W, 20cm) for 30 min. And (3) breaking the wall of each test sample before and after irradiation, diluting and culturing in a PCA culture medium, recording the colony count, and calculating the survival rate.
(3) Method for measuring particle size of microspheres
The prepared microspheres are taken at random, 50 particles are measured by a vernier caliper, and the average value is taken.
2. Results and analysis of the experiments
The relevant performance indexes of the bacillus thuringiensis sustained-release microspheres are measured and shown in table 1.
TABLE 1 comparison of the Performance of biochar-based Bacillus thuringiensis microspheres with Bt inoculants
| Sample (I) | Particle size (mm) | Embedding Rate (%) | Survival rate (%) |
| Example 1 | 1.52 | 87.4 | 60.2 |
| Example 2 | 1.64 | 91.3 | 64.7 |
| Example 3 | 1.75 | 90.6 | 62.3 |
| Comparative example 1 | 1.66 | 84.3 | 51.8 |
| Comparative example 2 | 1.41 | 80.2 | 47.5 |
| Bt bacterial liquid | - | - | 5.31 |
As can be seen from Table 1, the particle size of the microspheres can be increased after the biochar is added, and the embedding rate of the biochar also has an influence on the embedding rate of the Bacillus thuringiensis, wherein the highest embedding rate in example 2 is up to 91.3 percent, which is higher than that of biochar of plant source and microspheres without biochar. The survival rate result shows that the ultraviolet resistance of the bacillus thuringiensis can be improved by carrying out the microsphericization on the bacillus thuringiensis, and the ultraviolet resistance of the bacillus thuringiensis can be further improved by adding the biochar. The survival rate of the bacillus thuringiensis after ultraviolet irradiation treatment shows that the animal source biochar is superior to the plant source biochar and the microspheres without biochar, wherein the survival rate of the embodiment 2 is the highest and reaches 64.7 percent.
Example 5 pharmacodynamic test of biochar-based Bacillus thuringiensis sustained-release microspheres
1. Experimental methods and procedures
Firstly, the biochar-based bacillus thuringiensis microspheres prepared in example 2 are subjected to a drug effect test on grubs in a field, and meanwhile, a control test is carried out by taking commercially available bacillus thuringiensis wettable powder as a control medicament. The specific experimental method is as follows:
the experimental field is peanut field of Shandong province, Pingdu city. The soil is sandy loam, the fertility is moderate, the biochar-based bacillus thuringiensis microspheres (prepared in example 2) and bacillus thuringiensis wettable powder prepared by the method are respectively used for treating the soil before peanut sowing, and the dosage of the agents is 3000g/hm2。
Secondly, referring to GB/T17980.72-2004 part 72 of pesticide field efficacy test criterion (II): the insecticide is used for controlling the soil insects in the dry land, 3 times of insect population investigation are carried out, and the investigation is carried out 1 time respectively at 10d, 20d and 40d after the insecticide is applied. Sampling at 5 points in each cell, each point 50cm x 50cm (0.25 m)2) The depth of the dug soil is about 30 cm. During the test, the peanut plants are observed whether to lose green and wilting after the peanut plants are applied, if the peanut plants do not have the symptoms, the peanut plants do not have phytotoxicity, and the test results are shown in table 2.
TABLE 2B-Y7-1 slow-release microsphere field drug effect experiment effect for controlling peanut grubs
2. Results and analysis of the experiments
The results in table 1 show that, compared with the existing Bt wettable powder, the bacillus thuringiensis slow release microspheres have better persistence, the average prevention and control effect after being applied for 40 days reaches 85.72%, and the bacillus thuringiensis slow release microspheres are safe to peanuts.
It should be understood that the technical solutions and concepts of the present invention may be equally replaced or changed by those skilled in the art, and all such changes or substitutions should fall within the protection scope of the appended claims.
Claims (8)
1. A preparation method of a biochar-based bacillus thuringiensis slow-release microsphere is characterized by comprising the following steps:
(1) adding biochar and bacillus thuringiensis raw powder into sterilized distilled water respectively, shaking, standing, centrifuging, drying the precipitate obtained by centrifuging at normal temperature in vacuum, and pulverizing to obtain biochar powder adsorbed and fixed with bacillus thuringiensis;
(2) uniformly mixing the charcoal powder adsorbed and fixed with the bacillus thuringiensis with a sodium alginate solution with the mass fraction of 10-30 g/L to obtain a medicine mixed suspension;
(3) dripping the obtained medicine mixed suspension into a crosslinking agent solution with the concentration of 10-40 g/L, crosslinking and solidifying, filtering and separating to obtain the biochar-based bacillus thuringiensis/sodium alginate microspheres;
(4) and (4) transferring the microspheres obtained in the step (3) into a chitosan acetic acid solution with the concentration of 1-10 g/L for film covering, filtering, separating, washing, and drying at normal temperature in vacuum to obtain the biochar-based bacillus thuringiensis slow-release microspheres.
2. The biochar-based bacillus thuringiensis sustained release microspheres of claim 1, wherein the bacillus thuringiensis raw powder comprises spores and insecticidal crystal protein.
3. The biochar-based bacillus thuringiensis slow-release microspheres according to claim 1, wherein the biochar is nano-biochar prepared from animal bone meal.
4. The biochar-based bacillus thuringiensis slow-release microspheres according to claim 1, wherein the mass ratio of biochar to bacillus thuringiensis raw powder is 1: 1-3: 1.
5. The biochar-based bacillus thuringiensis sustained release microspheres of claim 1, wherein the cross-linking agent is calcium chloride.
6. A biochar-based Bacillus thuringiensis sustained-release microsphere, which is prepared by the preparation method of any one of claims 1 to 5.
7. A biological agent characterized by comprising the biochar-based Bacillus thuringiensis sustained-release microspheres according to claim 1 as an active ingredient.
8. The use of the biochar-based bacillus thuringiensis slow release microspheres according to claim 6 as a biopesticide.
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| GB2617905A (en) * | 2022-02-25 | 2023-10-25 | Doff Portland Ltd | Mollusc barrier |
| CN117126011A (en) * | 2023-08-30 | 2023-11-28 | 临沂市农业科学院 | Coated composite microbial agent and preparation method thereof |
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