CN112921026B - Granular composite microbial agent and preparation method thereof - Google Patents

Granular composite microbial agent and preparation method thereof Download PDF

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CN112921026B
CN112921026B CN202110180697.5A CN202110180697A CN112921026B CN 112921026 B CN112921026 B CN 112921026B CN 202110180697 A CN202110180697 A CN 202110180697A CN 112921026 B CN112921026 B CN 112921026B
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CN112921026A (en
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徐坚麟
付源
王俊滔
向粤琴
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Hangzhou Nanda Environmental Protection Technology Co Ltd
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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    • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
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Abstract

The invention discloses a particle type composite microbial agent and a preparation method thereof, belonging to the field of microbial agent preparation, and comprising the following steps: a particle carrier, wherein the surface of the carrier comprises a cross-linked polymer, and the cross-linked polymer is formed by connecting PVA and XG through a cross-linking agent; and a composite microorganism and a functional agent supported on the particulate carrier; the composite microorganism and the functional agent are coated on the particle carrier in a mixed form. The invention adopts the high-density microorganism prepared by fermenting the microorganism with the additive containing the corn starch malate, adopts the particle carrier obtained by crosslinking PVA and XG on the particle mixture, adopts the functional agent containing anthocyanin rhamnoside and dextrin palmitate, and prepares the particle type composite microorganism agent together.

Description

Granular composite microbial agent and preparation method thereof
Technical Field
The invention belongs to the field of microbial agent preparation, and in particular relates to a granular composite microbial agent and a preparation method thereof.
Background
In the use process of the microorganism, the single strain has limited functions, and the compound microbial agent can be developed and optimized by utilizing the mutual cooperation among different types of microorganism, so that the use effect is improved. The stability and the effect of the microbial agent are very important in popularization and application, the yield and the activity are improved in the aspect of fermentation production, the preparation process is optimized in the aspect of product post-treatment, and the stability in use is improved.
The carrier materials of microbial agents can be classified into inorganic carrier materials, natural organic carrier materials, synthetic polymer carrier materials and composite carrier materials according to properties. The inorganic carrier material has the characteristics of high mechanical strength, good chemical, thermal and biological stability, wide material source, low cost, good mass transfer performance, environmental friendliness, no toxicity to cells and the like, and is widely applied. Common microorganism immobilized natural inorganic carrier materials mainly comprise sand grains, zeolite, vermiculite, swelling/diatomite and the like; the inorganic materials for synthesis processing mainly comprise active carbon, porous ceramics, biomass carbon, porous glass, foam metal, silica gel and the like. The synthetic polymer material can regulate and control the microcosmic appearance, specific surface area and other characteristics of the material in design, thereby meeting the requirements of different immobilized microorganisms in practical application and obtaining better microorganism immobilization effect and pollutant degradation efficiency. Common synthetic polymeric carrier materials include polyurethane foam, polyvinyl alcohol, polypropylene fibers, polyacrylamide, sponge, polyvinylpyrrolidone, and the like.
In the process of preparing the microbial agent by the microbial bacteria and the carrier, the adsorbed microorganisms are easy to leak due to unstable weak acting force between the microorganisms and the carrier material, so that the improvement of the stability of the microbial agent is of great practical significance.
Disclosure of Invention
The invention aims to provide a granular composite microbial agent with high water removal rate, high survival rate of dried microbial bacteria and good acid resistance.
The technical scheme adopted by the invention for achieving the purpose is as follows:
a granular composite microbial agent comprising:
a particle carrier, wherein the surface of the carrier comprises a cross-linked polymer, and the cross-linked polymer is formed by connecting PVA and XG (xanthan gum) through a cross-linking agent; the method comprises the steps of,
composite microorganism bacteria and functional agents loaded on the particle carrier;
the composite microorganism and the functional agent are coated on the particle carrier in a mixed form.
Preferably, the mixed form of the compound microorganism and the functional agent plays a role in protecting the compound microorganism.
Preferably, the crosslinking agent is epichlorohydrin.
Preferably, the particle carrier comprises at least one of an inorganic material and a biological material inside.
More preferably, the biological material is at least one of corn flour, straw stalk powder, corn stalk powder and rice bran.
Preferably, the functional agent is at least one of maltose, calcium carbonate, soluble starch, gelatin, maltodextrin, polyvinylpyrrolidone and glycerol.
More preferably, the inorganic material is at least one of perlite, fine clay, bentonite, diatomaceous earth, vermiculite powder and yellow cotton soil.
More preferably, the fineness of the inorganic material is not less than 100 meshes.
The invention aims to provide a preparation method of a granular composite microbial agent with high microbial growth activity, quick microbial propagation time, high water removal rate, high microbial survival rate after drying and good acid resistance.
The technical scheme adopted by the invention for achieving the purpose is as follows:
a preparation method of a granular composite microbial agent comprises the following steps: the composite microbial bacteria liquid is prepared by fermentation culture, the composite microbial bacteria liquid and the functional agent are mixed to prepare composite microbial bacteria slurry, and the composite microbial bacteria slurry and the particle carrier are mixed to prepare the particle type composite microbial bacteria agent.
Preferably, in the preparation of the compound microorganism bacterial liquid, the basic culture medium is mixed with the additive, the compound microorganism bacterial is inoculated, the inoculation amount is 2-6wt%, the constant temperature culture is carried out for 12-48 hours at 15-37 ℃, and the solid content of the compound microorganism in the high-density fermentation liquid is adjusted to 20-70%, so as to obtain the compound microorganism bacterial liquid. The basic culture medium is mixed with the additive, so that the culture effect on the composite microorganism can be improved, the microorganism is stimulated, the activity of the microorganism is improved, and the time for reaching the end of logarithmic growth is shortened.
More preferably, the additive is corn peptide and xylo-oligosaccharide, and the mass ratio of the corn peptide to the xylo-oligosaccharide is 1: mixing at a ratio of 0.3-1.
More preferably, the additive is added in an amount of 1 to 5wt%.
More preferably, the basal medium: the yeast powder is added in an amount of 0.3 to 0.7 weight percent, the beef extract is added in an amount of 0.5 to 1.5 weight percent, the lactose is added in an amount of 1 to 3 weight percent g, the glucose is added in an amount of 1 to 4 weight percent, the sodium acetate is added in an amount of 3 to 8 weight percent, the dipotassium hydrogen phosphate is added in an amount of 1 to 3 weight percent, the tween 80 is added in an amount of 0.05 to 0.1 weight percent, and the balance is distilled water, the mixture is boiled and dissolved, the pH value is adjusted to 6 to 7, the mixture is subpackaged at 110 to 130 ℃ and sterilized for 10 to 60 minutes.
More preferably, the additive contains corn starch malate, wherein the content of the corn starch malate is 3-15wt%. The corn starch malate is added into the additive, and acts on microorganism together with the corn peptide and the xylooligosaccharide in the original additive, so that the effect of the corn peptide and the xylooligosaccharide is improved, the stimulation to the microorganism is further improved on the basis, the time for reaching the end of logarithmic growth is further shortened, and the microorganism growth is improved.
Preparation of corn starch malate: mixing corn starch and malic acid solution, stirring uniformly, standing at room temperature for 10-24h, then drying in a baking oven at 40-60 ℃, stirring in the drying process, pulverizing after drying, and then placing in a baking oven at 130-150 ℃ for reaction for 3-12h. Taking out after the reaction is finished, repeatedly washing for a plurality of times by using a large amount of distilled water, carrying out suction filtration, drying and crushing to obtain the corn starch malate, wherein the adding amount of the corn starch is 2-6wt% of that of the malic acid solution, and the amount of the malic acid in the malic acid solution is 6-15wt%.
Preferably, the compound microorganism is nitritification bacteria and pseudomonas putida is prepared by the following steps of 1: mixing viable count of 0.1-1.
Preferably, the number of viable microorganisms in the composite microorganism bacterial liquid reaches 3×10 12 -12×10 12 CFU/mL。
Preferably, the preparation of the composite microbial slurry comprises the following steps: and mixing the compound microorganism bacterial liquid with a functional agent to prepare the compound microorganism bacterial slurry. The functional agent is calcium carbonate and maltodextrin, and the mass ratio of the calcium carbonate to the maltodextrin is 1: mixing at a ratio of 0.5-3.
Preferably, the usage amount of the functional agent is 6-15wt% of the composite microbial liquid.
More preferably, the functional agent contains anthocyanin rhamnoside and dextrin palmitate, wherein the content of the anthocyanin rhamnoside is 1-3wt% and the content of the dextrin palmitate is 0.3-2wt%. The anthocyanin rhamnoside, the dextrin palmitate and the active ingredients in the functional agent are mixed with the microbial liquid to form bacterial slurry, when the bacterial slurry is mixed with the particle carrier, the bacterial slurry is coated on the particle carrier, the microbial bacteria are adsorbed by the particle carrier, the functional agent, the anthocyanin rhamnoside and the dextrin palmitate form a protective layer on the surface, and can form a compact entangled composite structure with a cross-linked object on the surface of the particle carrier, so that a protective effect is provided for the microbial agent, the water removal rate of the microbial agent is improved, the survival rate of the microbial bacteria in the microbial agent after drying is improved, and the acid-resistant survival rate of the microbial agent is improved.
Preferably, the particle carrier is obtained by adding the mixture of particles after PVA and XG are pre-crosslinked with a crosslinking agent in alkali liquor, and then continuously crosslinking, drying and crushing.
Preferably, in the preparation of the carrier, PVA, XG and sodium hydroxide are added into distilled water, stirred and dissolved at the temperature of 80-100 ℃, cooled to 25-40 ℃, then a cross-linking agent is added, after 3-12 hours of reaction, the particle mixture is added, the mixture is treated for 18-36 hours under the condition of stirring for 0.5-2 minutes every 0.5-1 hour, and the mixture is washed and soaked in distilled water until the mixture is neutral, dried and crushed to obtain the particle carrier. PVA and XG are pre-crosslinked in advance, then a particle mixture is added to enable crosslinking to continue, a crosslinking material is introduced into the particle mixture, the prepared particle carrier containing the crosslinking substance provides more sites for the adsorption of microorganism bacteria, the loading effect on the microorganism bacteria is improved together, meanwhile, the adsorption strength of the microorganism bacteria can be further enhanced by adding a functional agent, the leakage of the microorganism bacteria is avoided, and due to the drying combination with the functional agent, the crosslinking network is filled, the affinity to water molecules is weakened, the moisture removal rate is improved during drying, and the influence of environmental acid-base change on the microorganism bacteria is reduced.
More preferably, the PVA is added in an amount of 5 to 15wt% of distilled water.
More preferably, XG is added in an amount of 0.1 to 0.5wt% of distilled water.
More preferably, the amount of sodium hydroxide added is 2 to 5wt%.
More preferably, the cross-linking agent is epichlorohydrin, and the addition amount of the cross-linking agent is 3-8wt% of distilled water.
More preferably, the particle mixture is an inorganic material and a biological material in a mass ratio of 1: mixing at a ratio of 0.1-10, wherein the inorganic material is diatomite, the fineness of the inorganic material is more than or equal to 100 meshes, and the biological material is straw stalk powder.
Preferably, in the preparation of the granular composite microbial agent, the granular composite microbial agent is obtained by mixing the composite microbial slurry with a granular carrier, uniformly stirring and mixing, granulating and drying.
Preferably, the mass ratio of the composite microbial slurry to the particle carrier is 1: 2-5.
The invention adopts the high-density microorganism prepared by fermenting the microorganism with the additive containing the corn starch malate, adopts the particle carrier obtained by crosslinking PVA and XG on the particle mixture, and adopts the functional agent containing anthocyanin rhamnoside and dextrin palmitate to prepare the particle type composite microorganism microbial agent, thus having the following beneficial effects: the growth activity of the microorganism is high, and the growth quantity is improved by more than 10 percent; the time for the growth of the microorganism to reach the end stage of logarithmic growth is short, and the time is reduced by more than 8 percent; the water removal rate is high, and reaches more than 88%; the survival rate of the dried microorganism is high and reaches more than 80 percent; the acid resistance is good, and the acid-resistant survival rate reaches more than 90%. Therefore, the preparation method of the granular composite microbial agent has the advantages of high microbial growth activity, quick microbial propagation time, high water removal rate, high microbial survival rate after drying and good acid resistance.
Drawings
FIG. 1 is a graph showing the results of microbial growth;
FIG. 2 is a graph showing the results of the time period from the growth of microbial cells to the end of logarithmic growth;
FIG. 3 is a graph showing the results of the moisture removal rate of the granular composite microbial agent;
FIG. 4 is a graph showing the result of the survival rate of microbial bacteria after drying the granular composite microbial agent;
FIG. 5 is a graph showing the acid-resistant survival rate of the dried granular composite microbial agent.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to the specific embodiments and the attached drawings:
nitrosation bacteria Nitrosomonas europaea (ATCC 19718) and Pseudomonas putida Pseudomonas putida (ATCC 49128) used in the examples of the present invention.
Example 1:
a preparation method of a granular composite microbial agent,
basal medium: the yeast powder is 0.5wt%, the beef extract is 1wt%, the lactose is 2wt%, the glucose is 2wt%, the sodium acetate is 5wt%, the dipotassium hydrogen phosphate is 2wt%, the Tween 80 is 0.08wt%, and the rest is distilled water, boiling and dissolving are performed, the pH value is adjusted to 6.9, and the packaging is performed at 120 ℃ for 15min for sterilization.
Preparing a composite microbial bacterial liquid: mixing the basic culture medium with the additive, inoculating the compound microorganism with the inoculum size of 5wt%, culturing for 24 hours at a constant temperature of 36 ℃, and adjusting the solid content of the compound microorganism in the high-density fermentation broth to 35% to obtain the compound microorganism bacterial liquid. The additive is corn peptide and xylo-oligosaccharide, and the mass ratio of the corn peptide to the xylo-oligosaccharide is 1:1, the additive is added in an amount of 4wt%, the composite microorganism is nitrosate bacteria and pseudomonas putida is added in an amount of 1: viable count of 0.5.
Preparing composite microbial bacterial slurry: and mixing the compound microorganism bacterial liquid with a functional agent to prepare the compound microorganism bacterial slurry. The functional agent is calcium carbonate and maltodextrin, and the mass ratio of the calcium carbonate to the maltodextrin is 1:2, and the usage amount of the functional agent is 9wt% of the composite microbial liquid.
And (3) preparing a carrier: adding PVA, XG and sodium hydroxide into distilled water, stirring and dissolving at 90 ℃, cooling to 30 ℃, adding a cross-linking agent, reacting for 6 hours, adding a particle mixture, treating for 24 hours under the condition of stirring for 0.5min every 0.5 hour, washing and soaking in distilled water until the mixture is neutral, drying and crushing to obtain a particle carrier, wherein the addition amount of PVA is 12wt% of distilled water, the addition amount of XG is 0.25wt% of distilled water, the addition amount of sodium hydroxide is 4wt%, the cross-linking agent is epichlorohydrin, the addition amount of the cross-linking agent is 5wt% of distilled water, and the particle mixture is inorganic material and biological material according to the mass ratio of 1:3, the inorganic materials are diatomite, the fineness of the inorganic materials is 100 meshes, and the biological materials are straw stalk powder.
Preparing a granular composite microbial agent: mixing the composite microbial slurry with a particle carrier, uniformly stirring and mixing, granulating, and drying to obtain the particle type composite microbial agent, wherein the mass ratio of the composite microbial slurry to the particle carrier is (1): 3.5.
Example 2:
the difference between this example and example 1 is that the additive contains 8wt% of corn starch malate in the preparation of the composite microbial liquid.
Preparation of corn starch malate: mixing corn starch and malic acid solution, stirring uniformly, standing at room temperature for 12h, then drying in a 50 ℃ oven, stirring in the drying process, crushing after drying, and then placing in a 140 ℃ oven for reaction for 6h. Taking out after the reaction is finished, repeatedly washing for a plurality of times by using a large amount of distilled water, carrying out suction filtration, drying and crushing to obtain the corn starch malate, wherein the adding amount of the corn starch is 3wt% of that of the malic acid solution, and the amount of the malic acid in the malic acid solution is 6wt%.
Example 3:
the difference between this example and example 2 is that the additive contains 12wt% of corn starch malate in the preparation of the composite microbial liquid.
Example 4:
the present example differs from example 3 only in that in the carrier treatment, the functional agent solution contains anthocyanin rhamnoside and dextrin palmitate, the content of anthocyanin rhamnoside is 1.2wt%, and the content of dextrin palmitate is 0.5wt%.
Example 5:
the present example differs from example 3 only in that in the carrier treatment, the functional agent solution contains anthocyanin rhamnoside and dextrin palmitate, the content of anthocyanin rhamnoside is 2.3wt%, and the content of dextrin palmitate is 1.2wt%.
Example 6:
this example differs from example 5 only in that the functional agent solution does not contain dextrin palmitate in the carrier treatment.
Example 7:
this example differs from example 5 only in that the functional agent solution does not contain anthocyanin rhamnoside in the carrier treatment.
Test example 1:
1. post fermentation thallus concentration test
Test sample: the fermented microbial liquid in each example.
Cell Optical Density (OD) 600 ): the fermentation broth was diluted with deionized water by an appropriate factor and absorbance was measured at a wavelength of 600nm in an ultraviolet-visible spectrophotometer.
And (3) testing the concentration of the thalli every 6min in the test, and counting the time required for the thalli to grow to reach the end stage of logarithmic growth after fermentation.
The results of the test of the cell concentration after fermentation culture of the microorganism are shown in FIG. 1, wherein the OD of example 1 600 The OD of example 2 was 21.8 600 The OD of example 3 was 24.3 600 The value of 25.6 is compared with example 1, and examples 2-3 show that the use of the corn starch malate improves the cell concentration after fermentation of the microbial bacterial liquid, namely, the growth amount of the cells is improved, the cell concentration is improved by 11.47% compared with example 1 in example 2, the cell concentration is improved by 17.43% compared with example 1 in example 3, and the cell growth amount can be further improved by increasing the use amount of the corn starch malate compared with example 2 in example 3.
The time required for the bacterial growth to reach the end of logarithmic growth after fermentation of the microbial bacteria is shown in fig. 2, wherein the end of logarithmic growth time of example 1 is 12.3 hours, the end of logarithmic growth time of example 2 is 11.2 hours, the end of logarithmic growth time of example 3 is 10.7 hours, and the use of the corn starch malate in examples 2-3 compared with example 1 shows that the time required for the bacterial growth to reach the end of logarithmic growth after fermentation of the microbial bacterial liquid is shortened, namely, the bacterial activity is improved, the time required for the bacterial growth to reach the end of logarithmic growth is reduced by 8.94% compared with example 1, the time required for the bacterial growth to reach the end of logarithmic growth is reduced by 13.01% compared with example 1, and the use of the corn starch malate in example 3 compared with example 2 shows that the time required for the bacterial growth to reach the end of logarithmic growth can be further reduced, and the bacterial activity is improved.
The concentration of the thallus obtained by fermentation is increased by more than 10%, the time for the growth of the microorganism to reach the end stage of logarithmic growth is short, and the time is reduced by more than 8%.
Test example 2:
1. moisture removal rate of dried granular composite microbial agent
Test sample: the granular composite microbial agent prepared in each example.
The water removal rate of the granular composite microbial agent is calculated by the mass of the granular composite microbial agent after the composite microbial agent is dried and the use amount of the carrier.
The results of the moisture removal rate test of the granular composite microbial agent are shown in fig. 3, wherein the moisture removal rate of example 3 is 89.37%, the moisture removal rate of example 4 is 92.52%, the moisture removal rate of example 5 is 93.48%, examples 4-5 show that the use of anthocyanin rhamnoside and dextrin palmitate improves the moisture removal rate of the granular composite microbial agent compared with example 3, and example 5 shows that the use of anthocyanin rhamnoside and dextrin palmitate together is superior to the use of anthocyanin rhamnoside or dextrin palmitate alone compared with examples 6-7.
The particle type composite microorganism obtained by the invention has high water removal rate, and the water removal rate reaches more than 88%;
2. viability of microbial bacteria in granular composite microbial inoculants
Test sample: the granular microbial agent prepared in each example.
The method for testing the viable count in the microbial inoculum comprises the following steps: in a sterile environment, adding a test sample into distilled water, stirring to decompose particles, releasing microorganism bacteria into the solution, carrying out 10-time gradient dilution on the bacteria solution, sucking 1mL of the bacteria solution into a sterile plate at a proper dilution, pouring into a PCA solid culture medium precooled to below 50 ℃, and uniformly mixing. The culture was carried out at 37℃for 36 hours, and plates with colony numbers between 30 and 300 were selected for colony count, and three plates were used for one dilution and averaged.
Acid resistance test: an acidic aqueous solution with a pH of 2 was prepared, and the test sample was added to the solution and allowed to stand for 3 hours. Taking out, washing, and testing according to the viable count testing method.
The viability of the microbial cells after drying of the granular composite microbial agent is shown in fig. 4, wherein the viability of the microbial cells after drying of example 3 is 81.84%, the viability of the microbial cells after drying of example 4 is 85.81%, the viability of the microbial cells after drying of example 5 is 87.12%, examples 4-5 compared with example 3 show that the use of anthocyanin rhamnoside and dextrin palmitate improves the viability of the granular composite microbial agent, and example 5 compared with examples 6-7 show that the co-use of anthocyanin rhamnoside and dextrin palmitate is better than the single use of anthocyanin rhamnoside or dextrin palmitate.
The acid-resistant survival rate of the dried granular composite microbial agent is shown in fig. 5, wherein the acid-resistant survival rate of the dried microbial agent in example 3 is 90.52%, the acid-resistant survival rate of the dried microbial agent in example 4 is 94.89%, the acid-resistant survival rate of the dried microbial agent in example 5 is 96.36%, the use of the anthocyanin rhamnoside and the dextrin palmitate is improved compared with the use of example 3, and the use of the anthocyanin rhamnoside and the dextrin palmitate is better than the use of the anthocyanin rhamnoside or the dextrin palmitate alone in comparison with the use of examples 6-7.
The microbial bacteria after drying has high survival rate, and the survival rate reaches more than 80 percent; the acid resistance is good, and the acid-resistant survival rate reaches more than 90%.
The above embodiments are merely for illustrating the present invention and not for limiting the same, and various changes and modifications may be made by one of ordinary skill in the art without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions are also within the scope of the present invention, which is defined by the claims.

Claims (7)

1. A granular composite microbial agent comprising:
a particulate support, the support surface comprising a cross-linked polymer, the cross-linked polymer being PVA and XG linked by a cross-linking agent; the method comprises the steps of,
composite microorganism bacteria and functional agents loaded on the particle carrier;
the composite microorganism bacteria and the functional agent are coated on the particle carrier in a mixed form; the usage amount of the functional agent is 6-15wt% of the composite microbial liquid;
the functional agent is at least one of maltose, calcium carbonate, soluble starch, gelatin, maltodextrin, polyvinylpyrrolidone and glycerin, and anthocyanin rhamnoside and dextrin palmitate; the content of anthocyanin rhamnoside is 1-3wt% and the content of dextrin palmitate is 0.3-2wt%.
2. The granular composite microbial agent according to claim 1, wherein: the cross-linking agent is epichlorohydrin.
3. The granular composite microbial agent according to claim 1, wherein: the particle carrier includes at least one of an inorganic material and a biological material inside.
4. A preparation method of a granular composite microbial agent comprises the following steps: mixing the composite microbial liquid with a functional agent to prepare composite microbial slurry, and mixing the composite microbial slurry with a particle carrier to prepare a particle type composite microbial agent; the particle carrier is obtained by adding a particle mixture after PVA and XG are pre-crosslinked with a crosslinking agent in alkali liquor, and then continuously crosslinking, drying and crushing; the functional agent is at least one of maltose, calcium carbonate, soluble starch, gelatin, maltodextrin, polyvinylpyrrolidone and glycerin, and anthocyanin rhamnoside and dextrin palmitate; the content of anthocyanin rhamnoside is 1-3wt% and the content of dextrin palmitate is 0.3-2wt%.
5. The method for preparing the granular composite microbial agent according to claim 4, which is characterized in that: the compound microorganism is nitritification bacteria and pseudomonas putida, the ratio of which is 1: mixing viable count of 0.1-1.
6. The method for preparing the granular composite microbial agent according to claim 4, which is characterized in that: the usage amount of the functional agent is 6-15wt% of the composite microbial liquid.
7. The method for preparing the granular composite microbial agent according to claim 4, which is characterized in that: the mass ratio of the composite microbial slurry to the particle carrier is 1: 2-5.
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