A method for remediation of heavy metal contaminated soil using microbial inoculants

Abstract

This invention relates to the field of soil remediation technology, specifically disclosing a method for remediating heavy metal-contaminated soil using microbial agents. The invention uses hydroxyapatite as a carrier, utilizing its adsorption properties to load licorice extract onto its surface. To further improve the stability of the licorice extract, a carboxymethyl cellulose (CMC) solution is added. After stirring, mixing, and drying, the excellent film-forming properties of CMC help improve the stability of the licorice extract, while also increasing the specific surface area and adsorption sites of the hydroxyapatite. The introduction of licorice extract and CMC not only enhances the chelating ability of hydroxyapatite for heavy metals but also provides an additional carbon source for microorganisms, promoting their growth and reproduction. The metabolic activity of *Trichoderma solani* and *Bacillus licheniformis* is significantly enhanced, resulting in a substantial increase in remediation efficiency.

Description

Technical Field

[0001] This invention relates to the field of soil remediation technology, and specifically to a method for remediating heavy metal-contaminated soil using microbial agents. Background Technology

[0002] Heavy metal contamination of soil is mainly caused by factors such as industrial wastewater discharge, mining activities, and the long-term use of agricultural fertilizers and pesticides, and is one of the most significant environmental problems facing the world today. Heavy metals in soil, such as lead, cadmium, mercury, and arsenic, are highly toxic, persistent, and bioaccumulative, not only disrupting the soil's ecological balance but also potentially harming human health through the food chain, causing various diseases. With the acceleration of industrialization and the continuous expansion of pollution, the remediation of heavy metal-contaminated soil has become an urgent task in the field of environmental governance.

[0003] Remediation technologies for heavy metal contaminated soil mainly include physical remediation, chemical remediation, and bioremediation. Physical remediation methods, such as topsoil replacement and electroremediation, are costly and prone to secondary pollution. Chemical remediation, through stabilization or leaching techniques, is fast-acting but may damage soil structure and introduce new chemical risks. Microbial technologies have gradually gained attention due to their advantages of being ecological, low-cost, simple, efficient, and widely applicable. However, existing microbial remediation technologies for heavy metals also have some problems, such as poor stability of microorganisms and limited remediation capacity for heavy metals.

[0004] Chinese patent document CN201710556915.4 discloses a soil heavy metal remediation biological amendment and its preparation method. It is prepared by compounding microbial agents, biochar, organosilicon, nitrogen raw materials, fast-acting calcium, phosphate rock powder and amino acids in a specific ratio. It can effectively remediate soil heavy metal pollution, improve soil physicochemical properties, improve soil microbial environment, balance soil nutrients, improve soil fertility, promote crop growth, and thus improve grain yield and quality. However, the prepared biological amendment has limited effect on the remediation of heavy metal pollution and still needs further improvement. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for remediating heavy metal contaminated soil using microbial agents.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for remediating heavy metal contaminated soil using microbial agents, the method comprising inoculating the heavy metal contaminated soil with microbial agents, tilling the soil to mix the microbial agents and soil evenly, and curing for 3-5 days, wherein the microbial agents include *Trichoderma terrestris*, *Bacillus licheniformis*, and modified hydroxyapatite material.

[0008] In the technical solution disclosed in this invention, the classification name of *Comamonasterrigena* is *Comamonasterrigena* 003, and its accession number is CGMCC No. 9423; the depositary institution is the China General Microbiological Culture Collection Center (CGMCC); the address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the deposit date is July 7, 2014.

[0009] In the technical solution disclosed in this invention, Bacillus licheniformis is classified as Bacillus licheniformis 005, with accession number CGMCC No. 9420; depositary institution: China General Microbiological Culture Collection Center (CGMCC); address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; deposit date: July 7, 2014.

[0010] In the technical solution disclosed in this invention, the preparation method of the modified hydroxyapatite material is as follows: under stirring conditions, a hydroalcoholic solution of licorice extract and a disodium hydrogen phosphate solution are simultaneously added dropwise to a calcium chloride solution and stirred evenly. Then, the pH of the solution is adjusted to 10-11, and the reaction is heated and stirred. After the reaction is completed, the solution is cooled to room temperature, and then a carboxymethyl cellulose solution is added. The mixture is stirred evenly again, the solvent is removed by rotary evaporation, and the mixture is freeze-dried and ground to obtain the modified hydroxyapatite material.

[0011] In the technical solution disclosed in this invention, the mass ratio of the licorice extract aqueous alcohol solution, disodium hydrogen phosphate solution, calcium chloride solution and carboxymethyl cellulose solution is 20-30:20-30:30:15-25.

[0012] In the technical solution disclosed in this invention, the mass fraction of licorice extract in the aqueous alcoholic solution of licorice extract is 1-3%, for example, 1%, 1.5%, 2%, 2.5%, 3%, but not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0013] In the technical solution disclosed in this invention, the aqueous alcohol solution is a mixed solution of ethanol and water, and the volume ratio of ethanol to water is 1-2:1-3. For example, 1:1, 1:2, 1:3, 2:1, 2:2, and 2:3 can be selected, but it is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0014] In the technical solution disclosed in this invention, the molar ratio of calcium chloride and disodium hydrogen phosphate in the reaction system is 5:3.

[0015] In the technical solution disclosed in this invention, the mass fraction of the carboxymethyl cellulose solution is 10-15%, for example, 10%, 11%, 12%, 13%, 14%, or 15%, but is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0016] In the technical solution disclosed in this invention, the temperature of the heating and stirring reaction is 60-75℃, for example, 60℃, 65℃, 70℃, or 75℃ can be selected; the heating and stirring reaction time is 2-4h, for example, 2h, 2.5h, 3h, 3.5h, or 4h can be selected, but it is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0017] In the technical solution disclosed in this invention, the preparation method of the microbial agent is as follows:

[0018] The modified hydroxyapatite material was dispersed in LB medium, and then the bacterial suspensions of Trichoderma solani and Bacillus licheniformis were resuspended in the medium for culture. After the culture was completed, the microbial agent was obtained by centrifugation and freeze-drying.

[0019] In the technical solution disclosed in this invention, the ratio of the modified hydroxyapatite material to LB culture medium is 0.01-0.05g:100mL. For example, 0.01g:100mL, 0.02g:100mL, 0.03g:100mL, 0.04g:100mL, and 0.05g:100mL can be selected, but it is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0020] In the technical solution disclosed in this invention, the bacterial suspension of Trichoderma solani accounts for 1-3% of the volume of LB medium. For example, 1%, 1.5%, 2%, 2.5%, and 3% can be selected, but it is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0021] In the technical solution disclosed in this invention, the viable count of *Trichoderma solani* bacterial solution is not less than 1.0 × 10⁻⁶. 6 cfu / mL.

[0022] In the technical solution disclosed in this invention, the Bacillus licheniformis bacterial solution accounts for 1-3% of the volume of LB culture medium. For example, 1%, 1.5%, 2%, 2.5%, and 3% can be selected, but it is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0023] In the technical solution disclosed in this invention, the number of viable bacteria in the Bacillus licheniformis bacterial solution is not less than 1.0 × 10⁻⁶. 6 cfu / mL.

[0024] In the technical solution disclosed in this invention, the cultivation conditions are: culturing in a shaker at 30°C and 150-200 r / min for 24-36 h.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The terrestrial trichomoniasis and Bacillus licheniformis provided by the present invention exert synergistic effects through different metabolic pathways and physiological characteristics, and jointly reduce the toxicity of heavy metals. The combined use of terrestrial trichomoniasis and Bacillus licheniformis not only improves the remediation efficiency, but also enhances the stability of the system, enabling it to adapt to complex soil environments.

[0027] (2) The *Trichoderma terrestris* provided by this invention mainly transforms highly toxic heavy metal ions into low-toxicity or insoluble forms through enzymatic reduction, thereby reducing the mobility and bioavailability of heavy metals. Simultaneously, *Trichoderma terrestris* can also complex heavy metals by secreting organic acids, reducing their free concentration and further alleviating the toxic effects of heavy metals on the microbial community. The remediation mechanism of *Bacillus licheniformis* mainly manifests in the secretion of extracellular polymeric substances (EPS) and adsorption on the cell surface. The synergistic effect of *Trichoderma terrestris* and *Bacillus licheniformis* is mainly reflected in: *Trichoderma terrestris* reduces the oxidized state of heavy metals through reduction, making them more easily captured by the EPS of *Bacillus licheniformis*, forming insoluble precipitates; simultaneously, *Bacillus licheniformis* improves the soil microenvironment through the secretion of EPS, providing a protective barrier for *Trichoderma terrestris* and reducing the inhibition of its growth by heavy metals; furthermore, the common metabolic activities of the two bacteria may promote the decomposition of organic matter in the soil, releasing nutrients (such as nitrogen and phosphorus), thereby maintaining the activity of the microbial community and enhancing the sustainability of the remediation system.

[0028] (3) In this invention, hydroxyapatite is used as a carrier to load licorice extract onto the surface of hydroxyapatite by utilizing its adsorption properties. At the same time, in order to further improve the stability of licorice extract, carboxymethyl cellulose solution is added. After stirring, mixing and drying, the good film-forming properties of carboxymethyl cellulose help to improve the stability of licorice extract, while also increasing the specific surface area and adsorption sites of hydroxyapatite. The introduction of licorice extract and carboxymethyl cellulose (CMC) not only enhances the chelating ability of hydroxyapatite for heavy metals, but also provides an additional carbon source for microorganisms, promoting their growth and reproduction. The metabolic activity of Trichoderma solani and Bacillus licheniformis is significantly improved, and the repair efficiency is greatly improved. Detailed Implementation

[0029] The present invention will be further described in detail below through specific preferred embodiments, but the present invention is not limited to the following embodiments.

[0030] It should be noted that, unless otherwise specified, all chemical reagents involved in this invention were purchased through commercial channels.

[0031] The licorice extract used in this embodiment of the invention, CAS number 97676-23-8, was purchased from Nanjing Taigao Plant Extract Biotechnology Co., Ltd.; the viable count of *Trichoderma solani* bacterial suspension was 1.0 × 10⁻⁶. 6 CFU / mL; the viable count of Bacillus licheniformis in the bacterial culture was 1.0 × 10⁻⁶. 6 cfu / mL.

[0032] Example 1

[0033] A method for preparing a microbial inoculant includes the following steps:

[0034] Under stirring conditions, 20 parts of a 2 wt% aqueous alcoholic solution of licorice extract (ethanol and water volume ratio of 1:1) and 20 parts of a disodium hydrogen phosphate solution were simultaneously added dropwise to 30 parts of a calcium chloride solution. The molar ratio of calcium chloride to disodium hydrogen phosphate in the reaction system was 5:3, and the mass ratio of licorice extract to calcium chloride was 0.05:1. The mixture was stirred for 12 hours, and then the pH of the solution was adjusted to 10 with ammonia. The mixture was heated and stirred at 60°C for 4 hours. After the reaction was completed, the mixture was cooled to room temperature, and then 20 parts of a 10 wt% carboxymethyl cellulose solution were added. The mixture was stirred for another 6 hours, the solvent was removed by rotary evaporation, the mixture was freeze-dried, and then ground through a 400-mesh sieve to obtain the modified hydroxyapatite material.

[0035] 0.03 g of modified hydroxyapatite was dispersed in 100 mL of LB medium. Then, 2 mL of *Trichoderma solani* and 2 mL of *Bacillus licheniformis* were resuspended in the medium for culture. The culture conditions were: 30 °C and 200 r / min in a shaker for 24 h. After the culture was completed, the microbial agent was obtained by centrifugation and freeze-drying.

[0036] Example 2

[0037] A method for preparing a microbial inoculant includes the following steps:

[0038] Under stirring conditions, 20 parts of a 2 wt% aqueous alcoholic solution of licorice extract (ethanol and water volume ratio of 1:1) and 20 parts of a disodium hydrogen phosphate solution were simultaneously added dropwise to 30 parts of a calcium chloride solution. The molar ratio of calcium chloride to disodium hydrogen phosphate in the reaction system was 5:3, and the mass ratio of licorice extract to calcium chloride was 0.05:1. The mixture was stirred for 12 hours, and then the pH of the solution was adjusted to 10 with ammonia. The mixture was heated and stirred at 60°C for 4 hours. After the reaction was completed, the mixture was cooled to room temperature, and then 20 parts of a 10 wt% carboxymethyl cellulose solution were added. The mixture was stirred for another 6 hours, the solvent was removed by rotary evaporation, the mixture was freeze-dried, and then ground through a 400-mesh sieve to obtain the modified hydroxyapatite material.

[0039] 0.01 g of modified hydroxyapatite was dispersed in 100 mL of LB medium. Then, 1 mL of Trichoderma solani and 1 mL of Bacillus licheniformis were resuspended in the medium for culture. The culture conditions were: 30 °C and 200 r / min in a shaker for 24 h. After the culture was completed, the microbial agent was obtained by centrifugation and freeze-drying.

[0040] Example 3

[0041] A method for preparing a microbial inoculant includes the following steps:

[0042] Under stirring conditions, 20 parts of a 2 wt% aqueous alcoholic solution of licorice extract (ethanol and water volume ratio of 1:1) and 20 parts of a disodium hydrogen phosphate solution were simultaneously added dropwise to 30 parts of a calcium chloride solution. The molar ratio of calcium chloride to disodium hydrogen phosphate in the reaction system was 5:3, and the mass ratio of licorice extract to calcium chloride was 0.05:1. The mixture was stirred for 12 hours, and then the pH of the solution was adjusted to 10 with ammonia. The mixture was heated and stirred at 60°C for 4 hours. After the reaction was completed, the mixture was cooled to room temperature, and then 20 parts of a 10 wt% carboxymethyl cellulose solution were added. The mixture was stirred for another 6 hours, the solvent was removed by rotary evaporation, the mixture was freeze-dried, and then ground through a 400-mesh sieve to obtain the modified hydroxyapatite material.

[0043] 0.05 g of modified hydroxyapatite was dispersed in 100 mL of LB medium. Then, 3 mL of Trichoderma solani and 3 mL of Bacillus licheniformis were resuspended in the medium for culture. The culture conditions were: 30 °C and 200 r / min in a shaker for 24 h. After the culture was completed, the microbial agent was obtained by centrifugation and freeze drying.

[0044] Comparative Example 1

[0045] A method for preparing a microbial inoculant includes the following steps:

[0046] Under stirring conditions, 20 parts of a 2 wt% aqueous alcoholic solution of licorice extract (ethanol and water volume ratio of 1:1) and 20 parts of a disodium hydrogen phosphate solution were simultaneously added dropwise to 30 parts of a calcium chloride solution. The molar ratio of calcium chloride to disodium hydrogen phosphate in the reaction system was 5:3, and the mass ratio of licorice extract to calcium chloride was 0.05:1. The mixture was stirred for 12 hours, and then the pH of the solution was adjusted to 10 with ammonia. The mixture was heated and stirred at 60°C for 4 hours. After the reaction was completed, the mixture was cooled to room temperature, and then 20 parts of a 10 wt% carboxymethyl cellulose solution were added. The mixture was stirred for another 6 hours, the solvent was removed by rotary evaporation, the mixture was freeze-dried, and then ground through a 400-mesh sieve to obtain the modified hydroxyapatite material.

[0047] 0.03 g of modified hydroxyapatite material was dispersed in 100 mL of LB medium, and then 4 mL of Bacillus licheniformis bacterial suspension was resuspended in the medium for culture. The culture conditions were: 30 °C and 200 r / min in a shaker for 24 h. After the culture was completed, the microbial agent was obtained by centrifugation and freeze drying.

[0048] Compared with Comparative Example 1, 2 mL of Trichomonas terrestris bacterial suspension was replaced with 2 mL of Bacillus licheniformis bacterial suspension.

[0049] Comparative Example 2

[0050] A method for preparing a microbial inoculant includes the following steps:

[0051] Under stirring conditions, 20 parts of a 2 wt% aqueous alcoholic solution of licorice extract (ethanol and water volume ratio of 1:1) and 20 parts of a disodium hydrogen phosphate solution were simultaneously added dropwise to 30 parts of a calcium chloride solution. The molar ratio of calcium chloride to disodium hydrogen phosphate in the reaction system was 5:3, and the mass ratio of licorice extract to calcium chloride was 0.05:1. The mixture was stirred for 12 hours, and then the pH of the solution was adjusted to 10 with ammonia. The mixture was heated and stirred at 60°C for 4 hours. After the reaction was completed, the mixture was cooled to room temperature, and then 20 parts of a 10 wt% carboxymethyl cellulose solution were added. The mixture was stirred for another 6 hours, the solvent was removed by rotary evaporation, the mixture was freeze-dried, and then ground through a 400-mesh sieve to obtain the modified hydroxyapatite material.

[0052] 0.03 g of modified hydroxyapatite material was dispersed in 100 mL of LB medium, and then 4 mL of Trichoderma solani bacterial suspension was resuspended in the medium for culture. The culture conditions were: 30 °C and 200 r / min in a shaker for 24 h. After the culture was completed, the microbial agent was obtained by centrifugation and freeze drying.

[0053] Compared with Example 1, 2 mL of Bacillus licheniformis bacterial solution was replaced with 2 mL of Trichodina terrestris bacterial solution.

[0054] Comparative Example 3

[0055] A method for preparing a microbial inoculant includes the following steps:

[0056] Under stirring conditions, 20 parts of a 2 wt% aqueous alcoholic solution of licorice extract (ethanol and water volume ratio of 1:1) and 20 parts of a disodium hydrogen phosphate solution were simultaneously added dropwise to 30 parts of a calcium chloride solution. The molar ratio of calcium chloride to disodium hydrogen phosphate in the reaction system was 5:3, and the mass ratio of licorice extract to calcium chloride was 0.05:1. The mixture was stirred for 12 hours, and then the pH of the solution was adjusted to 10 with ammonia. The mixture was heated and stirred at 60°C for 4 hours. After the reaction was completed, the mixture was cooled to room temperature, and then 20 parts of a 10 wt% carboxymethyl cellulose solution were added. The mixture was stirred for another 6 hours, the solvent was removed by rotary evaporation, the mixture was freeze-dried, and then ground through a 400-mesh sieve to obtain the modified hydroxyapatite material.

[0057] 0.03 g of modified hydroxyapatite material was dispersed in 100 mL of LB medium. Then, 2 mL of Bacillus subtilis culture and 2 mL of Bacillus licheniformis culture were resuspended in the medium for culture. The culture conditions were: 30 °C and 200 r / min in a shaker for 24 h. After the culture was completed, the microbial agent was obtained by centrifugation and freeze drying.

[0058] Compared with Example 1, Comparative Example 3 used Bacillus subtilis instead of Trichoderma terrestris. The Bacillus subtilis strain was HZB368880.

[0059] Comparative Example 4

[0060] A method for preparing a microbial inoculant includes the following steps:

[0061] Under stirring conditions, 20 parts of disodium hydrogen phosphate solution were added dropwise to 30 parts of calcium chloride solution. The molar ratio of calcium chloride to disodium hydrogen phosphate in the reaction system was 5:3. The mixture was stirred for 12 hours, and then the pH of the solution was adjusted to 10 with ammonia. The mixture was heated and stirred at 60°C for 4 hours. After the reaction was completed, the mixture was cooled to room temperature, and then 20 parts of 10 wt% carboxymethyl cellulose solution were added. The mixture was stirred for another 6 hours, the solvent was removed by rotary evaporation, the mixture was freeze-dried, and then ground through a 400-mesh sieve to obtain the modified hydroxyapatite material.

[0062] 0.03 g of modified hydroxyapatite was dispersed in 100 mL of LB medium. Then, 2 mL of *Trichoderma solani* and 2 mL of *Bacillus licheniformis* were resuspended in the medium for culture. The culture conditions were: 30 °C and 200 r / min in a shaker for 24 h. After the culture was completed, the microbial agent was obtained by centrifugation and freeze-drying.

[0063] Compared with Example 1, no licorice extract was added to the modified hydroxyapatite material in Comparative Example 4.

[0064] 0.1g of the microbial agents prepared in Examples 1-3 and Comparative Examples 1-4 were added to 100mL of simulated heavy metal-contaminated wastewater. The Cr in the heavy metal-contaminated wastewater... 6+ The concentration is 20 mg / L, Cd 2+ The concentration was 20 mg / L. After culturing in a shaker (25℃, 200 rpm) for 7 days, the supernatant was taken to measure the change in the concentration of heavy metal ions in the solution before and after adsorption. The test results are shown in Table 1.

[0065] Table 1. Results of heavy metal ion concentration changes in different groups.

[0066] <![CDATA[Cr 6+ Concentration (mg / L) <![CDATA[Cd 2+ Concentration (mg / L) Example 1 0.86 0.57 Example 2 0.93 0.64 Example 3 0.79 0.52 Comparative Example 1 3.24 2.08 Comparative Example 2 2.15 2.86 Comparative Example 3 1.98 1.23 Comparative Example 4 3.43 3.75

[0067] As can be seen from Table 1, the present invention can effectively reduce the content of heavy metal ions in the solution by using a combination of Trichoderma solani and Bacillus licheniformis.

[0068] Heavy metal ion solution was added to the air-dried and sieved soil, and deionized water was sprayed to maintain its moisture content at 80% of the field soil water holding capacity. After being kept at room temperature for 1 month, the soil was air-dried and sieved through a 100-mesh sieve to prepare soil samples with copper pollution concentration of 10 mg / kg, chromium (VI) pollution concentration of 10 mg / kg, and cadmium pollution concentration of 10 mg / kg. Subsequently, 1g of the microbial agent prepared in Example 1 and Comparative Examples 1-4 was added to 50g of soil sample, mixed evenly, and cured under natural conditions for 30 days. The content of heavy metal ions in the soil was then detected, and the results are shown in Table 2.

[0069] Table 2 Results of changes in heavy metal ion concentration in soil

[0070]

[0071]

[0072] Finally, it should be noted that the above embodiments do not limit the present invention in any way. Those skilled in the art can make modifications and improvements based on the present invention. Therefore, any modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A method for remediating heavy metal-contaminated soil using microbial inoculants, characterized in that, The method includes inoculating the soil with microbial agents into heavy metal contaminated soil, tilling the soil to mix the microbial agents and soil evenly, and curing for 3-5 days. The microbial agents include Trichoderma terrestris, Bacillus licheniformis, and modified hydroxyapatite material. The modified hydroxyapatite material is prepared as follows: under stirring conditions, licorice extract aqueous alcohol solution and disodium hydrogen phosphate solution are simultaneously added dropwise to calcium chloride solution and stirred evenly. Then, the pH of the solution is adjusted to 10-11, and the reaction is heated and stirred. After the reaction is completed, the mixture is cooled to room temperature, and then carboxymethyl cellulose solution is added. The mixture is stirred evenly again, the solvent is removed by rotary evaporation, freeze-dried, and ground to obtain the modified hydroxyapatite material.

2. The method according to claim 1, characterized in that, The mass ratio of the licorice extract aqueous alcohol solution, disodium hydrogen phosphate solution, calcium chloride solution, and carboxymethyl cellulose solution is 20-30:20-30:30:15-25.

3. The method according to claim 1, characterized in that, The mass fraction of licorice extract in the aqueous alcoholic solution of licorice extract is 1-3%.

4. The method according to claim 1, characterized in that, The molar ratio of calcium chloride to disodium hydrogen phosphate in the reaction system is 5:

3.

5. The method according to claim 1, characterized in that, The carboxymethyl cellulose solution has a mass fraction of 10-15%.

6. The method according to claim 1, characterized in that, The temperature for heating and stirring the reaction is 60-75℃, and the reaction time is 2-4 hours.

7. The method according to claim 1, characterized in that, The preparation method of the microbial agent is as follows: the modified hydroxyapatite material is dispersed in LB medium, and then the bacterial suspensions of Trichoderma solani and Bacillus licheniformis are resuspended in the medium for culture. After the culture is completed, the microbial agent is obtained by centrifugation and freeze drying.

8. The method according to claim 7, characterized in that, The viable count of *Trichoderma solani* bacterial suspension is not less than 1.0 × 10⁻⁶. 6 cfu / mL.

9. The method according to claim 7, characterized in that, The viable count of Bacillus licheniformis in the bacterial solution is not less than 1.0 × 10⁻⁶. 6 cfu / mL.

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