CN117903813B - A microbial soil activating agent and its preparation method and application - Google Patents

Abstract

The invention discloses a microbial soil activation microbial agent, a preparation method and application thereof, and belongs to the technical field of microbial materials. The microbial agent comprises functional microbial fermentation liquid, a microbial activator and a microbial carrier, wherein the mass ratio of the functional microbial fermentation liquid to the microbial activator to the microbial carrier is 1:0.5:5. The trichoderma harzianum strain obtained by screening has extracellular adsorption and metal complexation effects, realizes reduction and discharge of metal ions by regulating expression of reductase and related genes, has higher arsenic ion removal efficiency, has capabilities of phosphate dissolving, nitrogen fixing and IAA production, and has the effects of regulating soil and promoting crop growth. After the trichoderma harzianum, the azotobacter chroococcus and the pseudomonas fragi are combined in proportion, the interaction is obvious, the heavy metal removal efficiency can be improved, and the synergistic growth is promoted. The three microorganisms can also regulate the content of nutrient substances in the soil, particularly regulate the activities of various enzymes in the soil, restore the ecological activity of the soil and have remarkable social and economic benefits.

Description

A microbial soil activating agent and its preparation method and application

Technical Field

The invention belongs to the technical field of microbial materials, and specifically relates to a microbial soil activating agent and a preparation method and application thereof.

Background technique

Heavy metals refer to some metals and metalloids with biological toxicity, such as cadmium, mercury, arsenic, lead, chromium, etc. At present, the problem of heavy metal pollution in soil has attracted increasing attention. In urban heavy metal-contaminated soil, enzymes and microorganisms will be inhibited or even killed, which will seriously damage the soil ecological chain, affect the biological activity of the soil, and thus limit the growth of crops.

Heavy metal pollution in soil can be treated by physical, chemical or biological means. Physical and chemical methods are not only ineffective in repairing heavy metal-contaminated soil, but also pose a risk of secondary pollution to the soil. Currently, microorganisms are used to efficiently dissolve heavy metals in the soil, thereby promoting the absorption of heavy metal elements by plants; rhizosphere microorganisms are different from other microorganisms. While dissolving heavy metals, they can also secrete a large amount of plant hormones, thereby promoting plant production and development, thereby improving the efficiency of plant repair of heavy metals.

However, the current microbial treatment of heavy metal elements in soil is mostly focused on the repair of multiple metal elements, and the functions are single and the strains are complex, making it difficult to be effectively utilized and implemented. For example: the patent with application number CN201610252293.1 discloses a microbial agent for improving soil heavy metals, including photosynthetic bacteria, actinomycetes, lactic acid bacteria, yeast, Bacillus subtilis, Bacillus thuringiensis, Bacillus megaterium, nitrogen-fixing bacteria, citric acid bacteria, Bacillus licheniformis and a matrix that provides nutrition for the above-mentioned flora, and a microbial agent for improving soil heavy metals. The application number is CN202311153948.6, which discloses a microbial composite agent for repairing heavy metal contaminated soil, and each 100 parts of the microbial composite agent includes the following components in parts by weight: 5 parts of fecal cellulomonas, 10 parts of nitroreduced Pseudomonas, 30 parts of peptone, 35 parts of yeast extract and 20 parts of sodium chloride.

Therefore, how to develop an efficient microbial soil activation agent that can efficiently remove heavy metal elements in the soil while activating the soil environment and improving its ecological activity and soil fertility is a technical problem that needs to be solved urgently.

Summary of the invention

In view of the problems existing in the prior art, the present invention optimizes and separates a strain of Trichoderma harzianum, and combines it with screened brown spherical nitrogen-fixing bacteria and Pseudomonas amylovora to form a functional microbial strain. Under the joint action of a microbial activator and a carrier, the present invention can achieve efficient removal of heavy metal elements in the soil, and has the functions of promoting growth and regulating the soil.

In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

A microbial soil activating agent comprises a functional microbial fermentation liquid, a microbial activator and a microbial carrier, wherein the mass ratio of the three is 1:0.5:5.

Preferably, the microbial activator is obtained by mixing sodium alginate and glucose in a mass ratio of 1:(0.5-1.5).

Preferably, the microbial carrier is obtained by mixing bran, diatomaceous earth, biochar and fish bone powder in a mass ratio of (5-8): (3-5): (3-6): (1-3).

Preferably, the functional microbial fermentation broth comprises Trichoderma harzianum, Azotobacter chroococcum and Pseudomonas plecoglossicida.

Preferably, the Trichoderma harzianum has a deposit number of CGMCC No. 40702, and is deposited in the General Microbiology Center of the China Culture Collection Administration. The deposit date is June 16, 2023, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

The Trichoderma harzianum of the present invention is separated from an iron tailing in Weishan County, Jining City, Shandong Province, and the contents of various heavy metals including arsenic and lead in the sampling site are all exceeded.

The separation method is as follows: take 5g of soil sample and place it in sterilized PDA culture medium, add filtered and sterilized sodium arsenite solution to make the As ³⁺ concentration of 50mg/L, and after enrichment culture for 24h, transfer it to the culture medium with an As ⁵⁺ concentration of 100mg/L at a 2% inoculum, culture it under the same conditions, and gradually increase the As ⁵⁺ concentration. After transferring 5 times, dilute and spread it, separate and preserve the strain with the best growth, which is the target strain Trichoderma harzianum.

Morphological characteristics of the strain: The colony expands to 5 cm after culturing on a PDA plate at 25-28℃ for 4 days, and is dark gray-green; the hyphae are septate and branched. The conidiophores form a coniferous branching outline, and the conidia are nearly spherical or ovoid, green, and have smooth walls.

The deposit number of the brown ball nitrogen-fixing bacteria is CGMCC No. 1.5803, which was purchased from the General Microbiology Center of the China Microorganism Culture Collection Administration Committee, and the original deposit date is June 27, 2005; the deposit number of the Pseudomonas ayuensis is CGMCC No. 1.16111, which was purchased from the General Microbiology Center of the China Microorganism Culture Collection Administration Committee, and the original deposit date is March 29, 2017. Both the brown ball nitrogen-fixing bacteria and the Pseudomonas ayuensis can be purchased through the strain catalog of the collection center without the need for additional deposit.

A method for preparing a microbial soil activating agent comprises the following preparation steps:

(1) After thawing, Trichoderma harzianum, Azotobacter nitrofusus and Pseudomonas amylovora were inoculated in PDA medium for activation culture; after activation culture, the bacterial cakes of each strain were selected and added to the mixed fermentation liquid medium at an inoculation rate of 1.5% for fermentation culture, and when the effective viable bacteria count in each mixed fermentation liquid medium reached 4×10 ⁸ CFU/mL, the fermentation was stopped to obtain three fermentation broths, and then the three fermentation broths were mixed at a volume ratio of 1:1:1 to obtain a functional microbial fermentation broth;

(2) sterilizing bran, diatomaceous earth, biochar and fish bone powder in a mass ratio of 8:3:3:1, adding sodium alginate and glucose, stirring and mixing, and placing in a granulator for granulation to obtain solid particles with a particle size of 2-4 mm;

(3) The solid particles obtained in step (2) are added to the functional microbial fermentation liquid for adsorption, and then naturally dried to obtain the final product.

The activation culture method in step (1) is as follows: after thawing, Trichoderma harzianum, Azotobacter nitrofusus and Pseudomonas amylovora are inoculated into PDA culture medium respectively, and cultured at 25-28° C. When the mycelium grows to two-thirds of the plate, a 6 mm punch is used to punch the colonies in the basic PDA culture medium into bacterial cakes to complete the activation culture.

The formula and preparation method of the PDA culture medium are as follows: 200 grams of potatoes are added with water and boiled for at least 30 minutes, filtrated and the filtrate is taken, and then 20 grams of glucose and 15-20 grams of agar are added, and water is added to 1000 ml, the pH value is natural, and sterilized before use.

The mixed fermentation liquid culture medium is composed of: 5-7g glucose, 10-15g yeast powder, 0.1-0.15g _{KH 2} PO ₄ , 0.1-0.15g MgSO ₄ , 3-5g sodium chloride, 1000mL distilled water, and is sterilized at a high temperature of 121°C by steam for 20min to obtain a natural pH value.

The method for fermentation culture in the mixed fermentation liquid culture medium is: culture at 25-28°C and shaker culture at 200r/min.

Preferably, during granulation in step (2), maltose or starch is added as a binder to promote granulation.

An application of a microbial soil activation agent, wherein the agent is used for repairing and activating heavy metal contaminated soil.

More preferably, the bacterial agent is used to repair and activate arsenic-contaminated soil.

The raw materials used in the present invention are all commercially available.

The dosage of the soil microorganism activation agent of the present invention is 5-10kg/mu. The method of use is to evenly mix the soil repair agent with the soil to be repaired, spray water until the soil moisture is 60-70%, and maintain for 5-10 days.

Beneficial Effects

(1) The present invention screened out a strain of Trichoderma harzianum, which has extracellular adsorption and metal complexing functions, and can achieve the reduction and excretion of metal ions by regulating the expression of reductase and related genes. First, the functional groups and proteins on the surface can bind to heavy metal ions, and then the heavy metal ions are reduced to a low-valent state or a stable state through a biological reduction process, thereby fixing and removing heavy metal ions, especially for arsenic ions. The removal efficiency is high, and the strain also has the ability to solubilize phosphorus, fix nitrogen and produce IAA, and has the function of regulating soil and promoting crop growth;

(2) Brown ball nitrogen-fixing bacteria not only have good nitrogen-fixing ability and regulate the soil nutrient structure, but also can produce intracellular adsorption to combine heavy metal elements with chelators to form heat-stable proteins, so that the toxicity of heavy metal elements is weakened or eliminated in the process; Pseudomonas amygdaloides not only has metal chelation, but also can secrete organic acids, biosurfactants and other ingredients to enhance the activity of Trichoderma harzianum and brown ball nitrogen-fixing bacteria and promote their efficacy;

(3) After the Trichoderma harzianum of the present invention is combined with the brown ball nitrogen-fixing bacteria and the Pseudomonas amylovora in equal proportions, the interaction is significant, which can improve the removal efficiency of heavy metals. At the same time, the three microorganisms can also regulate the content of soil nutrients, especially the activities of various enzymes in the soil, and repair the ecological activity of the soil, with significant social and economic benefits.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing soil urease activity in Example 1 of the present invention and Comparative Examples 1-9;

FIG2 is a graph showing soil sucrase activity in Example 1 of the present invention and Comparative Examples 1-9;

FIG3 is a graph showing soil protease activity in Example 1 of the present invention and Comparative Examples 1-9;

FIG4 is a graph showing catalase activity in soil of Example 1 and Comparative Examples 1-9 of the present invention;

FIG5 is a graph showing the effect of the phosphate solubilization ability of Trichoderma harzianum, Azotobacter chlorosphaera and Pseudomonas ayutans of the present invention;

6 is a graph showing the growth of Trichoderma harzianum, Azotobacter sphaeroides and Pseudomonas ayuensis on an Axubei ammonia-fixing medium of the present invention, wherein A is a graph showing the growth of Trichoderma harzianum, B is a graph showing the growth of Pseudomonas ayuensis, and C is a graph showing the growth of Azotobacter sphaeroides;

FIG7 is a graph showing the siderophore production ability of Trichoderma harzianum, Azotobacter chlorosphaera and Pseudomonas ayutans of the present invention;

FIG8 is a graph showing the effect of the indoleacetic acid production ability of Trichoderma harzianum, Azotobacter chlorosphaera and Pseudomonas ayutans of the present invention.

Detailed ways

The technical solution of the present invention is further described below in conjunction with specific embodiments, but is not limited thereto.

Example 1

A microbial soil activating agent comprises a functional microbial fermentation liquid, a microbial activator and a microbial carrier, wherein the mass ratio of the three is 1:0.5:5.

The microbial activator is obtained by mixing sodium alginate and glucose in a mass ratio of 1:0.5.

The microbial carrier is obtained by mixing bran, diatomaceous earth, biochar and fish bone powder in a mass ratio of 8:3:3:1.

The functional microbial fermentation liquid contains Trichoderma harzianum, Azotobacter chroococcum and Pseudomonas plecoglossicida.

Preferably, the Trichoderma harzianum has a deposit number of CGMCC No. 40702, and is deposited in the General Microbiology Center of the China Culture Collection Administration. The deposit date is June 16, 2023, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

In this example, Trichoderma harzianum was isolated from an iron tailings in Weishan County, Jining City, Shandong Province. The contents of various heavy metals including arsenic and lead at the sampling site exceeded the standard.

The separation method is as follows: take 5g of soil sample and place it in sterilized PDA culture medium, add filtered and sterilized sodium arsenite solution to make the As ³⁺ concentration of 50mg/L, and after enrichment culture for 24h, transfer it to the culture medium with an As ³⁺ concentration of 100mg/L at a 2% inoculum, culture it under the same conditions, and gradually increase the As ³⁺ concentration. After transferring 5 times, dilute and spread it, separate and preserve the strain with the best growth, which is the target strain Trichoderma harzianum.

Morphological characteristics of the strain: The colony expands to 8 cm after culturing on a PDA plate at 25-28℃ for 4 days, and is dark gray-green; the hyphae are septate and branched. The conidiophores form a coniferous branching outline, and the conidia are nearly spherical or ovoid, green, and have smooth walls.

The deposit number of the brown ball nitrogen-fixing bacteria is CGMCC No. 1.5803, which was purchased from the General Microbiology Center of the China Microorganism Culture Collection Administration Committee, and the original deposit date is June 27, 2005; the deposit number of the Pseudomonas ayuensis is CGMCC No. 1.16111, which was purchased from the General Microbiology Center of the China Microorganism Culture Collection Administration Committee, and the original deposit date is March 29, 2017. Both the brown ball nitrogen-fixing bacteria and the Pseudomonas ayuensis can be purchased through the strain catalog of the collection center without the need for additional deposit.

A method for preparing a microbial soil activating agent comprises the following preparation steps:

(1) After thawing, Trichoderma harzianum, Azotobacter nitrofusus and Pseudomonas amylovora were inoculated into PDA medium for activation culture; after activation culture, the bacterial cakes of each strain were selected and added into mixed fermentation liquid medium at an inoculation rate of 1.5% for fermentation culture; when the effective viable bacteria count in each mixed fermentation liquid medium reached 4×10 ⁸ CFU/mL, the fermentation was stopped to obtain three fermentation broths, and then the three fermentation broths were mixed at a volume ratio of 1:1:1 to obtain a functional microbial fermentation broth;

(2) Sterilize bran, diatomaceous earth, biochar and fish bone powder in a mass ratio of 8:3:3:1, add sodium alginate and glucose, stir and mix well, and place in a granulator for granulation to obtain solid particles with a particle size of 2-4 mm;

(3) The solid particles obtained in step (2) are added to the functional microbial fermentation liquid for adsorption, and then naturally dried to obtain the final product.

The activation culture method in step (1) is as follows: after thawing, Trichoderma harzianum, Azotobacter nitrofusus and Pseudomonas amylovora are inoculated into PDA culture medium respectively, and cultured at 25-28° C. When the mycelium grows to two-thirds of the plate, a 6 mm punch is used to punch the colonies in the basic PDA culture medium into bacterial cakes to complete the activation culture.

The formula and preparation method of the PDA culture medium are as follows: 200 grams of potatoes are added with water and boiled for at least 30 minutes, filtrated and the filtrate is taken, and then 20 grams of glucose and 15-20 grams of agar are added, and water is added to 1000 ml, the pH value is natural, and sterilized before use.

The mixed fermentation liquid culture medium is composed of: 5-7g glucose, 10-15g yeast powder, 0.1-0.15g _{KH 2} PO ₄ , 0.1-0.15g MgSO ₄ , 3-5g sodium chloride, 1000mL distilled water, and is sterilized at a high temperature of 121°C by steam for 20min to obtain a natural pH value.

The method for fermentation culture in the mixed fermentation liquid culture medium is: culture at 25-28°C and shaker culture at 200r/min.

During granulation in step (2), starch is added as a binder to promote granulation.

An application of a microbial soil activation agent, wherein the agent is used for repairing and activating heavy metal contaminated soil.

The bacterial agent is used for repairing and activating arsenic-contaminated soil.

All the raw materials used are commercially available.

The dosage of soil microbial activation agent is 5-10kg/mu. The method of use is to mix the soil remediation agent with the soil to be remediated evenly, spray water until the soil moisture is 60-70%, and maintain for 5-10 days.

Example 2

A microbial soil activating agent comprises a functional microbial fermentation liquid, a microbial activator and a microbial carrier, wherein the mass ratio of the three is 1:0.5:5.

The microbial activator is obtained by mixing sodium alginate and glucose in a mass ratio of 1:1.5.

The microbial carrier is obtained by mixing bran, diatomaceous earth, biochar and fish bone powder in a mass ratio of 5:5:4:2.

The functional microbial fermentation liquid contains Trichoderma harzianum, Azotobacter chroococcum and Pseudomonas plecoglossicida.

The Trichoderma harzianum has a preservation number of CGMCC No. 40702, and is preserved in the General Microbiology Center of the China Culture Collection Administration. The preservation date is June 16, 2023, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

Trichoderma harzianum was isolated from an iron tailing in Weishan County, Jining City, Shandong Province, and the isolation method was the same as in Example 1.

The brown spherical nitrogen-fixing bacteria and Pseudomonas ayutans are selected the same as those in Example 1.

A method for preparing a microbial soil activating agent. Same as Example 1.

During the granulation in step (2), maltose is added as a binder to promote granulation.

Example 3

A microbial soil activating agent comprises a functional microbial fermentation liquid, a microbial activator and a microbial carrier, wherein the mass ratio of the three is 1:0.5:5.

The microbial activator is obtained by mixing sodium alginate and glucose in a mass ratio of 1:1.

The microbial carrier is obtained by mixing bran, diatomaceous earth, biochar and fish bone powder in a mass ratio of 6:4:6:3.

The functional microbial fermentation liquid contains Trichoderma harzianum, Azotobacter chroococcum and Pseudomonas plecoglossicida.

The Trichoderma harzianum has a preservation number of CGMCC No. 40702, and is preserved in the General Microbiology Center of the China Culture Collection Administration. The preservation date is June 16, 2023, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

Trichoderma harzianum was isolated from an iron tailing in Weishan County, Jining City, Shandong Province, and the isolation method was the same as in Example 1.

The brown spherical nitrogen-fixing bacteria and Pseudomonas ayutans are selected the same as those in Example 1.

A method for preparing a microbial soil activating agent. Same as Example 1.

During granulation in step (2), starch is added as a binder to promote granulation.

Example 4

Study on the growth-promoting effect of bacterial strains

To explore whether the three selected strains all have growth-promoting effects, qualitative tests of phosphate solubilization, nitrogen fixation, siderophore production, and indoleacetic acid production were performed. The test methods are as follows:

1) Determination of phosphate solubilization capacity

Prepare PKO solid culture medium, dilute the bacterial solution activated to OD ₆₀₀ = 0.8~1.0 and spread it on the plate culture medium, and culture it at 28-30℃ for 3 days. The strains with phosphate solubilization function will form a transparent phosphate-dissolving circle around the colonies.

The composition of PKO medium is: glucose 10 g, (NH ₄ )SO ₄ 0.5 g, NaCl 0.3 g, KCl 0.3 g, MgSO ₄ ·7H ₂ O 0.3 g, FeSO ₄ ·7H ₂ O 0.03 g, MnSO ₄ ·4H ₂ O 0.03 g, Ca ₃ (PO ₄ ) ₂ 5 g, agar 20 g, distilled water 1000 mL, pH 7.0-7.2.

2) Determination of nitrogen fixation capacity

The activated bacterial solution was gradiently diluted and spread on Ashuber's ammonia-fixing medium, and cultured at a constant temperature of 28-30℃ for 3 days. The plate was observed for colonies formed. Single colonies were transferred three times in succession. The strains that can grow stably have the ability to fix nitrogen.

The composition of Ashurbein fixed ammonia medium is: K ₂ HPO ₄ 0.2 g, NaCl 0.2 g, CaCO ₃ 2.0 g, mannitol 10 g, CaSO ₄ 0.1 g, MgSO ₄ 0.2 g, agar 1 g, distilled water 1000 mL, pH 6.9.

3) Determination of siderophore production capacity

The isolated and purified strain was inoculated on a CAS detection plate by the partition point inoculation method, cultured at 20-30°C for 3 days, and the colonies were observed. If there was an obvious orange-yellow halo around the colonies, it proved that the strain had the ability to produce siderophores.

The composition of the CAS detection plate is: 20% sucrose solution, 10mL10% acid hydrolyzed casein 30mL, 1mmol/LCaCl ₂ 1mL, 1 mmol/LMgSO ₄ 20mL, 0.1mol/L buffer 5mL, CAS dye solution 5mL, agar 18g, and distilled water 1000mL.

4) Determination of indoleacetic acid (IAA) production capacity

The test strain was inoculated into 50 mL of King'B medium. After constant temperature culture for 3 days, the bacterial suspension was centrifuged at 8000r/min for 6 minutes. 500μL of supernatant and 500μL of Salkowski colorimetric solution were added to 1.5mL centrifuge tubes respectively, and left to stand in the dark for 30 minutes to wait for color development. In addition, the bacterial solution was replaced with an equal amount of blank medium without bacteria and 50 mg/L of IAA as negative and positive controls. The color change can be used to preliminarily judge whether the strain has the ability to produce IAA. The darker the color, the more IAA the strain secretes.

The composition of King'B medium is: 20.0 g of peptone, 1.5 g of K ₂ HPO ₄ , 1.5 g of MgSO ₄ ·7H ₂ O, 1000 mL of distilled water, pH 7.2.

Table 1 Qualitative determination of growth-promoting ability of strains

Note: “+” indicates activity, “-” indicates inactivity.

Comparative Examples 1-9

The composition of the functional microbial fermentation liquid, that is, the volume ratio of Trichoderma harzianum, Azotobacter nitrofusus and Pseudomonas ayutans, was changed to set a comparative example. The remaining raw materials and processes were the same as those in Example 1. The specific preparation method is:

After thawing, Trichoderma harzianum, Azotobacter chlororoscens and Pseudomonas ayutans were inoculated into PDA medium for activation culture. After activation culture, the bacterial cakes of each strain were picked up and added into mixed fermentation liquid medium at an inoculation rate of 1.5% for fermentation culture. When the effective viable bacteria count in each mixed fermentation liquid medium reached 4×10 ⁸ CFU/mL, the fermentation was stopped to obtain three fermentation broths, which were then mixed in different volume ratios to obtain functional microbial fermentation broth.

The volume ratios of Trichoderma harzianum, Azotobacter nitrofurantoides and Pseudomonas ayutans in the comparative example are shown in Table 2:

Table 2 Comparative Example Bacterial Mass Ratio

Determination of heavy metal ion removal ability of strains

The functional microbial fermentation liquid obtained in Example 1 and Comparative Examples 1-9 of the present invention was inoculated with 1% addition into 100 mL of liquid culture medium (NB) containing 150 mg/L Pb ²⁺ , 100 mg/L As ⁵⁺ , and 200 mg/L Cd ²⁺ , and cultured at 25-28°C, 180 r/min shaking for 48 h, and centrifuged at 5000 r/min for 10 min. The supernatant was taken and the concentrations of Pb ²⁺ , As ⁵⁺ , and Cd ²⁺ in the supernatant were determined by ICP-MS. The heavy metal ion removal rate was calculated using the formula:

Wherein, C ₀ is the concentration of the original metal ions in the culture medium, and _{Ce is} the concentration of the remaining metal ions after treatment. The units are all mg/L. The removal results are shown in Table 3:

Table 3 Removal experimental results

Soil remediation experiment:

Test soil: Soil contaminated by heavy metals near an electroplating factory in Hedong District, Linyi City

Test method: Take the contaminated soil, of which the blank group is directly put into a flower pot of about 20cm×20cm; the other experimental groups are added with the microbial activators obtained in Examples 1-3 and Comparative Examples 1-9 at 5kg/mu (about 1.4% of the soil weight), mixed evenly and put into flower pots, each pot is filled with 1kg of soil, and 3 parallel samples are set for each group of tests, and the results are averaged. Deionized water is added by weighing method to maintain the soil moisture content of about 60%. After one week of maintenance, soil samples are collected, naturally air-dried after removing impurities, ground and sieved through 10 mesh and 100 mesh sieves for storage.

Analytical test methods:

Soil microorganisms were determined by dilution plate separation method. Soil urease activity was determined by indophenol colorimetry. Sucrase activity was determined by 3,5-dinitrosalicylic acid colorimetry. Catalase activity was determined by potassium permanganate titration. Protease activity was determined by modified ninhydrin colorimetry.

Soil samples were naturally air-dried and sieved, and then the soil-water ratio was 1:5 for leaching. The pH value of the soil was directly read using an acidometer and conductivity meter. The available nitrogen content was determined by alkaline diffusion method, the available phosphorus and available potassium content were determined by combined leaching-colorimetry, and the organic matter content was determined by potassium dichromate volumetric method. The content of heavy metal ions in rhizosphere soil was determined by atomic absorption spectrophotometer (PerkinElmer SIMMA6000, Norwalk, USA).

Table 4 Soil index test results

Table 5 Soil index test results

Table 6 Soil index test results

From the above performance data, we can see that the soil repaired by the embodiment of the present invention, under the activation of the three functional strains, the content of heavy metal elements in the soil is effectively controlled, and the effective activity of microorganisms, the content of soil nutrients, biomass, and enzyme activity levels are significantly improved. However, in Comparative Examples 1-9, which changed the composition and dosage of the strains, the synergistic balance between the strains was broken, the effect of the microbial activity process was weakened, and thus the soil repair effect was weakened.

It should be noted that the above embodiments are only some embodiments of the preferred methods of implementing the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

Claims (4)

1. A microbial soil activating agent, characterized in that it comprises a functional microbial fermentation liquid, a microbial activator and a microbial carrier, and the mass ratio of the three is 1:0.5:5; The microbial activator is obtained by mixing sodium alginate and glucose in a mass ratio of 1:(0.5-1.5); The microbial carrier is obtained by mixing bran, diatomaceous earth, biochar and fish bone powder in a mass ratio of (5-8): (3-5): (3-6): (1-3); The functional microbial fermentation liquid comprises Trichoderma harzianum, Azotobacter chroococcum and Pseudomonas plecoglossicida ; the Trichoderma harzianum has a deposit number of CGMCC No.40702 and is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration; the Azotobacter chroococcum has a deposit number of CGMCC No.1.5803 and is purchased from the General Microbiological Center of China National Microbiological Culture Collection Administration; the Pseudomonas plecoglossicida has a deposit number of CGMCC No.1.16111 and is purchased from the General Microbiological Center of China National Microbiological Culture Collection Administration; The preparation method of the microbial soil activating agent comprises the following preparation steps: (1) After thawing, Trichoderma harzianum, Azotobacter nitrofusus and Pseudomonas amylovora were inoculated in PDA medium for activation culture; after activation culture, the bacterial cakes of each strain were selected and added to the mixed fermentation liquid medium at an inoculation rate of 1.5% for fermentation culture, and when the effective viable bacteria count in the mixed fermentation liquid medium reached 4×10 ⁸ CFU/mL, the fermentation was stopped to obtain three fermentation broths, and then the three fermentation broths were mixed at a volume ratio of 1:1:1 to obtain a functional microbial fermentation broth; (2) sterilizing bran, diatomaceous earth, biochar and fish bone powder, adding microbial activators sodium alginate and glucose, stirring and mixing until uniform, and placing in a granulator for granulation to obtain solid particles with a particle size of 2-4 mm; (3) The solid particles obtained in step (2) are added to the functional microbial fermentation liquid for adsorption, and then naturally dried to obtain the final product. 2. The microbial soil activation agent according to claim 1, characterized in that during granulation in step (2), maltose or starch is added as a binder to promote granulation. 3. An application of the microbial soil activation agent according to claim 1 or 2, characterized in that the agent is used to repair and activate heavy metal contaminated soil. 4. The use of the microbial soil activation agent according to claim 3, characterized in that the agent is used to repair and activate arsenic-contaminated soil.