A soil remediation bacterial agent and a preparation method thereof

Abstract

The present application relates to the technical field of contaminated soil remediation, and particularly relates to a soil remediation bacterial agent and a preparation method thereof, the remediation bacterial agent comprises microbial bacterial slurry and biochar, wherein the microorganism bacteria in the microbial bacterial slurry comprise Pannonibacter phragmitetus BB bacteria, Bacillus subtilis, Trichoderma viride, Aspergillus niger and Pseudomonas, through mutual cooperation between several microorganisms, not only can the chromium element and pesticide DDT pollution in the soil be treated, but also mutual promotion can be achieved, the treatment effect of single microorganism on the soil can be optimized, the remediation efficiency can be ensured, the soil structure can be optimized and improved at the same time, and the remediated soil can be used as soon as possible.

Description

Technical Field

[0001] This invention relates to the field of contaminated soil remediation technology, specifically to a soil remediation microbial agent and its preparation method. Background Technology

[0002] Soil pollution in my country's arable land is mainly caused by heavy metals (cadmium, nickel, copper, arsenic, mercury, and lead) and organic pesticides (DDT and polycyclic aromatic hydrocarbons). Among these pollutants, chromium and DDT are the most representative and widely distributed pollutants in soil. Remediation methods for soils contaminated with both heavy metals and organic pesticides mainly include physicochemical remediation and microbial remediation. Physicochemical remediation methods require strict condition control, are costly, and involve complex procedures. While some soil conditioners can achieve the desired effect in a short period through adsorption or chelation, they only change the form of heavy metals or organic pollutants without completely removing them. Furthermore, the persistent nature of organic pollutants makes physicochemical methods less effective in remediating contaminated farmland.

[0003] Microbial soil remediation technology refers to the use of naturally occurring or cultured functional microbial communities under suitable environmental conditions to promote or enhance the metabolic functions of microorganisms, thereby reducing the activity of toxic pollutants or degrading them into non-toxic substances. Specifically, it utilizes the ability of microorganisms, under pollution stress, to secrete organic substances with the capacity to complex or decompose pollutants, reducing the mobility or altering the polarity of pollutants, thus making them less likely to enter the organism's body; or, under the action of extracellular enzymes secreted by microorganisms, pollutants are decomposed and transformed into non-toxic and harmless substances outside the body.

[0004] Existing microbial soil remediation technologies mainly focus on restoring soil ecosystems and primarily use single-purpose microbial soil remediation agents, such as those targeting only soil pesticide or heavy metal pollution. These are ill-suited for remediation environments in soils with combined heavy metal and organic pesticide pollution. Therefore, there is an urgent need to provide a multi-purpose microbial soil remediation agent. Summary of the Invention

[0005] To address the technical problem that existing microbial soil remediation agents have limited functionality and cannot effectively treat soils contaminated with both heavy metals and organic pesticides, this invention provides a soil remediation microbial agent that achieves efficient removal of heavy metal chromium and pesticide DDT from soil by combining different types of microorganisms with different functions.

[0006] To achieve the above-mentioned objectives, the embodiments of the present invention adopt the following technical solutions:

[0007] A soil remediation microbial agent comprising microbial sludge and biochar, wherein the microbial sludge comprises Pannonibacter phragmitetus BB, Bacillus subtilis, Trichoderma viride, Aspergillus niger, and Pseudomonas aeruginosa, wherein the preservation number of Pannonibacter phragmitetus BB is CGMCC No.3052, and it has been disclosed in patent application CN106834184B entitled "A composite microbial community and its application in the remediation of Cr(VI) contaminated soil".

[0008] Compared to existing technologies, the soil remediation microbial agent provided by this invention utilizes *Pannonibacter phragmitetus* BB, which has a prominent reducing ability for hexavalent chromium ions, combined with *Aspergillus niger* and *Trichoderma*, which have a prominent degradation effect on organic molecules. This combination enables the degradation of hexavalent chromium ions and organic pesticides in the soil. Furthermore, the addition of *Pseudomonas* and *Bacillus subtilis* not only decomposes cellulose, starch, and nitrified organic matter in the soil, improving soil structure, but the decomposed organic matter also serves as nutrients for *Pannonibacter phragmitetus* and *Aspergillus niger*, enhancing their activity and promoting their treatment effect on hexavalent chromium in the soil. The synergistic effect of these microorganisms not only treats chromium and DDT pollution in the soil but also promotes mutual enhancement, optimizing the treatment effect of individual microorganisms on the soil. This ensures remediation efficiency while improving soil structure, resulting in optimal remediation results.

[0009] Preferably, Bacillus subtilis, Trichoderma viride, Aspergillus niger, and Pseudomonas aeruginosa are commercial strains purchased from Beina Biotechnology, with Bacillus subtilis numbered BNCC185325, Trichoderma viride numbered BNCC341615, Aspergillus niger numbered BNCC185666, and Pseudomonas aeruginosa numbered BNCC223900.

[0010] Preferably, the mass ratio of *Alternaria panlonga*, *Bacillus subtilis*, *Trichoderma viride*, *Aspergillus niger*, and *Pseudomonas aeruginosa* in the microbial sludge is 0.3–0.5:1–2:1–2:0.5–1.0:0.8–1.5; and the effective viable count of each microorganism in the microbial sludge is ≥10. 9 cfu / g; the further preferred mass ratio of Panlongia albicans, Trichoderma viride, Aspergillus niger and Pseudomonas aeruginosa is 0.5:1.5:1.5:1.0:1.0.

[0011] Preferably, the biochar is obtained by pyrolysis carbonization of agricultural waste after crushing and drying. The specific carbonization process is as follows: agricultural organic waste is placed in a carbonization furnace and pyrolyzed at 400-450°C under closed conditions to obtain the biochar.

[0012] On the other hand, the present invention also improves the preparation method of the above-mentioned soil remediation microbial agent, which specifically includes the following steps:

[0013] S1: Microbial slant seeds of Alkali Lake Panronia, Bacillus subtilis, Trichoderma viride, Aspergillus niger and Pseudomonas aeruginosa were sequentially subjected to liquid seed culture and scale-up culture to obtain the corresponding fermentation broth;

[0014] S2: The fermentation broths of Alkali Lake Bacillus, Bacillus subtilis, Trichoderma viride, Aspergillus niger and Pseudomonas aeruginosa are centrifuged to obtain microbial sludge. The microbial sludge is mixed evenly with biochar according to the mass ratio, dried and granulated to obtain the soil remediation microbial agent.

[0015] Preferably, the method for culturing microbial slant seeds of *Alternaria panlonga*, *Bacillus subtilis*, *Trichoderma viride*, *Aspergillus niger*, and *Pseudomonas aeruginosa* is as follows: *Alternaria panlonga*, *Bacillus subtilis*, *Trichoderma viride*, *Aspergillus niger*, and *Pseudomonas aeruginosa* are inoculated onto solid culture medium under aseptic conditions and cultured, wherein *Alternaria panlonga*, *Trichoderma viride*, *Aspergillus niger*, and *Pseudomonas aeruginosa* are cultured at 30°C for 3 days, and *Bacillus subtilis* is cultured at 37°C for 2 days, thereby obtaining the corresponding microbial slant seeds.

[0016] Preferably, both the liquid seed culture and the scale-up culture process use liquid culture medium, wherein the liquid culture medium for Panlonia alkaline lake bacillus, Bacillus subtilis and Pseudomonas includes the following components in parts by weight: 10 parts tryptone, 5 parts yeast extract, 10 parts sodium chloride and 1000 parts distilled water.

[0017] The culture medium for *Trichoderma viride* comprises the following components in parts by weight: 10 parts potato extract, 20 parts glucose, 3 parts potassium dihydrogen phosphate, 1.5 parts magnesium sulfate, 15 parts agar, and 1000 parts distilled water.

[0018] The culture medium for Aspergillus niger comprises the following components in parts by weight: 130 parts malt extract, 0.1 parts chloramphenicol, 15 parts agar, and 1000 parts distilled water.

[0019] Preferably, during the liquid seed culture process, Aspergillus niger, Trichoderma viride, and Pseudomonas aeruginosa are cultured in a shaker at 30°C and 150 rpm; Alkali bacillus panlonga and Bacillus subtilis are cultured statically at 37°C; and the culture is stopped when the OD600 value of each strain in the liquid culture is 3.0 to 4.0.

[0020] Preferably, the expanded culture conditions are as follows: Aspergillus niger, Trichoderma viride, and Pseudomonas aeruginosa are cultured in a fermenter at 30°C with a stirring speed of 150–200 rpm and a sterile air-to-ventilation ratio of 1:1, until the effective viable count of each strain in the individual fermentation broth is >5 × 10⁻⁶. 9 CFU / mL;

[0021] Alkali bacillus and Bacillus subtilis were cultured in a fermenter at 37°C until the viable count of each strain in the individual fermentation broth was >8 × 10⁻⁶. 9 CFU / mL

[0022] Preferably, the biochar and microbial sludge in S2 are mixed at a mass ratio of 3-5:1, stirred evenly, granulated, and dried until the water content is ≤15% to obtain the soil remediation microbial agent. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0024] In the following examples, unless otherwise specified, the Pannonibacterphragmitetus BB strain used has the accession number CGMCC No. 3052. This strain has been disclosed in the patent application CN106834184B entitled "A composite microbial community and its application in the remediation of Cr(VI) contaminated soil". The other microbial strains are all commercial strains purchased from Beina Biotechnology, including Bacillus subtilis with accession number BNCC185325, Trichoderma viride with accession number BNCC341615, Aspergillus niger with accession number BNCC185666, and Pseudomonas aeruginosa with accession number BNCC223900.

[0025] Example 1

[0026] This embodiment provides a soil remediation microbial agent, which is made from microbial sludge and biochar. The microbial sludge includes *Alternaria panlonga*, *Bacillus subtilis*, *Trichoderma viride*, *Aspergillus niger*, and *Pseudomonas aeruginosa*, with a mass ratio of 0.5:1.5:1.5:1.0:1.0. The biochar is a product obtained by pyrolysis carbonization of agricultural organic waste such as straw, peanut shells, and corn cobs after crushing, drying, and processing. The specific carbonization process is as follows: agricultural organic waste is placed in a carbonization furnace and heated by an external heat source under closed conditions at 400-450℃ to obtain the biochar.

[0027] The preparation method of this soil remediation microbial agent specifically includes the following steps:

[0028] S1: Microbial slant seeds of *Alternaria panlonga*, *Bacillus subtilis*, *Trichoderma viride*, *Aspergillus niger*, and *Pseudomonas aeruginosa* were sequentially subjected to liquid seed culture and scale-up culture to obtain the corresponding fermentation broths:

[0029] The method for slant seed culture is as follows: Alkali bacillus panlonga, Bacillus subtilis, Trichoderma viride, Aspergillus niger, and Pseudomonas aeruginosa are inoculated into solid culture medium under aseptic conditions. Alkali bacillus panlonga, Trichoderma viride, Aspergillus niger, and Pseudomonas aeruginosa are cultured at 30°C for 3 days, and Bacillus subtilis is cultured at 37°C for 2 days to obtain the corresponding microbial slant seeds.

[0030] The liquid seed culture method is as follows: Under aseptic conditions, take 1 ml of the corresponding microbial slant seed liquid and inoculate it into a 250 mL container with a volume of 20 mL of the corresponding liquid culture medium. The liquid culture medium of Panlonia alkaline lake bacillus, Bacillus subtilis and Pseudomonas includes the following components by weight: 10 parts tryptone, 5 parts yeast extract, 10 parts sodium chloride and 1000 parts distilled water.

[0031] The culture medium for Trichoderma viride consists of the following components by weight: 10 parts potato extract, 20 parts glucose, 3 parts potassium dihydrogen phosphate, 1.5 parts magnesium sulfate, 15 parts agar, and 1000 parts distilled water.

[0032] The culture medium for Aspergillus niger consists of the following components in parts by weight: 130 parts malt extract, 0.1 parts chloramphenicol, 15 parts agar, and 1000 parts distilled water.

[0033] Aspergillus niger, Trichoderma viride, and Pseudomonas aeruginosa were cultured in a shaker at 150 rpm at 30°C; Alkali bacillus panlonga and Bacillus subtilis were cultured statically at 37°C; single-strain liquid culture was stopped when the OD600 value was between 3.0 and 4.0.

[0034] The method for scaling up the culture is as follows: Inoculate the bacterial broth obtained from the liquid seed culture into the fermenter at an inoculation rate of 10% of the liquid culture medium volume. The composition of the liquid culture medium in the fermenter should be consistent with that used in the liquid seed culture. *Aspergillus niger*, *Trichoderma viride*, and *Pseudomonas aeruginosa* are stirred at 180 rpm in the fermenter at 30°C with a sterile air-to-ventilation ratio of 1:1 until the effective viable count in the individual fermentation broth of each strain is >5 × 10⁻⁶. 9 CFU / mL;

[0035] Alkali bacillus and Bacillus subtilis were cultured in a fermenter at 37°C until the viable count of each strain in the individual fermentation broth was >8 × 10⁻⁶. 9 CFU / mL, which gives the scale-up fermentation broth of the corresponding strain;

[0036] S2: Centrifuge the fermentation broth separately to obtain microbial sludge; measure the effective viable count of each microorganism in the microbial sludge, all of which are ≥10. 9 The soil remediation agent is obtained by mixing microbial sludge with biochar at a mass ratio of 4:1, granulating, and drying until the moisture content is ≤15%.

[0037] Example 2

[0038] This embodiment provides a soil remediation microbial agent, which is made from microbial sludge and biochar. The microbial sludge includes *Alternaria panlonga*, *Bacillus subtilis*, *Trichoderma viride*, *Aspergillus niger*, and *Pseudomonas aeruginosa*, with a mass ratio of 0.3:2.0:1.0:0.5:0.8. The biochar is a product obtained by pyrolysis and carbonization of agricultural organic waste such as straw and fallen leaves after crushing and drying. The specific carbonization process is as follows: agricultural organic waste is placed in a carbonization furnace and heated by an external heat source under closed conditions at 400-450℃ to obtain the biochar.

[0039] The preparation method of this soil remediation microbial agent specifically includes the following steps:

[0040] S1: Microbial slant seeds of *Alternaria panlonga*, *Bacillus subtilis*, *Trichoderma viride*, *Aspergillus niger*, and *Pseudomonas aeruginosa* were sequentially subjected to liquid seed culture and scale-up culture to obtain the corresponding fermentation broths:

[0041] The method for slant seed culture is as follows: Alkali bacillus panlonga, Bacillus subtilis, Trichoderma viride, Aspergillus niger, and Pseudomonas aeruginosa are inoculated into solid culture medium under aseptic conditions. Alkali bacillus panlonga, Trichoderma viride, Aspergillus niger, and Pseudomonas aeruginosa are cultured at 30°C for 3 days, and Bacillus subtilis is cultured at 37°C for 2 days to obtain the corresponding microbial slant seeds.

[0042] The liquid seed culture method is as follows: Under aseptic conditions, take 1 ml of the corresponding microbial slant seed liquid and inoculate it into a 250 mL container with a volume of 20 mL of the corresponding liquid culture medium. The liquid culture medium of Panlonia alkaline lake bacillus, Bacillus subtilis and Pseudomonas includes the following components by weight: 10 parts tryptone, 5 parts yeast extract, 10 parts sodium chloride and 1000 parts distilled water.

[0043] The culture medium for Trichoderma viride consists of the following components by weight: 10 parts potato extract, 20 parts glucose, 3 parts potassium dihydrogen phosphate, 1.5 parts magnesium sulfate, 15 parts agar, and 1000 parts distilled water.

[0044] The culture medium for Aspergillus niger consists of the following components in parts by weight: 130 parts malt extract, 0.1 parts chloramphenicol, 15 parts agar, and 1000 parts distilled water.

[0045] Aspergillus niger, Trichoderma viride, and Pseudomonas aeruginosa were cultured in a shaker at 150 rpm at 30°C; Alkali bacillus panlonga and Bacillus subtilis were cultured statically at 37°C; single-strain liquid culture was stopped when the OD600 value was between 3.0 and 4.0.

[0046] The method for scaling up the culture is as follows: Inoculate the bacterial broth obtained from the liquid seed culture into the fermenter at an inoculation rate of 10% of the liquid culture medium volume. The composition of the liquid culture medium in the fermenter should be consistent with that used in the liquid seed culture. *Aspergillus niger*, *Trichoderma viride*, and *Pseudomonas aeruginosa* are stirred at 180 rpm in the fermenter at 30°C with a sterile air-to-ventilation ratio of 1:1 until the effective viable count in the individual fermentation broth of each strain is >5 × 10⁻⁶. 9 CFU / mL;

[0047] Alkali bacillus and Bacillus subtilis were cultured in a fermenter at 37°C until the viable count of each strain in the individual fermentation broth was >8 × 10⁻⁶. 9 CFU / mL. This yields the scale-up fermentation broth for the corresponding bacterial strain;

[0048] S2: Centrifuge the fermentation broth separately to obtain microbial sludge; measure the effective viable count of each microorganism in the microbial sludge, all of which are ≥10. 9 The soil remediation agent is obtained by mixing microbial sludge with biochar at a mass ratio of 3:1, granulating, and drying until the moisture content is ≤15%.

[0049] Example 3

[0050] This embodiment provides a soil remediation microbial agent, which is made from microbial sludge and biochar. The microbial sludge includes *Alternaria panlonga*, *Bacillus subtilis*, *Trichoderma viride*, *Aspergillus niger*, and *Pseudomonas aeruginosa*, with a mass ratio of 0.5:1.0:2.0:1.0:1.5. The biochar is a product obtained by pyrolysis and carbonization of agricultural organic waste after crushing and drying. The specific carbonization process is as follows: agricultural organic waste is placed in a carbonization furnace and heated by an external heat source under closed conditions at 400-450℃ to obtain the biochar.

[0051] The preparation method of this soil remediation microbial agent specifically includes the following steps:

[0052] S1: Microbial slant seeds of *Alternaria panlonga*, *Bacillus subtilis*, *Trichoderma viride*, *Aspergillus niger*, and *Pseudomonas aeruginosa* were sequentially subjected to liquid seed culture and scale-up culture to obtain the corresponding fermentation broths:

[0053] The method for slant seed culture is as follows: Alkali bacillus panlonga, Bacillus subtilis, Trichoderma viride, Aspergillus niger, and Pseudomonas aeruginosa are inoculated into solid culture medium under aseptic conditions. Alkali bacillus panlonga, Trichoderma viride, Aspergillus niger, and Pseudomonas aeruginosa are cultured at 30°C for 3 days, and Bacillus subtilis is cultured at 37°C for 2 days to obtain the corresponding microbial slant seeds.

[0054] The liquid seed culture method is as follows: Under aseptic conditions, take 1 ml of the corresponding microbial slant seed liquid and inoculate it into a 250 mL container with a volume of 20 mL of the corresponding liquid culture medium. The liquid culture medium of Panlonia alkaline lake bacillus, Bacillus subtilis and Pseudomonas includes the following components by weight: 10 parts tryptone, 5 parts yeast extract, 10 parts sodium chloride and 1000 parts distilled water.

[0055] The culture medium for Trichoderma viride consists of the following components by weight: 10 parts potato extract, 20 parts glucose, 3 parts potassium dihydrogen phosphate, 1.5 parts magnesium sulfate, 15 parts agar, and 1000 parts distilled water.

[0056] The culture medium for Aspergillus niger consists of the following components in parts by weight: 130 parts malt extract, 0.1 parts chloramphenicol, 15 parts agar, and 1000 parts distilled water.

[0057] Aspergillus niger, Trichoderma viride, and Pseudomonas aeruginosa were cultured in a shaker at 150 rpm at 30°C; Alkali bacillus panlonga and Bacillus subtilis were cultured statically at 37°C; single-strain liquid culture was stopped when the OD600 value was between 3.0 and 4.0.

[0058] The method for scaling up the culture is as follows: Inoculate the bacterial broth obtained from the liquid seed culture into the fermenter at an inoculation rate of 10% of the liquid culture medium volume. The composition of the liquid culture medium in the fermenter should be consistent with that used in the liquid seed culture. *Aspergillus niger*, *Trichoderma viride*, and *Pseudomonas aeruginosa* are stirred at 180 rpm in the fermenter at 30°C with a sterile air-to-ventilation ratio of 1:1 until the effective viable count in the individual fermentation broth of each strain is >5 × 10⁻⁶. 9 CFU / mL;

[0059] Alkali bacillus and Bacillus subtilis were cultured in a fermenter at 37°C until the viable count of each strain in the individual fermentation broth was >8 × 10⁻⁶. 9 CFU / mL. This yields the scale-up fermentation broth for the corresponding bacterial strain;

[0060] S2: Centrifuge the fermentation liquid separately to obtain microbial sludge; mix the microbial sludge with biochar at a mass ratio of 5:1, granulate and dry until the moisture content is ≤15% to obtain the soil remediation microbial agent.

[0061] Comparative Example 1

[0062] This comparative example provides a soil remediation microbial agent, which is made from microbial sludge and biochar. Except for not containing Trichoderma viride, the composition of the microbial sludge and the preparation process of the remediation microbial agent are the same as those in Example 1.

[0063] Comparative Example 2

[0064] This comparative example provides a soil remediation microbial agent, which is made from microbial sludge and biochar. Except for not containing Aspergillus niger, the composition of the microbial sludge and the preparation process of the remediation microbial agent are consistent with those in Example 1.

[0065] Comparative Example 3

[0066] This comparative example provides a soil remediation microbial agent, which is made from microbial sludge and biochar. Except for not containing Pseudomonas, the composition of the microbial sludge and the preparation process of the remediation microbial agent are consistent with those in Example 1.

[0067] Comparative Example 4

[0068] This comparative example provides a soil remediation microbial agent, which is made from microbial sludge and biochar. The mass ratio of Alternaria panlonga, Bacillus subtilis, Trichoderma viride, Aspergillus niger and Pseudomonas aeruginosa in the microbial sludge is 1.0:0.5:1.0:0.8:1.0. The remaining components and the preparation process of the remediation microbial agent are consistent with those in Example 1.

[0069] Example of detection:

[0070] Small-scale tests were conducted on the soil remediation microbial agents prepared in Examples 1-3 and Comparative Examples 1-4. The soil samples were selected from a soil in Hebei Province that needed to be remediated. The soil pH was >7.5 and the main pollutants were Cr (hexavalent) and pesticide DDT.

[0071] The concentrations of major pollutants in the soil before treatment are shown in Table 1 below:

[0072] content 400mg / kg 250μg / kg

[0073] During the treatment process, the area to be remediated was divided into 8 parts, numbered 1-8. Areas 1-7 were sequentially treated with the soil remediation microbial agents prepared in Examples 1-3 and Comparative Examples 1-4, with an application rate of 3 kg / m². 2 Area 8 served as a blank control group. After the remediation microbial agent was applied, the soil was tilled to ensure even mixing with the soil. Following this, the soil underwent routine maintenance, with all eight areas receiving consistent maintenance methods. Four months later, the Cr (hexavalent) and DDT contents in the soil were measured, and the removal rate was calculated. The results are shown in Table 2 below.

[0074] Table 2

[0075]

[0076] It can be seen that the soil remediation microbial agent prepared in this application has outstanding effects in removing hexavalent chromium pollution and pesticide DDT from the soil. It can increase the removal rate of hexavalent chromium to more than 60% and the removal rate of DDT to more than 70% within 4 months.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A soil remediation microbial agent, characterized in that, The soil remediation microbial agent is made from microbial sludge and biochar. The microbial sludge is composed of Pannonibacter phragmitetus, Bacillus subtilis, Trichoderma viride, Aspergillus niger, and Pseudomonas aeruginosa. The preservation number of Pannonibacter phragmitetus is CGMCC No. 3052. The mass ratio of Pannonibacter phragmitetus, Bacillus subtilis, Trichoderma viride, Aspergillus niger, and Pseudomonas aeruginosa in the microbial sludge is 0.3~0.5:1~2:1~2:0.5~1.0:0.8~1.

5.

2. The soil remediation microbial agent as described in claim 1, characterized in that, The effective viable count of each microorganism in the microbial sludge is ≥10. 9 cfu / g.

3. The soil remediation microbial agent as described in claim 1, characterized in that, The biochar is produced by pyrolysis carbonization of agricultural organic waste after crushing and drying.

4. The soil remediation microbial agent as described in claim 3, characterized in that, The agricultural organic waste is placed in a carbonization furnace and pyrolyzed at 400–450°C under closed conditions to obtain the biochar.

5. The method for preparing the soil remediation microbial agent according to any one of claims 1 to 4, characterized in that, Specifically, the following steps are included: S1: Microbial slant seeds of Alkali Lake Panronia, Bacillus subtilis, Trichoderma viride, Aspergillus niger and Pseudomonas aeruginosa are sequentially subjected to liquid seed culture and scale-up culture to obtain the fermentation broth of the corresponding microorganisms; S2: The fermentation broths of *Alternaria panlonga*, *Bacillus subtilis*, *Trichoderma viride*, *Aspergillus niger*, and *Pseudomonas aeruginosa* are centrifuged to obtain the microbial sludge. The microbial sludge is then mixed evenly with the biochar at a specific mass ratio, dried, and granulated to obtain the soil remediation microbial agent.

6. The method for preparing the soil remediation microbial agent as described in claim 5, characterized in that, The method for culturing the microbial slant seeds of Alternaria panlonga, Bacillus subtilis, Trichoderma viride, Aspergillus niger, and Pseudomonas aeruginosa is as follows: Alternaria panlonga, Bacillus subtilis, Trichoderma viride, Aspergillus niger, and Pseudomonas aeruginosa are inoculated onto solid culture medium under aseptic conditions and cultured separately. Alternaria panlonga, Trichoderma viride, Aspergillus niger, and Pseudomonas aeruginosa are cultured at 30°C for 3 days, and Bacillus subtilis is cultured at 37°C for 2 days to obtain the corresponding microbial slant seeds.

7. The method for preparing the soil remediation microbial agent as described in claim 5, characterized in that, Both the liquid seed culture and the scale-up culture process use liquid culture medium, wherein the liquid culture medium for Bacillus paeonii, Bacillus subtilis and Pseudomonas pyrenoidosa includes the following components in parts by weight: 10 parts tryptone, 5 parts yeast extract, 10 parts sodium chloride and 1000 parts distilled water. The culture medium for *Trichoderma viride* comprises the following components in parts by weight: 10 parts potato extract, 20 parts glucose, 3 parts potassium dihydrogen phosphate, 1.5 parts magnesium sulfate, 15 parts agar, and 1000 parts distilled water. The culture medium for Aspergillus niger comprises the following components in parts by weight: 130 parts malt extract, 0.1 parts chloramphenicol, 15 parts agar, and 1000 parts distilled water.

8. The method for preparing the soil remediation microbial agent as described in claim 5, characterized in that, During the liquid seed culture process, Aspergillus niger, Trichoderma viride, and Pseudomonas aeruginosa were cultured in a shaker at 30°C and 150 rpm; while Alternaria panlonga and Bacillus subtilis were cultured statically at 37°C. The culture was stopped when the OD600 value of each single-strain liquid culture was between 3.0 and 4.

0.

9. The method for preparing the soil remediation microbial agent as described in claim 5, characterized in that, The expanded culture conditions were as follows: Aspergillus niger, Trichoderma viride, and Pseudomonas aeruginosa were cultured in a fermenter at 30°C with a stirring speed of 150-200 rpm and a sterile air-to-ventilation ratio of 1:1, until the viable count of each strain in the individual fermentation broth was >5 × 10⁻⁶. 9 CFU / mL; Alkali bacillus and Bacillus subtilis were cultured in a fermenter at 37°C until the viable count of each strain in the individual fermentation broth was >8 × 10⁻⁶. 9 CFU / mL.

10. The method for preparing the soil remediation microbial agent as described in claim 5, characterized in that, The biochar and microbial sludge described in S2 are mixed at a mass ratio of 3~5:1, stirred evenly, granulated, and dried until the water content is ≤15% to obtain the soil remediation microbial agent.

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