Microbial remediation agent for heavy metal contaminated soil and preparation method of microbial remediation agent

Abstract

The invention relates to the technical field of soil remediation agents, in particular to a microbial remediation agent for heavy metal contaminated soil and a preparation method of the microbial remediation agent. The preparation method comprises the following steps: S1, uniformly mixing an inorganic carrier and modified charcoal to obtain a composite carrier; s2, respectively placing the composite carriers in a bacillus megaterium fermentation broth and a bacillus cereus fermentation broth, oscillating for 3-6 hours, separating, and drying to obtain a composite carrier containing bacillus megaterium and a composite carrier containing bacillus cereus; s3, uniformly mixing the composite carrier containing the bacillus megatherium and the composite carrier containing the bacillus cereus to obtain the microbial remediation agent. The prepared microbial remediation agent can efficiently remove various heavy metals in polluted soil, and the effect of soil remediation is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of soil remediation agents, and particularly to a microbial remediation agent for heavy metal contaminated soil and a preparation method thereof. Background Art

[0002] Heavy metal pollution is one of the main problems faced in the current environmental protection field. Especially under the background of the accelerating industrialization process, the heavy metal pollution in the soil is becoming increasingly serious. Heavy metals such as lead, cadmium, arsenic, and mercury are not only harmful to plant growth but also affect human health through the food chain. Traditional treatment methods such as physical and chemical methods are often costly and may cause further damage to the soil ecosystem. Therefore, microbial remediation technology, as a new emerging green remediation method, has received extensive attention in recent years.

[0003] Microbial remediation refers to the use of the metabolic characteristics of microorganisms to promote the removal or stabilization of heavy metals, thereby achieving the remediation of contaminated soil. Some microorganisms can convert heavy metals into less toxic forms or reduce their bioavailability through bioadsorption, biotransformation, and biosorption. However, microbial remediation technology is affected by environmental factors for the growth and colonization of microorganisms, and there will be competition with indigenous microorganisms. If they cannot adapt to the environment, they will be eliminated. Therefore, it is necessary to find a suitable carrier for microorganisms to improve the survival rate of microorganisms in contaminated soil and enhance the microbial remediation efficiency.

[0004] Based on this, we propose a microbial remediation agent for heavy metal contaminated soil and a preparation method thereof. Summary of the Invention

[0005] The purpose of the present invention is to provide a microbial remediation agent for heavy metal contaminated soil and a preparation method thereof to solve the problems raised in the prior art.

[0006] To achieve the above purpose, the present invention provides the following technical solutions: A preparation method of a microbial remediation agent for heavy metal contaminated soil, comprising the following steps: Step S1: Mix an inorganic carrier and modified biochar evenly to obtain a composite carrier; Step S2: Place the composite carrier in the fermentation broth of Bacillus megaterium and the fermentation broth of Bacillus cereus respectively, shake for 3 - 6 h, separate and dry to obtain a composite carrier containing Bacillus megaterium and a composite carrier containing Bacillus cereus; Step S3: Mix the composite carrier containing Bacillus megaterium and the composite carrier containing Bacillus cereus evenly to obtain a microbial remediation agent.

[0007] Further, the mass ratio of the inorganic carrier to the modified biochar is 1:(1 - 3).

[0008] Further, the preparation method of the modified biochar is as follows: Step (1): Under nitrogen protection, mix the aqueous alcohol solution and ferrous sulfate heptahydrate evenly, add the humic acid solution, stir evenly, then add the biochar, and dropwise add the sodium borohydride solution. Finish dropping in 30 - 40 min, stir for 1 - 2 h, stand for aging for 4 - 6 h, centrifuge, wash, and dry to obtain the substrate; Step (2): Mix concentrated ammonia water and epichlorohydrin evenly, react at 60 - 70 °C for 3 - 5 h, add the substrate and sodium hydroxide, react at 105 - 115 °C for 8 - 10 h, then filter, wash, and dry to obtain the aminated substrate; Step (3): Add the aminated substrate to the ethanol solution of glutaraldehyde, react for 6 - 8 h, filter, wash, and dry to obtain the aldehyde - based substrate; Step (4): Add the aldehyde - based substrate to the lysine solution, react at 40 - 50 °C for 6 - 12 h, cool to room temperature, add sodium borohydride, continue to react for 3 - 5 h, then add NaOH and carbon disulfide, first react at 20 - 30 °C for 20 - 40 min, then transfer to a water bath at 40 - 50 °C and continue to react for 4 - 8 h, cool to room temperature, filter, wash, and dry to obtain the modified biochar.

[0009] In the above technical solution, first, through the complexation of humic acid and Fe 2+ complex, then use the liquid - phase reduction method to load zero - valent iron (nZVI) onto the biochar, effectively increasing the specific surface area of the biochar; then use the reaction of concentrated ammonia water and epichlorohydrin to generate (S) - 1 - amino - 3 - chloropropan - 2 - ol, and then use - Cl to undergo a substitution reaction with the hydroxyl groups on the surface of the substrate to graft the amino group onto the surface of the substrate to obtain the aminated substrate; then, use the cross - linking effect of glutaraldehyde to combine the aminated substrate and lysine, introduce the amino group, and finally react with carbon disulfide to introduce the dithiocarboxyl group to obtain the aldehyde - based substrate.

[0010] Further, in the step (1), the mass ratio of the aqueous alcohol solution, ferrous sulfate heptahydrate, and the humic acid solution is 1:(0.02 - 0.05):(0.25 - 0.50).

[0011] Further, the aqueous alcohol solution is composed of deionized water and ethanol mixed according to a mass ratio of 7:3.

[0012] Further, the preparation method of the humic acid solution is as follows: Mix the humic acid and sodium hydroxide solution evenly, and use hydrochloric acid solution to adjust the pH = 7 to obtain the humic acid solution.

[0013] Further, the mass ratio of the humic acid and the sodium hydroxide solution is 1:(200 - 500).

[0014] Furthermore, the concentration of the sodium hydroxide solution is 0.1 mol / L, and the concentration of the hydrochloric acid solution is 0.1 mol / L.

[0015] Furthermore, the mass of the biochar is 0.2 - 0.4 times the mass of ferrous sulfate heptahydrate, the concentration of the sodium borohydride solution is 0.5 mol / L, and its dosage is 0.8 - 1.0 times that of the aqueous alcohol solution.

[0016] Furthermore, in the step (2), the mass ratio of concentrated ammonia water, epichlorohydrin, matrix, and sodium hydroxide is 1:(3 - 4):(0.2 - 0.4):(0.03 - 0.05).

[0017] Furthermore, in the step (3), the mass ratio of the aminated matrix to the ethanol solution of glutaraldehyde is 1:(50 - 100).

[0018] Furthermore, the concentration of glutaraldehyde in the ethanol solution of glutaraldehyde is 3 - 5 g / L.

[0019] Furthermore, in the step (4), the mass ratio of the aldehyde - functionalized matrix to the lysine solution is 1:(15 - 30).

[0020] Furthermore, the concentration of the lysine solution is 20 - 40 wt%, and the solvent is deionized water.

[0021] Furthermore, the dosage of sodium borohydride is 0.3 - 0.5 times the mass of the aldehyde - functionalized matrix.

[0022] Furthermore, the molar ratio of amino groups, NaOH, and carbon disulfide in the lysine solution is 1:1:(1.0 - 1.2).

[0023] Furthermore, the inorganic carrier is prepared by calcining one or a mixture of diatomite, sepiolite, and dolomite.

[0024] Furthermore, the preparation methods of the Bacillus megaterium fermentation broth and the Bacillus cereus fermentation broth are as follows: Inoculate Bacillus megaterium and Bacillus cereus into solid media respectively, activate at 25 - 37 °C for 16 - 26 h, then inoculate into seed media, and culture on a shaker at 180 - 300 rmp at 25 - 37 °C for 16 - 36 h, and then perform liquid fermentation in liquid media for 3 - 5 d to obtain the Bacillus megaterium fermentation broth and the Bacillus cereus fermentation broth.

[0025] Furthermore, the solid medium is: beef extract 3 - 5 g / L, peptone 10 - 15 g / L, sodium chloride 3 - 5 g / L, agar 15 - 20 g / L, pH = 7.0 - 7.4.

[0026] Furthermore, the seed culture medium is: beef extract 3 - 5 g / L, peptone 10 - 15 g / L, sodium chloride 3 - 5 g / L, pH = 7.0 - 7.4.

[0027] Furthermore, the liquid culture medium is: glucose 10 - 15 g / L, yeast extract powder 2 - 3 g / L, ammonium sulfate 1 - 2 g / L, dipotassium hydrogen phosphate 2 - 3 g / L, magnesium sulfate heptahydrate 0.1 - 0.2 g / L, calcium chloride 0.1 - 0.2 g / L, pH = 7.0 - 7.4.

[0028] Furthermore, the liquid fermentation process conditions are: fermentation temperature 25 - 35 °C, stirring speed 120 - 600 rmp.

[0029] Furthermore, the mass ratio of the composite carrier containing Bacillus megaterium to the composite carrier containing Bacillus cereus is 1:(1 - 2).

[0030] Furthermore, the effective viable count of the microbial remediator in step S4 is 1×10 9 -1×10 10 CFU / g.

[0031] An application of a microbial remediator for heavy metal - contaminated soil, comprising the following steps: Sow ryegrass seeds into the heavy metal - contaminated soil, maintain appropriate humidity and temperature. After 10 - 20 days, when the plants germinate and grow, add the microbial remediator, and jointly remove heavy metals in the soil by utilizing the absorption of heavy metals by ryegrass and the strengthening effect of the microbial remediator.

[0032] Furthermore, the seeding rate of the ryegrass seeds is 2 - 4 g / m 2 .

[0033] Furthermore, the appropriate temperature is 15 - 35 °C, and water is sprayed and irrigated once every 10 - 24 h to keep the soil water content at 60 - 80% of the maximum water - holding capacity in the field.

[0034] Compared with the prior art, the beneficial effects of the present invention are: 1. For the microbial remediator for heavy metal - contaminated soil and its preparation method of the present invention, humic acid is introduced into biochar. Its rich carboxyl, phenolic hydroxyl and other functional groups form chemical bonding with the biochar surface. At the same time, through the complexation of humic acid with Fe 2+ nanoscale zero - valent iron (nZVI) particles are uniformly loaded on the biochar surface, reducing the agglomeration and oxidation of nZVI, effectively increasing the specific surface area of biochar; also, the rich functional groups of humic acid can complex metal ions, thereby enhancing the adsorption of heavy metals by zero - valent iron - modified biochar; React concentrated ammonia water with epichlorohydrin to generate (S)-1-amino-3-chloropropan-2-ol. Through the substitution reaction of -Cl with the hydroxyl groups on the substrate surface, graft the amino group onto the substrate surface to obtain an aminated substrate; utilize the aldehyde group of glutaraldehyde to react with the amino group, graft the aminated substrate and lysine onto glutaraldehyde, ensuring that each amino group reacts with only one glutaraldehyde molecule during the crosslinking process to avoid the problem of pore blockage caused by over-crosslinking; at the same time, retain the remaining aldehyde groups for subsequent lysine grafting. The introduced lysine (diamino compound) molecule serves as a flexible spacer arm, and through its remaining amino group, NaOH, and carbon disulfide (CS 2 ) reaction for modification, graft the sulfur-containing group dithiocarboxyl (—C(=S)—S - ) onto the molecular chain of lysine, which can chelate with heavy metal ions such as Cu 2+ , Pb 2+ etc., significantly improving the selective adsorption of highly toxic heavy metals.

[0035] 2. A microbial remediation agent for heavy metal contaminated soil and its preparation method according to the present invention, through the combination of an inorganic carrier and modified biochar, improves the survival rate and activity of microorganisms, making its remediation effect in contaminated soil more significant. At the same time, it can effectively adsorb and remove heavy metal ions in the soil, thereby reducing the pollution level of the soil and improving the soil environment. Detailed implementation manners

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0037] In this example, Bacillus megaterium: model BMZ20280, sourced from Ningbo Mingzhou Biotechnology Co., Ltd.; Bacillus cereus: model BMZ135313, sourced from Ningbo Mingzhou Biotechnology Co., Ltd.; biochar: corn straw biochar, with a particle size of 200 mesh, sourced from Henan Jiahe Water Purification Materials Co., Ltd.; diatomite: model CD01-140, with a particle size of 100 mesh, sourced from Shengzhou Huali Diatomite Products Co., Ltd.; ryegrass seeds are commercially available grass seeds; the heavy metal-contaminated soil is taken from the polluted soil near an industrial area in Nanjing, with the debris removed, passed through a 60-mesh sieve, sterilized, and the Cd content in the soil is detected to be 65.22 - 68.19 mg / kg, the Pb content is 166.34 - 169.60 mg / kg, the Hg content is 1.22 - 1.25 mg / kg, and the pH value is 6.71 - 6.73.

[0038] Unless otherwise specified, the following parts are by mass.

[0039] The preparation methods of the fermentation broth of Bacillus megaterium and Bacillus cereus used in this example are as follows: Inoculate Bacillus megaterium and Bacillus cereus into a solid medium respectively, activate at 35°C for 24 h, then inoculate into a seed medium, and culture on a shaker at 200 rmp at 35°C for 24 h, and then place in a liquid medium for liquid fermentation for 4 d, with a fermentation temperature of 30°C and a stirring speed of 400 rmp to obtain the fermentation broth of Bacillus megaterium and the fermentation broth of Bacillus cereus; The solid medium is: beef extract 3 g / L, peptone 10 g / L, sodium chloride 3 g / L, agar 15 g / L, pH = 7.0; The seed medium is: beef extract 3 g / L, peptone 10 g / L, sodium chloride 3 g / L, pH = 7.0; The liquid medium is: glucose 10 g / L, yeast extract powder 2 g / L, ammonium sulfate 1 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.1 g / L, calcium chloride 0.1 g / L, pH = 7.0.

[0040] Example 1: A preparation method of a microbial remediator for heavy metal-contaminated soil, including the following processes: Step S1: Calcinate diatomite at 1200°C for 1 h to obtain an inorganic carrier, and mix the inorganic carrier passed through a 200-mesh sieve and the modified biochar evenly according to a mass ratio of 1:1 to obtain a composite carrier; Step S2: Place the composite carrier into the fermentation broth of Bacillus megaterium and Bacillus cereus respectively. After shaking for 3 h, separate and dry to obtain a composite carrier containing Bacillus megaterium and a composite carrier containing Bacillus cereus; Step S3: Mix the composite carrier containing Bacillus megaterium and the composite carrier containing Bacillus cereus evenly at a mass ratio of 1:1 to obtain a microbial repair agent; The preparation method of the modified biochar is as follows: Step (1): Mix 0.2 parts of humic acid and 40 parts of 0.1 mol / L sodium hydroxide solution evenly, and adjust the pH to 7 with 0.1 mol / L hydrochloric acid solution to obtain a humic acid solution; Under nitrogen protection, mix 100 parts of an alcohol aqueous solution and 2 parts of ferrous sulfate heptahydrate evenly, add 25 parts of the humic acid solution, stir evenly, then add 0.4 parts of biochar, dropwise add 80 parts of 0.5 mol / L sodium borohydride solution, finish dropping in 30 min, stir for 1 h, stand and age for 4 h, and after centrifugation, washing and drying, obtain a matrix; Step (2): Mix 2 parts of 25 wt% concentrated ammonia water and 6 parts of epichlorohydrin evenly, react in a 60 °C water bath for 3 h, add 0.4 parts of the matrix and 0.06 parts of sodium hydroxide, react in a 105 °C oil bath for 8 h and then filter, wash and dry to obtain an aminated matrix; Step (3): Add 0.4 parts of the aminated matrix to 20 parts of an ethanol solution containing 3 g / L glutaraldehyde, react for 6 h, filter, wash and dry to obtain an aldehyde-grouped matrix; Step (4): Add 0.4 parts of the aldehyde-grouped matrix to 6 parts of a 20 wt% lysine solution, react at 40 °C for 6 h, cool to room temperature, add 0.12 parts of sodium borohydride, continue to react for 3 h, then add NaOH and carbon disulfide, first react at 20 °C for 20 min, then transfer to a 40 °C water bath and continue to react for 4 h, cool to room temperature, filter, wash and dry to obtain the modified biochar; The molar ratio of the amino group in the lysine solution, NaOH and carbon disulfide is 1:1:1; The application of a microbial repair agent for heavy metal contaminated soil includes the following steps: Sow ryegrass seeds into the heavy metal contaminated soil, and the seeding rate is 2 g / m 2 , maintain appropriate humidity and temperature (the appropriate temperature is 15 °C, spray and irrigate once every 10 h, and keep the soil water content at 60% of the maximum water holding capacity of the field). After 20 days, wait for the plants to germinate and grow, then add a microbial repair agent with an application rate of 80 kg / mu each. Utilize the absorption of heavy metals by ryegrass and the strengthening effect of the microbial repair agent to jointly remove heavy metals in the soil, and continue to culture for 45 days to obtain the treated soil.

[0041] Example 2: A preparation method of a microbial remediation agent for heavy metal contaminated soil, comprising the following processes: Step S1: Calcine diatomite at 1200 °C for 1 h to obtain an inorganic carrier, and uniformly mix the inorganic carrier passing through a 200-mesh sieve and modified biochar in a mass ratio of 1:2 to obtain a composite carrier; Step S2: Place the composite carrier in the fermentation broth of Bacillus megaterium and the fermentation broth of Bacillus cereus respectively, shake for 5 h, separate and dry to obtain a composite carrier containing Bacillus megaterium and a composite carrier containing Bacillus cereus; Step S3: Uniformly mix the composite carrier containing Bacillus megaterium and the composite carrier containing Bacillus cereus in a mass ratio of 1:1.5 to obtain a microbial remediation agent; The preparation method of the modified biochar is as follows: Step (1): Uniformly mix 0.1 part of humic acid and 40 parts of 0.1 mol / L sodium hydroxide solution, adjust the pH to 7 with 0.1 mol / L hydrochloric acid solution to obtain a humic acid solution; under nitrogen protection, uniformly mix 100 parts of alcohol aqueous solution and 3 parts of ferrous sulfate heptahydrate, add 40 parts of humic acid solution, stir evenly, then add 0.9 part of biochar, dropwise add 90 parts of 0.5 mol / L sodium borohydride solution, finish dropping in 35 min, stir for 1.5 h, stand and age for 5 h, centrifuge, wash and dry to obtain a matrix; Step (2): Uniformly mix 3 parts of 25 wt% concentrated ammonia water and 10.5 parts of epichlorohydrin, react in a 65 °C water bath for 4 h, add 1 part of the matrix and 0.12 part of sodium hydroxide, react in an 110 °C oil bath for 9 h and then filter, wash and dry to obtain an aminated matrix; Step (3): Add 1 part of the aminated matrix to 70 parts of an ethanol solution with a concentration of 4 g / L glutaraldehyde, react for 7 h, filter, wash and dry to obtain an aldehyde-grouped matrix; Step (4): Add 1 part of the aldehyde-grouped matrix to 25 parts of a 30 wt% lysine solution, react at 45 °C for 10 h, cool to room temperature, add 0.4 part of sodium borohydride, continue to react for 4 h, then add NaOH and carbon disulfide, first react at 25 °C for 30 min, then transfer to a 45 °C water bath and continue to react for 6 h, cool to room temperature, filter, wash and dry to obtain modified biochar; the molar ratio of amino group, NaOH and carbon disulfide in the lysine solution is 1:1:1.1; An application of a microbial remediation agent for heavy metal contaminated soil, comprising the following steps: Sow ryegrass seeds into the heavy metal contaminated soil, and the seeding rate is 3 g / m 2, maintain appropriate humidity and temperature (the appropriate temperature is 30°C, spray and irrigate once every 16 hours, and keep the soil water content at 70% of the maximum field water holding capacity). After 15 days, wait for the plants to germinate and grow, then add a microbial remediation agent with a dosage of 80 kg / mu. Utilize the absorption of heavy metals by ryegrass and the strengthening effect of the microbial remediation agent to jointly remove heavy metals from the soil, and continue to culture for 45 days to obtain the treated soil.

[0042] Example 3: A preparation method of a microbial remediation agent for heavy metal contaminated soil, including the following processes: Step S1: Calcinate diatomite at 1200°C for 1 hour to obtain an inorganic carrier, and mix the inorganic carrier passing through a 200-mesh sieve and modified biochar evenly according to a mass ratio of 1:3 to obtain a composite carrier; Step S2: Place the composite carrier in the fermentation broth of Bacillus megaterium and the fermentation broth of Bacillus cereus respectively, shake for 6 hours, separate and dry to obtain a composite carrier containing Bacillus megaterium and a composite carrier containing Bacillus cereus; Step S3: Mix the composite carrier containing Bacillus megaterium and the composite carrier containing Bacillus cereus evenly according to a mass ratio of 1:2 to obtain a microbial remediation agent; The preparation method of the modified biochar is as follows: Step (1): Mix 0.1 part of humic acid and 50 parts of 0.1 mol / L sodium hydroxide solution evenly, adjust the pH to 7 with 0.1 mol / L hydrochloric acid solution to obtain a humic acid solution; under nitrogen protection, mix 100 parts of alcohol aqueous solution and 5 parts of ferrous sulfate heptahydrate evenly, add 50 parts of the humic acid solution, stir evenly, then add 2 parts of biochar, dropwise add 100 parts of sodium borohydride solution, finish dropping in 40 minutes, stir for 2 hours, stand and age for 6 hours, centrifuge, wash and dry to obtain a matrix; Step (2): Mix 5 parts of 25 wt% concentrated ammonia water and 20 parts of epichlorohydrin evenly, react in a 70°C water bath for 5 hours, add 2 parts of the matrix and 0.25 part of sodium hydroxide, react in an 115°C oil bath for 10 hours, then filter, wash and dry to obtain an aminated matrix; Step (3): Add 2 parts of the aminated matrix to 200 parts of an ethanol solution with a concentration of 5 g / L glutaraldehyde, react for 8 hours, filter, wash and dry to obtain an aldehyde group-containing matrix; Step (4): Add 2 parts of aldehyde-functionalized matrix into 60 parts of 40 wt% lysine solution, react at 50 °C for 12 h. After cooling to room temperature, add 1 part of sodium borohydride and continue to react for 5 h. Then add NaOH and carbon disulfide, first react at 30 °C for 40 min, then transfer to a 50 °C water bath and continue to react for 8 h. After cooling to room temperature, filter, wash, and dry to obtain modified biochar; the molar ratio of amino groups in the lysine solution, NaOH, and carbon disulfide is 1:1:1.2; Application of a microbial remediation agent for heavy metal contaminated soil, comprising the following steps: Sow ryegrass seeds into the heavy metal contaminated soil, with a seeding rate of 4 g / m 2 , maintain appropriate humidity and temperature (the appropriate temperature is 35 °C, spray and irrigate once every 24 h, and keep the soil water content at 80% of the maximum field water holding capacity). After 10 days, when the plants germinate and grow, add a microbial remediation agent with an application rate of 80 kg / mu, and use the absorption of heavy metals by ryegrass and the strengthening effect of the microbial remediation agent to jointly remove heavy metals from the soil, and continue to culture for 45 days to obtain the treated soil.

[0043] Comparative Example 1: A preparation method of a microbial remediation agent for heavy metal contaminated soil, comprising the following process: Compared with Example 2, in Comparative Example 1, the modified biochar is replaced with biochar of the same mass, and other steps are the same as those in Example 2.

[0044] Comparative Example 2: A preparation method of a microbial remediation agent for heavy metal contaminated soil, comprising the following process: Compared with Example 2, Comparative Example 2 does not include steps (3) and (4), and the modified biochar is replaced with an amino-functionalized matrix, and other steps are the same as those in Example 2.

[0045] Comparative Example 3: A preparation method of a microbial remediation agent for heavy metal contaminated soil, comprising the following process: The preparation method of the modified biochar is as follows: Step (1): Mix 3 parts of 25 wt% concentrated ammonia water and 10.5 parts of epichlorohydrin evenly, react in a 65 °C water bath for 4 h, add 1 part of biochar and 0.12 part of sodium hydroxide, react in an 110 °C oil bath for 9 h and then filter, wash, and dry to obtain the amino-functionalized matrix; Step (2): Add 1 part of the amino-functionalized matrix into 70 parts of an ethanol solution with a concentration of 4 g / L glutaraldehyde, react for 7 h, filter, wash, and dry to obtain the aldehyde-functionalized matrix; Step (3): Add 1 part of aldehyde-functionalized matrix into 25 parts of 30 wt% lysine solution, react at 45 °C for 10 h. After cooling to room temperature, add 0.4 part of sodium borohydride and continue to react for 4 h. Then add NaOH and carbon disulfide, first react at 25 °C for 30 min, then transfer to a 45 °C water bath and continue to react for 6 h. After cooling to room temperature, filter, wash, and dry to obtain modified biochar; the molar ratio of amino group in the lysine solution, NaOH, and carbon disulfide is 1:1:1.1; Compared with Example 2, in Comparative Example 3, humic acid and ferrous sulfate heptahydrate are not used to modify the biochar, and other steps are the same as those in Example 2.

[0046] Experiment: 1. Determination of the number of viable bacteria: Take the microbial repair agents obtained in Example 2 and Comparative Examples 1-3, and test them according to the provisions of 6.3.2 in GB20287-2006. The results are shown in Table 1.

[0047] Table 1 Number of viable bacteria in the microbial repair agents of Example 2 and Comparative Examples 1-3

[0048] From the data in the above table, the following conclusions can be clearly obtained: Compared with Example 2, the number of viable bacteria in the products obtained in Comparative Examples 1-3 all decreased. It can be seen that in the present invention, by using modified biochar and inorganic carrier as a composite carrier, a synergistic effect is exerted to adsorb and immobilize the mixed microbial fermentation broth, thereby improving the stability and effectiveness of microorganisms in the soil remediation process.

[0049] 2. Determination of heavy metal removal rate: Take the treated soils obtained in Examples 1-3 and Comparative Examples 1-3, measure the contents of Cd, Pb, and Hg in the soil, calculate the removal rate to characterize the remediation effect of heavy metal-contaminated soil. Determination of cadmium: Detect according to the provisions of GB / T 17141-1997; determination of lead: Detect according to the provisions of GB / T 17141-1997; determination of mercury: Detect according to the provisions of GB / T 22105.1-2008 "Soil quality - Determination of total mercury, total arsenic, and total lead - Atomic fluorescence spectrometry - Part 1: Determination of total mercury in soil". The results are shown in Table 2.

[0050] Table 2 Remediation effects of heavy metal-contaminated soils in Examples 1-3 and Comparative Examples 1-3 (removal rate / %)

[0051] From the data in the above table, the following conclusions can be clearly obtained: 1. Compared with Examples 1 - 3, the repair effects of the products obtained in Comparative Example 1 and Comparative Example 2 both decreased, indicating that the modified biochar prepared by the present invention has more surface oxygen-containing functional groups and specific surface area compared with biochar, significantly enhancing the microbial colonization ability and heavy metal ion adsorption sites; at the same time, in this application, by introducing lysine as a bridge, a sulfur-containing group, dithiocarboxyl, was further introduced, which can form stable dithiocarboxylate chelates with heavy metal ions, improving the chemical fixation efficiency of heavy metals such as cadmium and lead, thereby enhancing the repair effect.

[0052] 2. Compared with Examples 1 - 3, the repair effect of the product obtained in Comparative Example 3 decreased to some extent, which indicates that the modification of biochar with humic acid and ferrous sulfate heptahydrate in the present invention can enhance the ability of biochar in aspects such as soil improvement and pollutant adsorption, thus playing a better role in the application of contaminated soil remediation.

[0053] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention.

Claims

1. A method for preparing a microbial remediation agent for heavy metal contaminated soil, characterized in that: The steps include: Step S1: uniformly mixing the inorganic carrier and the modified biochar to obtain a composite carrier; Step S2: placing the composite carrier in a Bacillus megaterium fermentation broth and a Bacillus cereus fermentation broth respectively, shaking for 3-6 hours, separating and drying to obtain a composite carrier containing Bacillus megaterium and a composite carrier containing Bacillus cereus; Step S3: uniformly mixing the composite carrier containing Bacillus megaterium and the composite carrier containing Bacillus cereus to obtain a microbial repair agent; The preparation process of the modified biochar is as follows: first, the biochar is modified by nano zero-valent iron to obtain a matrix; then, amino groups are grafted by substitution reaction to obtain an amino matrix; the amino matrix and lysine are cross-linked by glutaraldehyde, and finally, the remaining amino groups in lysine are reacted with NaOH and carbon disulfide to graft dithiocarboxyl groups to obtain the modified biochar.

2. The method for preparing a microbial remediation agent for heavy metal contaminated soil according to claim 1, characterized in that: The preparation method of the modified biochar is as follows: Step (1): Under nitrogen protection, the alcohol aqueous solution and ferrous sulfate heptahydrate are mixed evenly, the humic acid solution is added, stirred evenly, the biochar is added, and the sodium borohydride solution is added dropwise, and the dripping is completed within 30-40 minutes, stirred for 1-2 hours, and aged for 4-6 hours. After centrifugation, washing, and drying, a matrix is ​​obtained; Step (2): evenly mix concentrated ammonia water and epichlorohydrin, react at 60-70°C for 3-5h, add the substrate and sodium hydroxide, react at 105-115°C for 8-10h, filter, wash and dry to obtain an amino substrate; Step (3): adding the amino substrate to an ethanol solution of glutaraldehyde, reacting for 6-8 hours, filtering, washing and drying to obtain the aldehyde-modified substrate; Step (4): Add the aldehyde-modified substrate to the lysine solution, react at 40-50°C for 6-12 hours, cool to room temperature, add sodium borohydride, continue to react for 3-5 hours, then add NaOH and carbon disulfide, first react at 20-30°C for 20-40 minutes, then transfer to a 40-50°C water bath and continue to react for 4-8 hours, cool to room temperature, filter, wash and dry to obtain modified biochar.

3. The method for preparing a microbial remediation agent for heavy metal contaminated soil according to claim 2, characterized in that: The preparation method of the humic acid solution is as follows: The humic acid and sodium hydroxide solution were mixed evenly, and the pH value was adjusted to 7 with hydrochloric acid solution to obtain a humic acid solution.

4. The method for preparing a microbial remediation agent for heavy metal contaminated soil according to claim 2, characterized in that: In the step (2), the mass ratio of concentrated aqueous ammonia, epichlorohydrin, substrate and sodium hydroxide is 1:(3-4):(0.15-0.3):(0.03-0.05).

5. The method for preparing a microbial remediation agent for heavy metal contaminated soil according to claim 2, characterized in that: In the step (3), the mass ratio of the amino substrate to the ethanol solution of glutaraldehyde is 1:(50-100).

6. The method for preparing a microbial remediation agent for heavy metal contaminated soil according to claim 2, characterized in that: In the step (4), the molar ratio of amino group, NaOH and carbon disulfide in the lysine solution is 1:1:(1.0-1.2).

7. The method for preparing a microbial remediation agent for heavy metal contaminated soil according to claim 1, characterized in that: The preparation methods of the Bacillus megaterium fermentation broth and the Bacillus cereus fermentation broth are as follows: Bacillus megaterium and Bacillus cereus are inoculated into solid culture medium respectively, activated at 25-37°C for 16-26 hours, then inoculated into seed culture medium, cultured at 25-37°C and 180-300 rpm for 16-36 hours, and then placed in liquid culture medium for liquid fermentation for 3-5 days to obtain Bacillus megaterium fermentation liquid and Bacillus cereus fermentation liquid.

8. The method for preparing a microbial remediation agent for heavy metal contaminated soil according to claim 1, characterized in that: The mass ratio of the composite carrier containing Bacillus megaterium to the composite carrier containing Bacillus cereus is 1:(1-2).

9. A microbial remediation agent for heavy metal contaminated soil prepared according to the preparation method according to any one of claims 1 to 8.

10. An application of the microbial remediation agent for heavy metal contaminated soil according to claim 9, characterized in that: The following steps are involved: Sow ryegrass seeds in heavy metal contaminated soil, maintain suitable humidity and temperature, wait for the plants to germinate and grow after 10-20 days, then add microbial remediation agents, and use the ryegrass's absorption of heavy metals and the strengthening effect of the microbial remediation agents to jointly remove heavy metals from the soil.

Images