A microbial remediation agent for heavy metal contaminated soil and its preparation method

Abstract

The present invention relates to the technical field of soil remediation agents, and specifically relates to a microbial remediation agent for heavy metal-contaminated soil and a preparation method thereof. The present invention comprises the following steps: Step S1: uniformly mixing an inorganic carrier and modified biochar to obtain a composite carrier; Step S2: placing the composite carrier into a fermentation broth of Bacillus megaterium and a fermentation broth of Bacillus cereus 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 remediation agent. The microbial remediation agent prepared by the present invention can efficiently remove various heavy metals in contaminated soil, achieving the effect of soil remediation.

Description

Technical Field

[0001] The present invention relates to the technical field of soil remediation agents, and specifically 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. Certain microorganisms can convert heavy metals into less toxic forms or reduce their bioavailability through biological adsorption, biotransformation, and biological precipitation. 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:

[0007] A preparation method of a microbial remediation agent for heavy metal contaminated soil includes the following steps:

[0008] Step S1: Mix an inorganic carrier and modified biochar evenly to obtain a composite carrier;

[0009] Step S2: Place the composite carrier in the fermentation broth of Bacillus megaterium and the fermentation broth of Bacillus cereus respectively. After shaking for 3 - 6 h, separate and dry to obtain a composite carrier containing Bacillus megaterium and a composite carrier containing Bacillus cereus;

[0010] Step S3: Mix the composite carrier containing Bacillus megaterium and the composite carrier containing Bacillus cereus evenly to obtain a microbial remediation agent.

[0011] Further, the mass ratio of the inorganic carrier to the modified biochar is 1:(1 - 3).

[0012] Further, the preparation method of the modified biochar is as follows:

[0013] 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, finishing the dropping in 30 - 40 min, stir for 1 - 2 h, stand for aging for 4 - 6 h, and after centrifugation, washing, and drying, obtain the matrix;

[0014] Step (2): Mix concentrated ammonia water and epichlorohydrin evenly, react at 60 - 70 °C for 3 - 5 h, add the matrix and sodium hydroxide, react at 105 - 115 °C for 8 - 10 h and then filter, and after washing and drying, obtain the aminated matrix;

[0015] Step (3): Add the aminated matrix into the ethanol solution of glutaraldehyde, react for 6 - 8 h, filter, wash, and dry to obtain the aldehyde - group - containing matrix;

[0016] Step (4): Add the aldehyde - group - containing matrix into 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.

[0017] In the above - mentioned 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 the substitution reaction of - Cl and the hydroxyl groups on the surface of the matrix to graft the amino group onto the surface of the matrix to obtain the aminated matrix; then, use the cross - linking effect of glutaraldehyde to combine the aminated matrix and lysine, introduce the amino group, and finally react with carbon disulfide to introduce the dithiocarboxyl group to obtain the aldehyde - group - containing matrix.

[0018] 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).

[0019] Further, the aqueous alcohol solution is composed of deionized water and ethanol mixed in a mass ratio of 7:3.

[0020] Further, the preparation method of the humic acid solution is as follows: Mix humic acid and sodium hydroxide solution evenly, and adjust the pH to 7 with hydrochloric acid solution to obtain the humic acid solution.

[0021] Further, the mass ratio of the humic acid to the sodium hydroxide solution is 1:(200 - 500).

[0022] Further, 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.

[0023] Further, 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 alcohol aqueous solution.

[0024] Further, in the step (2), the mass ratio of concentrated ammonia water, epichlorohydrin, the matrix, and sodium hydroxide is 1:(3 - 4):(0.2 - 0.4):(0.03 - 0.05).

[0025] Further, in the step (3), the mass ratio of the amino-functionalized matrix to the ethanol solution of glutaraldehyde is 1:(50 - 100).

[0026] Further, the concentration of glutaraldehyde in the ethanol solution of glutaraldehyde is 3 - 5 g / L.

[0027] Further, in the step (4), the mass ratio of the aldehyde-functionalized matrix to the lysine solution is 1:(15 - 30).

[0028] Further, the concentration of the lysine solution is 20 - 40 wt%, and the solvent is deionized water.

[0029] Further, the dosage of sodium borohydride is 0.3 - 0.5 times the mass of the aldehyde-functionalized matrix.

[0030] Further, the molar ratio of amino groups, NaOH, and carbon disulfide in the lysine solution is 1:1:(1.0 - 1.2).

[0031] Further, the inorganic carrier is prepared by calcining one or a mixture of diatomite, sepiolite, and dolomite.

[0032] Further, the preparation methods of the Bacillus megaterium fermentation broth and the Bacillus cereus fermentation broth are as follows:

[0033] Bacillus megaterium and Bacillus cereus were respectively inoculated into solid medium and activated at 25 - 37°C for 16 - 26 h, then inoculated into seed medium and cultured on a shaker at 180 - 300 rmp at 25 - 37°C for 16 - 36 h, and then placed in liquid medium for liquid fermentation for 3 - 5 d to obtain Bacillus megaterium fermentation broth and Bacillus cereus fermentation broth.

[0034] 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.

[0035] Furthermore, the seed medium is: beef extract 3 - 5 g / L, peptone 10 - 15 g / L, sodium chloride 3 - 5 g / L, pH = 7.0 - 7.4.

[0036] Furthermore, the liquid 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.

[0037] Furthermore, the liquid fermentation process conditions are: fermentation temperature 25 - 35°C, stirring speed 120 - 600 rmp.

[0038] Furthermore, the mass ratio of the composite carrier containing Bacillus megaterium to the composite carrier containing Bacillus cereus is 1:(1 - 2).

[0039] Furthermore, the effective viable count of the microbial remediator in step S4 is 1×10 9 -1×10 10 CFU / g.

[0040] An application of a microbial remediator for heavy metal - contaminated soil, comprising the following steps:

[0041] Sow ryegrass seeds into 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 the absorption of heavy metals by ryegrass and the strengthening effect of the microbial remediator.

[0042] Furthermore, the seeding rate of the ryegrass seeds is 2 - 4 g / m 2 .

[0043] Furthermore, the appropriate temperature is 15 - 35°C, and water is sprayed and irrigated every 10 - 24 h to keep the soil water content at 60 - 80% of the maximum water - holding capacity of the field.

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0045] 1. For a microbial remediation agent for heavy metal contaminated soil and its preparation method according to the present invention, humic acid is introduced into biochar. Its abundant functional groups such as carboxyl groups and phenolic hydroxyl groups form chemical bonding with the biochar surface. At the same time, through the complexation of humic acid and Fe 2+ nanoscale zero-valent iron (nZVI) particles are evenly loaded on the biochar surface through complexation, reducing the agglomeration and oxidation of nZVI, effectively increasing the specific surface area of the biochar; the abundant functional groups of humic acid can also complex metal ions, thereby enhancing the adsorption of heavy metals by zero-valent iron modified biochar;

[0046] Then, by reacting concentrated ammonia water and epichlorohydrin, (S)-1-amino-3-chloropropan-2-ol is generated. Through the substitution reaction of -Cl and the hydroxyl groups on the matrix surface, the amino group is grafted onto the matrix surface to obtain an aminated matrix; by reacting the aldehyde group of glutaraldehyde with the amino group, the aminated matrix and lysine are grafted onto glutaraldehyde, ensuring that each amino group reacts with only one glutaraldehyde molecule during the crosslinking process, avoiding the problem of pore blockage caused by over-crosslinking; at the same time, the remaining aldehyde groups are reserved for subsequent lysine grafting. The introduced lysine (diamino compound) molecule is used as a flexible spacer arm, and through the reaction of its remaining amino group, NaOH and carbon disulfide (CS2), it is modified to 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+ and Pb 2+ and significantly improve the selective adsorption of highly toxic heavy metals.

[0047] 2. For 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, the survival rate and activity of microorganisms are improved, making the 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. Specific Embodiments

[0048] 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 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.

[0049] 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, particle size 200 mesh, sourced from Henan Jiahe Water Purification Materials Co., Ltd.; diatomite: model CD01-140, particle size 100 mesh, sourced from Shengzhou Huali Diatomite Products Co., Ltd.; ryegrass seeds are commercially available grass seeds; heavy metal-contaminated soil is taken from polluted soil near an industrial area in Nanjing, debris in the soil is removed, it is sieved through a 60-mesh sieve, sterilized, and the Cd content in the soil is detected to be 65.22 - 68.19 mg / kg, Pb content 166.34 - 169.60 mg / kg, Hg content 1.22 - 1.25 mg / kg, pH value 6.71 - 6.73.

[0050] Unless otherwise specified, the following parts are by mass.

[0051] The preparation methods of the fermentation broth of Bacillus megaterium and Bacillus cereus used in this example are as follows:

[0052] Bacillus megaterium and Bacillus cereus are respectively inoculated into a solid medium and activated at 35°C for 24 h, then inoculated into a seed medium and cultured on a shaker at 200 rmp at 35°C for 24 h, and then placed in a liquid medium for liquid fermentation for 4 d, fermentation temperature 30°C, stirring speed 400 rmp, to obtain the fermentation broth of Bacillus megaterium and the fermentation broth of Bacillus cereus;

[0053] 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;

[0054] The seed medium is: beef extract 3 g / L, peptone 10 g / L, sodium chloride 3 g / L, pH = 7.0;

[0055] 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.

[0056] Example 1: A preparation method of a microbial remediator for heavy metal-contaminated soil, comprising the following processes:

[0057] Step S1: Calcinate diatomite at 1200°C for 1 h to obtain an inorganic carrier, and uniformly mix the inorganic carrier sieved through a 200-mesh sieve and modified biochar in a mass ratio of 1:1 to obtain a composite carrier;

[0058] Step S2: Place the composite carrier in the fermentation broth of *Bacillus megaterium* and the fermentation broth of *Bacillus cereus* respectively. After shaking for 3 h, separate and dry to obtain the composite carrier containing *Bacillus megaterium* and the composite carrier containing *Bacillus cereus*;

[0059] Step S3: Mix the composite carrier containing *Bacillus megaterium* and the composite carrier containing *Bacillus cereus* evenly according to the mass ratio of 1:1 to obtain the microbial remediator;

[0060] The preparation method of the modified biochar is as follows:

[0061] 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 the humic acid solution; Under nitrogen protection, mix 100 parts of 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 the matrix;

[0062] 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 the aminated matrix;

[0063] Step (3): Add 0.4 parts of the aminated matrix to 20 parts of an ethanol solution with a concentration of 3 g / L glutaraldehyde, react for 6 h, filter, wash and dry to obtain the aldehyde-grouped matrix;

[0064] Step (4): Add 0.4 parts of the aldehyde-grouped matrix to 6 parts of 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 amino group, NaOH and carbon disulfide in the lysine solution is 1:1:1;

[0065] The application of a microbial remediator for heavy metal contaminated soil includes the following steps:

[0066] 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 hours, and keep the soil water content at 60% of the maximum field water holding capacity). After 20 days, wait for the plants to germinate and grow, and then add a microbial remediation 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 remediation agent to jointly remove heavy metals from the soil, and continue to cultivate for 45 days to obtain the treated soil.

[0067] Example 2: A preparation method of a microbial remediation agent for heavy metal contaminated soil, including the following processes:

[0068] Step S1: Calcinate diatomite at 1200°C for 1 hour to obtain an inorganic carrier. Mix the inorganic carrier passing through a 200-mesh sieve and modified biochar evenly according to a mass ratio of 1:2 to obtain a composite carrier;

[0069] Step S2: Place the composite carrier in the fermentation broth of Bacillus megaterium and the fermentation broth of Bacillus cereus respectively. After shaking for 5 hours, separate and dry to obtain a composite carrier containing Bacillus megaterium and a composite carrier containing Bacillus cereus;

[0070] Step S3: Mix the composite carrier containing Bacillus megaterium and the composite carrier containing Bacillus cereus evenly according to a mass ratio of 1:1.5 to obtain a microbial remediation agent;

[0071] The preparation method of the modified biochar is as follows:

[0072] Step (1): Mix 0.1 part of humic acid and 40 parts of 0.1 mol / L sodium hydroxide solution evenly, and adjust the pH = 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 3 parts of ferrous sulfate heptahydrate evenly, add 40 parts of the 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 minutes, stir for 1.5 hours, stand and age for 5 hours, centrifuge, wash and dry to obtain a matrix;

[0073] Step (2): 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 hours, add 1 part of the matrix and 0.12 part of sodium hydroxide, react in an 110°C oil bath for 9 hours, then filter, wash and dry to obtain an aminated matrix;

[0074] 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 hours, filter, wash and dry to obtain an aldehyde-based matrix;

[0075] Step (4): Add 1 part of aldehyde-functionalized matrix to 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, 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 groups, NaOH, and carbon disulfide in the lysine solution is 1:1:1.1;

[0076] Application of a microbial remediation agent for heavy metal contaminated soil, comprising the following steps:

[0077] Sow ryegrass seeds into the heavy metal contaminated soil, the seeding rate is 3 g / m 2 , maintain suitable humidity and temperature (the suitable temperature is 30 °C, spray and irrigate once every 16 h, keep the soil water content at 70% of the maximum water holding capacity of the field), after 15 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 in the soil, continue to cultivate for 45 days to obtain the treated soil.

[0078] Example 3: A preparation method of a microbial remediation agent for heavy metal contaminated soil, comprising the following processes:

[0079] Step S1: Calcinate diatomite at 1200 °C for 1 h to obtain an inorganic carrier, mix the inorganic carrier passing through a 200-mesh sieve and the modified biochar evenly according to a mass ratio of 1:3 to obtain a composite carrier;

[0080] Step S2: Place the composite carrier in the fermentation broth of Bacillus megaterium and the fermentation broth of Bacillus cereus respectively, shake for 6 h, separate and dry to obtain a composite carrier containing Bacillus megaterium and a composite carrier containing Bacillus cereus;

[0081] 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;

[0082] The preparation method of the modified biochar is as follows:

[0083] Step (1): Mix 0.1 part of humic acid and 50 parts of 0.1 mol / L sodium hydroxide solution evenly, adjust the pH = 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 humic acid solution, stir evenly, then add 2 parts of biochar, dropwise add 100 parts of sodium borohydride solution, finish dropping in 40 min, stir for 2 h, stand and age for 6 h, centrifuge, wash, and dry to obtain a matrix;

[0084] 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 h, add 2 parts of the matrix and 0.25 part of sodium hydroxide, react in an 115 °C oil bath for 10 h, then filter. After washing and drying, an aminated matrix is obtained;

[0085] Step (3): Add 2 parts of the aminated matrix to 200 parts of an ethanol solution containing 5 g / L of glutaraldehyde, react for 8 h, filter, wash, and dry to obtain an aldehyde-group modified matrix;

[0086] Step (4): Add 2 parts of the aldehyde-group modified matrix to 60 parts of a 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 a modified biochar; the molar ratio of the amino group in the lysine solution, NaOH, and carbon disulfide is 1:1:1.2;

[0087] Application of a microbial remediation agent for heavy metal contaminated soil, comprising the following steps:

[0088] Sow ryegrass seeds into the heavy metal contaminated soil, with a seeding rate of 4 g / m 2 , maintain suitable humidity and temperature (the suitable 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. Utilize the absorption of heavy metals by ryegrass and the strengthening effect of the microbial remediation agent to jointly remove heavy metals in the soil, and continue to cultivate for 45 days to obtain the treated soil.

[0089] Comparative Example 1: A preparation method of a microbial remediation agent for heavy metal contaminated soil, comprising the following process:

[0090] 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.

[0091] Comparative Example 2: A preparation method of a microbial remediation agent for heavy metal contaminated soil, comprising the following process:

[0092] Compared with Example 2, in Comparative Example 2, steps (3) and (4) are not included, and the modified biochar is replaced with the aminated matrix, and other steps are the same as those in Example 2.

[0093] Comparative Example 3: A preparation method of a microbial remediation agent for heavy metal contaminated soil, comprising the following process:

[0094] The preparation method of the modified biochar is as follows:

[0095] Step (1): Mix 3 parts of 25wt% 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, then filter, wash and dry to obtain an aminated matrix;

[0096] Step (2): Add 1 part of the aminated matrix to 70 parts of an ethanol solution containing 4 g / L of glutaraldehyde, react for 7 h, filter, wash and dry to obtain an aldehyde-grouped matrix;

[0097] Step (3): Add 1 part of the aldehyde-grouped matrix to 25 parts of a 30wt% 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 the modified biochar; the molar ratio of the amino group, NaOH and carbon disulfide in the lysine solution is 1:1:1.1;

[0098] 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.

[0099] Experiment:

[0100] 1. Determination of the effective viable count: Take the microbial repair agents obtained in Example 2 and Comparative Examples 1-3, and test them according to the regulations in 6.3.2 of GB20287-2006. The results are shown in Table 1.

[0101] Table 1 Effective viable counts of the microbial repair agents in Example 2 and Comparative Examples 1-3

[0102]

[0103] According to the data in the above table, the following conclusions can be clearly obtained:

[0104] Compared with Example 2, the effective viable counts of the products obtained in Comparative Examples 1-3 all decreased. It can be seen that in the present invention, by using the modified biochar and the 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 the microorganisms in the soil remediation process.

[0105] 2. Determination of heavy metal removal rate: Take the treated soil 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.

[0106] Table 2 Remediation effects of heavy - metal - contaminated soil in Examples 1 - 3 and Comparative Examples 1 - 3 (removal rate / %)

[0107]

[0108] According to the data in the above table, the following conclusions can be clearly obtained:

[0109] 1. Compared with Examples 1 - 3, the remediation effects of the products obtained in Comparative Examples 1 and 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 the present application, by introducing lysine as a bridge, a sulfur - containing group dithiocarboxyl is 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 remediation effect.

[0110] 2. Compared with Examples 1 - 3, the remediation effect of the product obtained in Comparative Example 3 decreased, which indicates that the present invention uses humic acid and ferrous sulfate heptahydrate to modify biochar, which 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.

[0111] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above - mentioned exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non - restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, it is intended to embrace all changes falling within the meaning and scope of the equivalent elements of the claims in the present invention.

Claims

1. A preparation method of a microbial remediation agent for heavy metal contaminated soil, characterized in that: It includes the following steps: Step S1: Mix the inorganic carrier and the modified biochar evenly to obtain a composite carrier; Step S2: Place the composite carrier into the fermentation broth of Bacillus megaterium and the fermentation broth of Bacillus cereus respectively. After shaking 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 repair agent; The preparation process of the modified biochar is as follows: First, modify the biochar with nano zero-valent iron to obtain a matrix; then graft amino groups through a substitution reaction to obtain an aminated matrix; crosslink the aminated matrix and lysine with glutaraldehyde, and finally react the remaining amino groups in lysine with NaOH and carbon disulfide to graft dithiocarboxyl groups, thus obtaining it; 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 a matrix; Step (2): Mix concentrated ammonia water and epichlorohydrin evenly, react at 60 - 70 °C for 3 - 5 h, add the matrix and sodium hydroxide, react at 105 - 115 °C for 8 - 10 h, then filter, wash, and dry to obtain an aminated matrix; Step (3): Add the aminated matrix to the ethanol solution of glutaraldehyde, react for 6 - 8 h, filter, wash, and dry to obtain an aldehyde-grouped matrix; Step (4): Add the aldehyde-grouped matrix 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 40 - 50 °C water bath and continue to react for 4 - 8 h, cool to room temperature, filter, wash, and dry to obtain the modified biochar.

2. The preparation method of a microbial remediation agent for heavy metal contaminated soil according to claim 1, characterized in that: The preparation method of the humic acid solution is as follows: Mix humic acid and sodium hydroxide solution evenly, and use hydrochloric acid solution to adjust the pH = 7 to obtain a humic acid solution.

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

4. The preparation method of a microbial remediation agent for heavy metal contaminated soil according to claim 1, characterized in that: In the said step (3), the mass ratio of the aminated matrix to the ethanol solution of glutaraldehyde is 1:(50 - 100).

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

6. The preparation method of a microbial remediation agent for heavy metal contaminated soil according to claim 1, characterized in that: The preparation methods of the fermentation broth of Bacillus megaterium and the fermentation broth of Bacillus cereus are as follows: Bacillus megaterium and Bacillus cereus were respectively inoculated into solid medium and activated at 25 - 37°C for 16 - 26 h, then inoculated into seed medium and cultured on a shaker at 180 - 300 rmp at 25 - 37°C for 16 - 36 h, and then subjected to liquid fermentation in liquid medium for 3 - 5 d to obtain Bacillus megaterium fermentation broth and Bacillus cereus fermentation broth.

7. The preparation method of 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).

8. A microbial remediation agent for heavy metal contaminated soil prepared by the preparation method according to any one of claims 1 - 7.

9. Use of the microbial remediation agent for heavy metal contaminated soil according to claim 8, characterized in that: Comprising the following steps: Sow ryegrass seeds into heavy metal contaminated soil, maintain appropriate humidity and temperature, after 10 - 20 days when the plants germinate and grow, add the microbial remediation agent, and jointly remove heavy metals in the soil by utilizing the absorption of heavy metals by ryegrass and the strengthening effect of the microbial remediation agent.

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