Microbial-biochar composite microsphere material and preparation method and application thereof

Abstract

The application discloses a kind of microbial-biochar composite microsphere materials and its preparation method and application, belong to soil remediation technical field.The method includes: preparation oyster shell and chicken manure mixed biochar powder;Then bacteria liquid is strengthened culture with nanometer silicon dioxide;Then mixed biochar powder is added to strengthen bacteria liquid and adsorbed, the obtained mixture is mixed with sodium alginate solution, and is crosslinked with calcium chloride solution Solidification, a kind of microbial-biochar composite microsphere material is prepared.The material realizes the synchronous passivation of Cd and As by mineralization precipitation, ion exchange, surface complexation and microbial extracellular polymer combination mechanism, with the advantages of easy recovery, high stability, strong applicability and the like.The application provides an environment-friendly and economical soil remediation method, which can effectively fix cadmium and arsenic in soil, improve soil fertility and reduce environmental pollution risk.

Description

Technical Field

[0001] This invention relates to the field of soil remediation technology, specifically to a microbial-biochar composite microsphere material, its preparation method, and its application. Background Technology

[0002] Cadmium (Cd) and arsenic (As) are widely distributed and difficult to remediate in soil as a compound pollution. Furthermore, they may exhibit synergistic or antagonistic effects under different soil conditions, making the remediation of compound pollution significantly more challenging than that of single heavy metal pollution. Particularly in terms of chemical form and environmental behavior, cadmium exists primarily as a cationic form, and its bioavailability typically decreases and tends to become fixed as soil pH increases. Arsenic, on the other hand, exists primarily as anionic forms, and is more easily activated and dissolved at higher pH levels. Therefore, the traditional strategy of passivating cadmium by increasing pH often induces arsenic release while reducing cadmium activity, creating a typical contradiction between cadmium reduction and arsenic release in remediation. This makes the simultaneous fixation of Cd and As a key challenge in the development of current remediation materials.

[0003] Although biochar materials have shown some potential in heavy metal remediation, single biochar still has significant limitations in dealing with Cd / As combined pollution. While the strong alkalinity of oyster shell biochar is beneficial for reducing cadmium availability, it may increase arsenic activity due to increased pH, bringing the risk of cadmium reduction and arsenic release. Phosphate and arsenate in chicken manure biochar have similar chemical properties and easily compete for adsorption sites, thereby weakening the arsenic fixation effect.

[0004] Furthermore, while microbial remediation technology possesses unique advantages in heavy metal speciation, microorganisms are susceptible to oxidative stress damage under Cd / As combined stress. Heavy metal ions can invade cells and bind to proteins and enzymes, leading to structural and functional impairment. They also induce the accumulation of reactive oxygen species such as superoxide anions, hydrogen peroxide, and hydroxyl radicals, causing lipid peroxidation, protein denaturation, and DNA damage, ultimately inhibiting microbial growth and reproduction. Simultaneously, existing research and literature indicate that biochar systems may experience reduced microbial survival rates and oxidative stress due to high pH environments and heavy metal accumulation, resulting in decreased microbial survival and limited remediation functions, thus affecting the synergistic remediation effect of the composite system.

[0005] Therefore, there is an urgent need to develop a novel composite material system that can synergistically regulate the speciation of cadmium and arsenic, enhance the stress resistance of microorganisms, and achieve simultaneous, efficient, and stable Cd / As. Summary of the Invention

[0006] To address the challenges of simultaneous Cd / As passivation, including pH inconsistencies, differences between anions and cations, limitations of single-mechanism materials, and susceptibility to oxidative stress inhibition of microorganisms, this invention aims to provide a method for preparing microbial-biochar composite microspheres, specifically comprising the following steps: (1) Preparation of mixed biochar powder of oyster shell and chicken manure: oyster shell is pyrolyzed and ground to obtain oyster shell biochar powder; chicken manure is pyrolyzed and ground to obtain chicken manure biochar powder; oyster shell biochar powder and chicken manure biochar powder are mixed evenly to obtain mixed biochar powder.

[0007] (2) Preparation of nano-silica-enhanced bacterial culture: The bacterial strain was inoculated into liquid LB medium and cultured to the mid-logarithmic growth stage to obtain the logarithmic growth stage bacterial culture; nano-silica aqueous solution was added to the mid-logarithmic growth stage bacterial culture and cultured for a longer period; after the culture was completed, the culture was washed to obtain the washed bacterial strain, and the washed bacterial strain was added to liquid LB medium to obtain the nano-silica-enhanced bacterial culture.

[0008] (3) Biochar loaded with bacterial solution: The mixed biochar powder obtained in step (1) is added to the bacterial solution reinforced with nano-silica obtained in step (2) for adsorption to obtain a microbial-biochar mixture; the microbial-biochar mixture is added to sodium alginate aqueous solution to obtain a mixed colloidal solution; the mixed colloidal solution is injected into calcium chloride aqueous solution for crosslinking to obtain microbial-biochar composite microsphere material.

[0009] Preferably, the pyrolysis conditions for oyster shells in step (1) of the present invention are: pyrolysis at 350~450℃ for 1~3h.

[0010] Preferably, the pyrolysis conditions for chicken manure in step (1) of the present invention are: pyrolysis at 450~500℃ for 1~3h.

[0011] Preferably, in step (1) of the present invention, the mass ratio of oyster shell biochar powder to chicken manure biochar powder is 1~2:3.

[0012] Preferably, the particle size of the oyster shell biochar powder and the chicken manure biochar powder in step (1) of the present invention is 100~120 mesh.

[0013] Preferably, the bacterial strain in step (2) of the present invention is Bacillus belyssus or Bacillus subtilis.

[0014] Preferably, the conditions for culturing to the mid-logarithmic growth stage in step (2) of the present invention are: culturing at 30°C and 180 rpm with shaking until the mid-logarithmic growth stage; the culturing time is 12 hours.

[0015] Preferably, the OD600 of the logarithmic growth phase bacterial solution in step (2) of the present invention is 0.4~0.6.

[0016] Preferably, the concentration of the nano-silica aqueous solution in step (2) of the present invention is 5~10 mg / L.

[0017] Preferably, in step (2) of the present invention, the volume ratio of the logarithmic growth phase bacterial solution to the nano silica aqueous solution is 1:3~4.

[0018] Preferably, the OD600 of the bacterial solution enhanced with nano-silica in step (2) of the present invention is 1.2~1.3.

[0019] Preferably, in step (3) of the present invention, the solid-liquid ratio of the bacterial solution after mixing biochar powder and nano-silica reinforcement is 1g:5~10mL.

[0020] Preferably, in step (3) of the present invention, the volume ratio of the microbial-biochar mixture to the sodium alginate aqueous solution is 1:1.

[0021] Preferably, the mass percentage concentration of sodium alginate aqueous solution in step (3) of the present invention is 2% to 4%.

[0022] Preferably, the mass percentage concentration of the calcium chloride aqueous solution in step (3) of the present invention is 2%.

[0023] Preferably, the adsorption time in step (3) of the present invention is 2 hours.

[0024] Preferably, the crosslinking conditions in step (3) of the present invention are: static crosslinking at 4~20℃ for 12~24h.

[0025] Another object of the present invention is to provide a microbial-biochar composite microsphere material prepared by the method described above.

[0026] Another objective of this invention is to provide an application of microbial-biochar composite microsphere material in the remediation of cadmium and arsenic contaminated soil.

[0027] Mechanism of this invention: This invention prepares a microbial-biochar composite microsphere material by loading bacterial solution onto calcium-phosphorus dual-source biochar. The material utilizes oyster shells to provide a calcium source primarily composed of calcium carbonate, and chicken manure to provide a phosphorus source carrier rich in phosphorus and a porous carbon skeleton. Furthermore, *Bacillus belyssioides* or *Bacillus subtilis* is further cultured with nano-SiO2 during the logarithmic growth phase to enhance the antioxidant capacity of the bacteria, thereby increasing their activity. This allows them to maintain biofilm / EPS secretion and cell wall binding sites even under the stress of high pH and metal accumulation at the biochar interface. This results in a stable "carboxyl / hydroxyl-protein / EPS" composite complex layer formed with the functional groups on the biochar surface, transferring metal ions from a migratory state to a more stable bound state. Simultaneously, sodium alginate provides a mass transfer buffer and shock-resistant microenvironment, while the microsphere morphology avoids bacterial inactivation and site collapse caused by direct exposure to biochar. This invention achieves synergistic effects through multiple mechanisms, including calcium source supply, alkalinity regulation, phosphate precipitation, and site competition, ultimately realizing simultaneous passivation of cadmium and arsenic while reducing migration and bioavailability.

[0028] This invention provides a microbial-biochar composite microsphere material, its preparation method, and its application, which have the following beneficial effects: (1) This invention uses nano-silica to induce antioxidant bacteria-biochar sodium alginate embedded composite material, and achieves simultaneous passivation of cadmium and arsenic and reduces their mobility and bioavailability through multiple synergistic mechanisms such as calcium and phosphorus dual-source biochar mineralization precipitation, biochar pores and functional group adsorption / complexation, and Bacillus cell wall and extracellular polymer (EPS) binding and fixation.

[0029] (2) This invention prepares mixed biochar from oyster shells and chicken manure. The raw materials are cheap and readily available, and waste resources are utilized. The combination of oyster shell biochar and chicken manure biochar forms a multifunctional matrix that combines precipitation, adsorption, ion exchange and pH regulation, thereby enhancing the ability to simultaneously fix cadmium and arsenic from the source. Nano-silica induces and enhances the anti-oxidation system of microorganisms, significantly improving the survival and stability of bacteria under heavy metal stress and alkaline environment, overcoming the problems of easy inactivation and difficulty in simultaneously reducing cadmium and releasing arsenic in traditional direct loading. The coupling of mixed biochar and enhanced microorganisms achieves simultaneous passivation of cadmium and arsenic.

[0030] (3) The present invention uses sodium alginate encapsulation technology to solidify the microbial-biochar mixture into microspheres. This design not only facilitates the addition, recycling and reuse of materials and reduces the risk of secondary pollution, but also provides additional physical protection for microorganisms through the encapsulation structure, further enhancing the stability of the material. The material prepared by the present invention is suitable for various engineering scenarios such as polluted water bodies, soil leachate and in-situ soil remediation, and has good operability and adaptability. Attached Figure Description

[0031] Figure 1 These are the infrared spectra of the composite materials prepared in Examples 1, 2, 1, and 2 of this invention.

[0032] Figure 2 This is a graph showing the arsenic removal efficiency of the composite materials prepared in Examples 1, 2, 4, and 5 of this invention.

[0033] Figure 3 The graph shows the cadmium removal efficiency of the composite materials prepared in Examples 1, 2, 4, and 5 of this invention. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] The Bacillus subtilis used in the embodiments and comparative examples of this invention was obtained from the China General Microbiological Culture Collection Center (https: / / www.cgmcc.net / ), with accession number CGMCC1.821 and accession date of March 1, 1972; the Bacillus belyes used was obtained from the China General Microbiological Culture Collection Center (https: / / www.cgmcc.net / ), with accession number 1.12669 and accession date of October 9, 2013.

[0036] Example 1 A method for preparing microbial-biochar composite microspheres, the specific steps of which are as follows: (1) Preparation of oyster shell and chicken manure mixed biochar powder: Washed and dried oyster shells were pyrolyzed at 400℃ for 2 hours, then crushed and ground to obtain oyster shell biochar powder (100 120 mesh); the dried chicken manure was pyrolyzed at 500℃ for 2 hours, then pulverized and ground to obtain chicken manure biochar powder (100 mesh). (120 mesh); Mix oyster shell biochar powder and chicken manure biochar powder at a mass ratio of 1:3 to obtain mixed biochar powder.

[0037] (2) Preparation of nano-silica-enhanced bacterial culture: The liquid LB medium was autoclaved (120℃, 15 minutes) and set aside for use; Bacillus belye (10 μL) was inoculated into liquid LB medium (30 mL) and cultured to mid-logarithmic growth to obtain a bacterial culture in the logarithmic growth phase (OD600≈0.5); a nano-silica aqueous solution with a concentration of 5 mg / L was added to the bacterial culture in the mid-logarithmic growth phase and cultured for another 12 h (OD600≈1), wherein the volume ratio of the bacterial culture in the mid-logarithmic growth phase to the nano-silica aqueous solution was 1:3; after the culture was completed, the culture was washed with PBS buffer to obtain the washed bacterial culture, and the washed bacterial culture was added to liquid LB medium to obtain a nano-silica-enhanced bacterial culture (OD600≈1.2).

[0038] (3) Biochar-loaded bacterial solution: The mixed biochar powder obtained in step (1) was added to the bacterial solution enhanced with nano-silica obtained in step (2) and shaken for 2 hours for adsorption (the solid-liquid ratio of the mixed biochar powder and the bacterial solution enhanced with nano-silica was 1 g: 10 mL) to obtain a microbial-biochar mixture; the microbial-biochar mixture was added to a sodium alginate aqueous solution with a mass percentage concentration of 2% (the volume ratio of the microbial-biochar mixture and the sodium alginate aqueous solution was 1:1) for encapsulation to obtain a mixed slurry; the mixed slurry was injected into a CaCl2 aqueous solution with a mass percentage concentration of 2% using a 5 mL syringe and allowed to stand for crosslinking at 20°C for 12 hours to obtain a microbial-biochar composite microsphere material.

[0039] The microbial-biochar composite microsphere material prepared in this embodiment was used for the remediation of cadmium and arsenic contaminated soil. Specifically, the material was added to the contaminated soil at a ratio of 8g:1kg (the soil pH was neutral, specifically 6.5-8, and the pH could be adjusted with hydrochloric acid or sodium hydroxide), and placed at 37°C for 30 days. The contents of DTPA-extractable cadmium and TCLP-leached arsenic in the soil before and after treatment were measured, and the cadmium fixation rate was calculated to be 80.03% and the arsenic fixation rate was 58.38%.

[0040] Example 2 A method for preparing microbial-biochar composite microspheres, the specific steps of which are as follows: (1) Preparation of oyster shell and chicken manure mixed biochar powder: Washed and dried oyster shells were pyrolyzed at 400℃ for 2 hours, then crushed and ground to obtain oyster shell biochar powder (100 120 mesh); the dried chicken manure was pyrolyzed at 500℃ for 2 hours, then pulverized and ground to obtain chicken manure biochar powder (100 mesh). (120 mesh); Mix oyster shell biochar powder and chicken manure biochar powder at a mass ratio of 1:3 to obtain mixed biochar powder.

[0041] (2) Preparation of nano-silica-enhanced bacterial culture: The liquid LB medium was autoclaved (120℃, 15 minutes) and set aside for use; Bacillus belye (10 μL) was inoculated into liquid LB medium (30 mL) and cultured to mid-logarithmic growth to obtain a bacterial culture in the logarithmic growth phase (OD600≈0.4); 8 mg / L nano-silica aqueous solution was added to the mid-logarithmic growth bacterial culture and cultured for another 12 h (OD600≈1), wherein the volume ratio of the mid-logarithmic growth bacterial culture to the nano-silica aqueous solution was 1:4; after the culture was completed, the culture was washed with PBS buffer to obtain the washed bacterial culture, and the washed bacterial culture was added to liquid LB medium to obtain a nano-silica-enhanced bacterial culture (OD600≈1.2).

[0042] (3) Biochar-loaded bacterial solution: The mixed biochar powder obtained in step (1) was added to the bacterial solution enhanced with nano-silica obtained in step (2) and shaken for 2 hours for adsorption (the solid-liquid ratio of the mixed biochar powder and the bacterial solution enhanced with nano-silica was 1 g: 5 mL) to obtain a microbial-biochar mixture; the microbial-biochar mixture was added to a sodium alginate aqueous solution with a mass percentage concentration of 3% (the volume ratio of the microbial-biochar mixture and the sodium alginate aqueous solution was 1:1) for encapsulation to obtain a mixed slurry; the mixed slurry was injected into a CaCl2 aqueous solution with a mass percentage concentration of 2% using a 5 mL syringe and allowed to stand for crosslinking at 20°C for 12 hours to obtain a microbial-biochar composite microsphere material.

[0043] The microbial-biochar composite microsphere material prepared in this embodiment was used for the remediation of cadmium and arsenic contaminated soil. Specifically, the material was added to the contaminated soil at a ratio of 8g:1kg (the soil pH was neutral, specifically 6.5-8, and the pH could be adjusted with hydrochloric acid or sodium hydroxide), and placed at 37°C for 30 days. The contents of DTPA-extractable cadmium and TCLP-leached arsenic in the soil before and after treatment were measured, and the fixation rate of cadmium was calculated to be 78.16%, and the fixation rate of arsenic was 55.92%.

[0044] Example 3 A method for preparing microbial-biochar composite microspheres, the specific steps of which are as follows: (1) Preparation of oyster shell and chicken manure mixed biochar powder: Washed and dried oyster shells were pyrolyzed at 450℃ for 1 hour, then crushed and ground to obtain oyster shell biochar powder (100 120 mesh); the dried chicken manure was pyrolyzed at 450℃ for 1 hour, then pulverized and ground to obtain chicken manure biochar powder (100 mesh). (120 mesh); Mix oyster shell biochar powder and chicken manure biochar powder at a mass ratio of 1.5:3 to obtain mixed biochar powder.

[0045] (2) Preparation of nano-silica-enhanced bacterial culture: The liquid LB medium was autoclaved (120℃, 15 minutes) and set aside for use; Bacillus belye (10 μL) was inoculated into liquid LB medium (30 mL) and cultured to mid-logarithmic growth to obtain a bacterial culture in the logarithmic growth phase (OD600≈0.6); 10 mg / L nano-silica aqueous solution was added to the mid-logarithmic growth bacterial culture and cultured for another 12 h (OD600≈1), wherein the volume ratio of the mid-logarithmic growth bacterial culture to the nano-silica aqueous solution was 1:3.5; after the culture was completed, the culture was washed with PBS buffer to obtain the washed bacterial culture, and the washed bacterial culture was added to liquid LB medium to obtain a nano-silica-enhanced bacterial culture (OD600≈1.3).

[0046] (3) Biochar-loaded bacterial solution: The mixed biochar powder obtained in step (1) was added to the bacterial solution enhanced with nano-silica obtained in step (2) and shaken for 2 hours for adsorption (the solid-liquid ratio of the mixed biochar powder and the bacterial solution enhanced with nano-silica was 1 g: 7.5 mL) to obtain a microbial-biochar mixture; the microbial-biochar mixture was added to a sodium alginate aqueous solution with a mass percentage concentration of 4% (the volume ratio of the microbial-biochar mixture and the sodium alginate aqueous solution was 1:1) for encapsulation to obtain a mixed slurry; the mixed slurry was injected into a CaCl2 aqueous solution with a mass percentage concentration of 2% using a 5 mL syringe and allowed to stand for crosslinking at 4 °C for 20 hours to obtain a microbial-biochar composite microsphere material.

[0047] The microbial-biochar composite microsphere material prepared in this embodiment was used for the remediation of cadmium and arsenic contaminated soil. Specifically, the material was added to the contaminated soil at a mass ratio of 10g:1kg (the soil pH was neutral, specifically 6.5-8, and the pH could be adjusted with hydrochloric acid or sodium hydroxide), and placed at 37°C for 30 days. The contents of DTPA-extractable cadmium and TCLP-leached arsenic in the soil before and after treatment were measured, and the fixation rate of cadmium was calculated to be 77.63%, and the fixation rate of arsenic was 55.82%.

[0048] Example 4 A method for preparing microbial-biochar composite microspheres, the specific steps of which are as follows: (1) Preparation of oyster shell and chicken manure mixed biochar powder: Washed and dried oyster shells were pyrolyzed at 350℃ for 3 hours, then crushed and ground to obtain oyster shell biochar powder (100 120 mesh); the dried chicken manure was pyrolyzed at 475℃ for 3 hours, then pulverized and ground to obtain chicken manure biochar powder (100 mesh). (120 mesh); Mix oyster shell biochar powder and chicken manure biochar powder evenly at a mass ratio of 2:3 to obtain mixed biochar powder.

[0049] (2) Preparation of nano-silica-enhanced bacterial culture: The liquid LB medium was autoclaved (120℃, 15 minutes) and set aside for use; Bacillus belye (10 μL) was inoculated into liquid LB medium (30 mL) and cultured to mid-logarithmic growth to obtain a bacterial culture in the logarithmic growth phase (OD600≈0.4); 8 mg / L nano-silica aqueous solution was added to the mid-logarithmic growth bacterial culture and cultured for another 12 h (OD600≈1), wherein the volume ratio of the mid-logarithmic growth bacterial culture to the nano-silica aqueous solution was 1:3; after the culture was completed, the culture was washed with PBS buffer to obtain the washed bacterial culture, and the washed bacterial culture was added to liquid LB medium to obtain a nano-silica-enhanced bacterial culture (OD600≈1.3).

[0050] (3) Biochar-loaded bacterial solution: The mixed biochar powder obtained in step (1) was added to the bacterial solution enhanced with nano-silica obtained in step (2) and shaken for 2 hours for adsorption (the solid-liquid ratio of the mixed biochar powder and the bacterial solution enhanced with nano-silica was 1 g: 10 mL) to obtain a microbial-biochar mixture; the microbial-biochar mixture was added to a sodium alginate aqueous solution with a mass percentage concentration of 2% (the volume ratio of the microbial-biochar mixture and the sodium alginate aqueous solution was 1:1) for encapsulation to obtain a mixed slurry; the mixed slurry was injected into a CaCl2 aqueous solution with a mass percentage concentration of 2% using a 5 mL syringe and allowed to stand for crosslinking at 15 °C for 24 hours to obtain a microbial-biochar composite microsphere material.

[0051] The microbial-biochar composite microsphere material prepared in this embodiment was used for the remediation of cadmium and arsenic contaminated soil. Specifically, the material was added to the contaminated soil at a mass ratio of 12g:1kg (the soil pH was neutral, specifically 6.5-8, and the pH could be adjusted with hydrochloric acid or sodium hydroxide), and placed at 37°C for 30 days. The contents of DTPA-extractable cadmium and TCLP-leached arsenic in the soil before and after treatment were measured, and the fixation rate of cadmium was calculated to be 78.53% and the fixation rate of arsenic was 56.86%.

[0052] Example 5 A method for preparing microbial-biochar composite microspheres, the specific steps of which are as follows: (1) Preparation of oyster shell and chicken manure mixed biochar powder: Washed and dried oyster shells were pyrolyzed at 350℃ for 3 hours, then crushed and ground to obtain oyster shell biochar powder (100 120 mesh); the dried chicken manure was pyrolyzed at 475℃ for 3 hours, then pulverized and ground to obtain chicken manure biochar powder (100 mesh). (120 mesh); Mix oyster shell biochar powder and chicken manure biochar powder evenly at a mass ratio of 2:3 to obtain mixed biochar powder.

[0053] (2) Preparation of nano-silica-enhanced bacterial culture: The liquid LB medium was autoclaved (120℃, 15 minutes) and set aside for use; Bacillus belye (10 μL) was inoculated into liquid LB medium (30 mL) and cultured to mid-logarithmic growth to obtain a bacterial culture in the logarithmic growth phase (OD600≈0.5); a nano-silica aqueous solution with a concentration of 5 mg / L was added to the bacterial culture in the mid-logarithmic growth phase and cultured for another 12 h (OD600≈1), wherein the volume ratio of the bacterial culture in the mid-logarithmic growth phase to the nano-silica aqueous solution was 1:3; after the culture was completed, the culture was washed with PBS buffer to obtain the washed bacterial culture, and the washed bacterial culture was added to liquid LB medium to obtain a nano-silica-enhanced bacterial culture (OD600≈1.2).

[0054] (3) Biochar-loaded bacterial solution: The mixed biochar powder obtained in step (1) was added to the bacterial solution enhanced with nano-silica obtained in step (2) and shaken for 2 hours for adsorption (the solid-liquid ratio of the mixed biochar powder and the bacterial solution enhanced with nano-silica was 1 g: 5 mL) to obtain a microbial-biochar mixture; the microbial-biochar mixture was added to a sodium alginate aqueous solution with a mass percentage concentration of 2% (the volume ratio of the microbial-biochar mixture and the sodium alginate aqueous solution was 1:1) for encapsulation to obtain a mixed slurry; the mixed slurry was injected into a CaCl2 aqueous solution with a mass percentage concentration of 2% using a 5 mL syringe and allowed to stand for crosslinking at 15°C for 24 hours to obtain a microbial-biochar composite microsphere material.

[0055] The microbial-biochar composite microsphere material prepared in this embodiment was used for the remediation of cadmium and arsenic contaminated soil. Specifically, the material was added to the contaminated soil at a mass ratio of 12g:1kg (the soil pH was neutral, specifically 6.5-8, and the pH could be adjusted with hydrochloric acid or sodium hydroxide), and placed at 37°C for 30 days. The contents of DTPA-extractable cadmium and TCLP-leached arsenic in the soil before and after treatment were measured, and the fixation rate of cadmium was calculated to be 79.29%, and the fixation rate of arsenic was 58.51%.

[0056] Comparative Example 1 A method for preparing composite microsphere materials, the specific steps of which are as follows: (1) Preparation of oyster shell and chicken manure mixed biochar powder: Washed and dried oyster shells were pyrolyzed at 400℃ for 2 hours, then crushed and ground to obtain oyster shell biochar powder (100 120 mesh); the dried chicken manure was pyrolyzed at 500℃ for 2 hours, then pulverized and ground to obtain chicken manure biochar powder (100 mesh). (120 mesh); Mix oyster shell biochar powder and chicken manure biochar powder at a mass ratio of 1:3 to obtain mixed biochar powder.

[0057] (2) Inoculate Bacillus belye (10 μL) into liquid LB medium (30 mL) and culture for 18 h to obtain Bacillus belye bacterial suspension (OD600≈1.2).

[0058] (3) Biochar loaded with bacterial solution: The mixed biochar powder obtained in step (1) was added to the Bacillus vesiculosus bacterial solution and shaken for 2 hours for adsorption (the solid-liquid ratio of the mixed biochar powder and the bacterial solution reinforced with nano-silica was 1 g: 10 mL) to obtain a microbial-biochar mixture; the microbial-biochar mixture was added to a sodium alginate aqueous solution with a mass percentage concentration of 2% (the volume ratio of the microbial-biochar mixture and the sodium alginate aqueous solution was 1:1) for encapsulation to obtain a mixed slurry; the mixed slurry was injected into a CaCl2 aqueous solution with a mass percentage concentration of 2% using a 5 mL syringe and allowed to stand for crosslinking at 20°C for 12 hours to obtain a composite microsphere material.

[0059] The composite microsphere material prepared in this comparative example was used for the remediation of cadmium and arsenic contaminated soil. Specifically, the material was added to the contaminated soil at a ratio of 8g:1kg of microbial-biochar composite microsphere material to dry soil (the soil pH was neutral, specifically 6.5-8, which could be adjusted with hydrochloric acid or sodium hydroxide), and placed at 37℃ for 30 days. The contents of DTPA-extractable cadmium and TCLP-leached arsenic in the soil before and after treatment were measured, and the fixation rate of cadmium was calculated to be 72.84%, and the fixation rate of arsenic was 43.91%.

[0060] Comparative Example 2 A method for preparing composite microsphere materials, the specific steps of which are as follows: (1) Preparation of oyster shell and chicken manure mixed biochar powder: Washed and dried oyster shells were pyrolyzed at 400℃ for 2 hours, then crushed and ground to obtain oyster shell biochar powder (100 120 mesh); the dried chicken manure was pyrolyzed at 500℃ for 2 hours, then pulverized and ground to obtain chicken manure biochar powder (100 mesh). (120 mesh); Mix oyster shell biochar powder and chicken manure biochar powder at a mass ratio of 1:3 to obtain mixed biochar powder.

[0061] (2) Inoculate Bacillus belye (10 μL) into liquid LB medium (30 mL) and culture for 18 h to obtain Bacillus belye bacterial suspension (OD600≈1.2).

[0062] (3) Biochar loaded with bacterial solution: The mixed biochar powder obtained in step (1) was added to the Bacillus beleices bacterial solution and shaken for 2 hours for adsorption (the solid-liquid ratio of the mixed biochar powder and the bacterial solution reinforced with nano-silica was 1 g: 5 mL) to obtain a microbial-biochar mixture; the microbial-biochar mixture was added to a sodium alginate aqueous solution with a mass percentage concentration of 2% (the volume ratio of the microbial-biochar mixture and the sodium alginate aqueous solution was 1:1) for encapsulation to obtain a mixed slurry; the mixed slurry was injected into a CaCl2 aqueous solution with a mass percentage concentration of 2% using a 5 mL syringe and allowed to stand for crosslinking at 20°C for 12 hours to obtain a composite microsphere material.

[0063] The composite microsphere material prepared in this comparative example was used for the remediation of cadmium and arsenic contaminated soil. Specifically, the material was added to the contaminated soil at a ratio of 8g:1kg of microbial-biochar composite microsphere material to dry soil (the soil pH was neutral, specifically 6.5-8, which could be adjusted with hydrochloric acid or sodium hydroxide), and placed at 37℃ for 30 days. The contents of DTPA-extractable cadmium and TCLP-leached arsenic in the soil before and after treatment were measured, and the fixation rate of cadmium was calculated to be 73.68%, and the fixation rate of arsenic was 43.27%.

[0064] The external spectra of the materials prepared in Examples 1, 2, Comparative Examples 1 and 2 of this invention are as follows: Figure 1 As shown, Figure 1 In 3432-3435cm -1 The (-OH) group exhibits a consistently broad peak, indicating that the encapsulation system possesses stable hydrogen bonds and available hydroxyl sites for coordination; simultaneously, the peak at 1620-1645 cm⁻¹... -1 With 1421cm -1 This constitutes a typical pairwise signal of asymmetric / symmetric stretching of carboxylate (-COO-), superimposed at 1523 cm⁻¹. -1 The protein characteristics indicate that the bacteria and their EPS did indeed form a carboxyl-protein complex layer on the material surface, meaning the bacteria were successfully loaded onto the biochar; this indicates that Cd 2+ In this system, adsorption is not solely physical; internal coordination complexation and adsorption by the embedded network also occur at the -COO- / -OH sites. Furthermore, at 1000-1100 cm⁻¹... -1The peak enhancement was more likely to occur in the SiO2-enhanced group (Example 1 and Example 2), indicating that nano-SiO2 did indeed enter and change the microenvironment of the polysaccharide-silica network, making the carboxyl / hydroxyl sites in a more stable structure (less susceptible to metal impact). The material finally prepared by nano-silica enhancement is conducive to the formation of complex fixation interface, which is beneficial to the simultaneous fixation of cadmium and arsenic in polluted soil.

[0065] Comparative Example 3 A method for preparing microbial microsphere materials, the specific steps of which are as follows: (1) Preparation of nano-silica-enhanced bacterial culture: The liquid LB medium was autoclaved (120℃, 15 minutes) and set aside for use; Bacillus belye (10 μL) was inoculated into liquid LB medium (30 mL) and cultured to mid-logarithmic growth to obtain a bacterial culture in the logarithmic growth phase (OD600≈0.5); 5 mg / L nano-silica aqueous solution was added to the mid-logarithmic growth bacterial culture and cultured for another 12 h (OD600≈1), wherein the volume ratio of the mid-logarithmic growth bacterial culture to the nano-silica aqueous solution was 1:3; after the culture was completed, the culture was washed with PBS buffer to obtain the washed bacterial culture, and the washed bacterial culture was added to liquid LB medium to obtain a nano-silica-enhanced bacterial culture (OD600≈1.2).

[0066] (2) Microbial microsphere material: The bacterial solution obtained in step (1) was added to a sodium alginate aqueous solution with a mass percentage concentration of 2% (the volume ratio of the bacterial solution after nano-silica reinforcement to the sodium alginate aqueous solution was 1:1) for encapsulation to obtain a mixed slurry; the mixed slurry was injected into a CaCl2 aqueous solution with a mass percentage concentration of 2% using a 5mL syringe, and allowed to stand at 20℃ for 12h for crosslinking to obtain microbial microsphere material.

[0067] The microbial-biochar composite microsphere material prepared in this comparative example was used for the remediation of cadmium and arsenic contaminated soil. Specifically, the material was added to the contaminated soil at a ratio of 8g:1kg (the soil pH was neutral, specifically 6.5-8, and could be adjusted with hydrochloric acid or sodium hydroxide), and placed at 37℃ for 30 days. The contents of DTPA-extractable cadmium and TCLP-leached arsenic in the soil before and after treatment were measured, and the cadmium fixation rate was calculated to be 20.59%, and the arsenic fixation rate was 12.24%.

[0068] The microbial-biochar composite microspheres (40 mg) prepared in Examples 1, 2, 4, and 5 were used for the removal of heavy metals from 20 mL of cadmium solution (concentration 40 mg / L), 20 mL of arsenic solution (concentration 40 mg / L), and a mixed cadmium-arsenic solution (cadmium concentration 40 mg / L, arsenic-cadmium concentration 40 mg / L). The arsenic salt in the solutions was disodium arsenate, and the cadmium salt was cadmium nitrate. After the solutions containing the composite microspheres were placed in an incubator at 30℃ / 180 rpm and shaken for 12 h, the concentrations of cadmium and arsenic in the solutions were measured. The removal rates are as follows: Figure 2 and Figure 3 As shown, the composite material achieved a cadmium removal rate of 96.2%–98.2% and an arsenic removal rate of 91.1%–96.5% in cadmium-arsenic solutions.

[0069] In summary, this invention prepares a functional composite material based on calcium-phosphorus complementary oyster shell-chicken manure biochar, synergistically loaded with Bacillus subtilis and Bacillus belye, enhanced with nano-silica for antioxidant properties. This system achieves efficient simultaneous immobilization of cadmium and arsenic through multiple synergistic mechanisms, including biochar mineralization precipitation, adsorption complexation, pH regulation, and microbial arsenic oxidation and extracellular polymer binding. The final material is encapsulated in sodium alginate to form stable microspheres, exhibiting advantages such as high passivation efficiency (cadmium and arsenic removal rates of 98.2% and 96.5% in solution, respectively), strong engineering applicability, and environmental friendliness. This effectively overcomes the technical challenges of low passivation efficiency, easy bacterial inactivation, and the difficulty in simultaneously reducing cadmium and releasing arsenic in traditional methods.

[0070] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a microbial-biochar composite microsphere material, characterized in that, Specifically, the following steps are included: (1) Preparation of mixed biochar powder of oyster shell and chicken manure: oyster shell is pyrolyzed and ground to obtain oyster shell biochar powder; chicken manure is pyrolyzed and ground to obtain chicken manure biochar powder; oyster shell biochar powder and chicken manure biochar powder are mixed evenly to obtain mixed biochar powder. (2) Preparation of nano-silica-enhanced bacterial culture: The bacterial strain was inoculated into liquid LB medium and cultured to the mid-logarithmic growth phase to obtain the logarithmic growth phase bacterial culture; nano-silica aqueous solution was added to the mid-logarithmic growth phase bacterial culture and cultured for a longer period; after the culture was completed, the culture was washed to obtain the washed bacterial strain, and the washed bacterial strain was added to liquid LB medium to obtain the nano-silica-enhanced bacterial culture. (3) Biochar loaded with bacterial solution: The mixed biochar powder obtained in step (1) is added to the bacterial solution reinforced with nano-silica obtained in step (2) for adsorption to obtain a microbial-biochar mixture; the microbial-biochar mixture is added to sodium alginate aqueous solution to obtain a mixed colloidal solution; the mixed colloidal solution is injected into calcium chloride aqueous solution for crosslinking to obtain microbial-biochar composite microsphere material.

2. The method for preparing microbial-biochar composite microspheres according to claim 1, characterized in that, In step (1), the pyrolysis conditions for oyster shells are: pyrolysis at 350~450℃ for 1~3h; the pyrolysis conditions for chicken manure are: pyrolysis at 450~500℃ for 1~3h; and the mass ratio of oyster shell biochar powder to chicken manure biochar powder is 1~2:

3.

3. The method for preparing microbial-biochar composite microspheres according to claim 1, characterized in that, The particle size of the oyster shell biochar powder and chicken manure biochar powder mentioned in step (1) is 100~120 mesh.

4. The method for preparing microbial-biochar composite microspheres according to claim 1, characterized in that, The bacterial strain mentioned in step (2) is Bacillus belyss or Bacillus subtilis.

5. The method for preparing microbial-biochar composite microspheres according to claim 1, characterized in that, The conditions for culturing to the mid-logarithmic growth stage in step (2) are: culturing at 30°C and 180 rpm with shaking until the mid-logarithmic growth stage; the culturing time is 12 hours.

6. The method for preparing microbial-biochar composite microspheres according to claim 1, characterized in that, In step (2), the OD600 of the logarithmic growth phase bacterial solution is 0.4~0.6; the concentration of the nano silica aqueous solution is 5~10 mg / L; the volume ratio of the logarithmic growth phase bacterial solution to the nano silica aqueous solution is 1:3~4; and the OD600 of the nano silica-enhanced bacterial solution is 1.2~1.

3.

7. The method for preparing microbial-biochar composite microspheres according to claim 1, characterized in that, In step (3), the solid-liquid ratio of the mixed biochar powder and the nano-silica-enhanced bacterial solution is 1g:5~10mL; the volume ratio of the microbial-biochar mixture to the sodium alginate aqueous solution is 1:1; the mass percentage concentration of the sodium alginate aqueous solution is 2%~4%; and the mass percentage concentration of the calcium chloride aqueous solution is 2%.

8. The method for preparing microbial-biochar composite microspheres according to claim 1, characterized in that, The adsorption time in step (3) is 2 hours; the cross-linking conditions are: static cross-linking at 4~20℃ for 12~24 hours.

9. Microbial-biochar composite microspheres prepared by the method according to any one of claims 1 to 8.

10. The application of the microbial-biochar composite microsphere material according to claim 9 in the remediation of cadmium and arsenic contaminated soil.

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