Preparation process of iron salt-chitosan composite modified microbial high-adaptability biochar

Abstract

The present application belongs to the technical field of sewage treatment, and particularly relates to a preparation process of an iron salt-chitosan composite modified microbial high-adaptation type biochar. The present application combines an iron salt-chitosan composite modified biochar carrier with arsenic and antimony resistance functional bacteria, and the composite modified biochar not only retains excellent pore structure and specific surface area, but also forms a micro-ecosystem of nutrient release and micro-environment buffering through the synergistic effect between materials. The immobilized functional bacteria further degrade or transform As and Sb forms through biological transformation, realize the synergistic effect of multiple mechanisms, and greatly improve the removal efficiency of arsenic and antimony composite pollution. Moreover, the raw material biochar of the present application is widely available and low in cost, chitosan and iron salt are both environmentally compatible materials, the overall preparation process is green and environmentally friendly, and the prepared biochar can be applied to the treatment of high-concentration and complex-component arsenic and antimony contaminated wastewater, and has good application value.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment technology, specifically relating to a preparation process of iron salt-chitosan composite modified microbial highly adaptable biochar. Background Technology

[0002] With the rapid development of modern industry, heavy metal pollution has become increasingly serious, with arsenic (As) and antimony (Sb) posing a particularly prominent threat to the aquatic environment. Antimony and arsenic are highly harmful to the human body; long-term exposure can induce liver and kidney dysfunction, pulmonary edema, and even tumors.

[0003] Common technologies for removing antimony and arsenic from water include precipitation, membrane technology, adsorption, ion exchange, biological treatment, and phytoremediation. While chemical precipitation is relatively simple and low-cost, it consumes large amounts of chemicals and generates significant quantities of heavy metal-containing sludge that is difficult to treat. This sludge is classified as hazardous waste, making its treatment challenging and costly. Furthermore, this method has low removal rates for low concentrations or complexed As and Sb, making it difficult to ensure consistently high effluent quality. Membrane separation methods (such as reverse osmosis and nanofiltration) can achieve good effluent quality and partially enable water reuse; however, their investment and operating costs are extremely high, membrane modules are prone to fouling and clogging, maintenance is complex, and the concentrated liquid generated during treatment requires further treatment, increasing costs and environmental risks. Adsorption methods, using activated carbon and other adsorbents, exhibit poor selectivity for heavy metals such as As and Sb, have limited adsorption capacity, are difficult and costly to regenerate, and can even become hazardous waste after adsorption saturation.

[0004] Biochar has attracted attention in the field of heavy metal adsorption due to its wide availability, low cost, and porosity. However, ordinary biochar has a weak adsorption capacity for As(III) / Sb(III) in treating As and Sb wastewater, often requiring prior oxidation to a higher oxidation state. Traditional oxidants (such as chlorine) easily generate toxic byproducts during this oxidation process. Furthermore, biochar lacks sufficient mechanical strength in high-concentration As and Sb wastewater, making it prone to breakage and increasing loss rates. It also has poor resistance to interference, particularly with common SO4 in the wastewater. 2- SiO3 2- Plasma competes for adsorption sites, reducing its adsorption efficiency for As and Sb. Therefore, developing a highly efficient, stable, economical, and environmentally friendly technology for treating water contaminated with heavy metals such as As and Sb is urgently needed. Summary of the Invention

[0005] The purpose of this invention is to address existing problems by providing a process for preparing iron salt-chitosan composite modified microbial highly adaptable biochar.

[0006] This invention is achieved through the following technical solution:

[0007] A process for preparing iron salt-chitosan composite modified microbial highly adaptable biochar includes the following steps:

[0008] S1. The pretreated corn stalks are pyrolyzed under N2 atmosphere, then activated by superheated steam, and after natural cooling, impurities are removed, washed, dried and sieved to obtain high-strength activated biochar.

[0009] S2. Under N2 protection, high-strength activated biochar is immersed in a chelate solution, 0.1M EDTA is added and the pH is adjusted to 5.8. After full immersion, a microwave-assisted reaction is carried out, followed by carbothermic reduction. Then, it is immersed in a 3-mercaptopropionic acid solution and stirred at 50~55℃ for 5~6 hours. The mixture is then centrifuged, washed with water, and vacuum dried to obtain composite biochar.

[0010] S3. Soak the composite biochar in polydopamine solution and shake for 3-4 hours. Rinse three times with 0.1M PBS buffer, vacuum dry to constant weight, then immerse in the treatment solution and stir at room temperature for 2-3 hours. Centrifuge and set aside.

[0011] S4. Add the composite biochar treated in step S3 to the AI-2 inducer-LB mixture, shake for 2-3 hours, then add an equal volume of Bacillus subtilis suspension, perform gradient adsorption to obtain loaded biochar, and then perform biomineralization fixation.

[0012] Further, step S1 includes the following steps:

[0013] (1) Crush corn stalks to 100 mesh, soak them in 2~2.5M K2CO3 solution, control the solid-liquid ratio to 1:8~12, stir at 55~65℃ for 20~28h, and then dry at 100~110℃ until the moisture content is <10%;

[0014] (2) The pretreated corn stalks were heated to 450℃ in a N2 atmosphere at 10~15℃ / min for 2~3h, and then activated by superheated steam at 200℃. The flow rate of the superheated steam was 50~70mL / min and the activation time was 1~2h. At the same time as the activation treatment, 5% nano SiO2 sol was sprayed. The sol system contained 0.05M ZrOCl2. After natural cooling, impurities were removed by supercritical CO2. The conditions of supercritical CO2 were 40℃ and 10MPa. After acid washing with 1% HF, it was neutralized with 0.1M NaOH and then washed with distilled water until neutral. After drying at 65~75℃, it was passed through a 100-mesh sieve to obtain high-strength activated biochar.

[0015] Furthermore, the mass-to-volume ratio of the high-strength activated biochar to the chelate solution in step S2 is 1g:15~20mL;

[0016] The pH adjustment is specifically achieved using a 0.1M NaOH and 0.1M HCl solution system.

[0017] The preparation of the chelate solution includes the following steps:

[0018] 1) Prepare a 0.3M FeCl3·6H2O solution, according to Fe 3+ NaBH4 = 1:1~2 (molar ratio), add 0.1M NaBH4 solution dropwise under ice bath conditions, stir the reaction at 400~500 rpm, and reduce 50% of Fe. 3+ nZVI, forming Fe 0 / Fe 3+ Mixture;

[0019] 2) According to chitosan: residual Fe 3+ Weigh out chitosan at a mass ratio of 1:1, dissolve the chitosan in 0.1M citrate buffer solution with a pH of 5.0, stir to dissolve, and then add the above-mentioned Fe. 0 / Fe 3+ After mixing the mixture thoroughly, add 0.05M Ce(NO3)3·6H2O and continue stirring at room temperature for 1~2 hours to form a chelate solution.

[0020] The 3-mercaptopropionic acid (MPA) solution is prepared by adding 3-mercaptopropionic acid to anhydrous ethanol at a solid-liquid ratio of 1:10, adjusting the pH to 8.0 with 0.1M NaOH, and stirring until homogeneous.

[0021] Furthermore, in step S2, the microwave-assisted reaction is controlled with a microwave power of 300-400W, a temperature of 60-70℃, and a time of 40-50min.

[0022] The carbothermal reduction is carried out at a temperature of 370~390℃ for 2~3 hours.

[0023] The centrifugation speed is 4000~6000 rpm, the centrifugation time is 10~15 min, and the mixture is washed with water until neutral.

[0024] The vacuum drying temperature is 60~70℃, and the drying time is 8~12h.

[0025] Further, the preparation method of the polydopamine solution in step S3 is as follows: dissolve polydopamine powder in deionized water at a ratio of 1~1.4g:100mL, stir until dissolved, adjust the pH to 8.5 with 0.1M Tris-HCl buffer, add 0.01M ZrO2 nanoparticles with a particle size of 20~50nm, and disperse evenly by magnetic stirring;

[0026] The preparation method of the treatment solution is as follows: MgCl2·6H2O and CaCl2 are dissolved in deionized water at a weight ratio of 2.03:0.74 and stirred until dissolved. The mass-volume ratio of CaCl2 to deionized water is 1g:140~160mL.

[0027] Furthermore, during the oscillation process described in step S3, the temperature inside the shaker is controlled at 25°C and the rotation speed is 100~200 rpm;

[0028] The temperature is controlled at 60~70℃ during vacuum drying.

[0029] Further, the mass-to-volume ratio of the composite biochar and the AI-2 inducer-LB mixture in step S4 is 1g:4~6mL;

[0030] The preparation method of the AI-2 inducer-LB mixture is as follows: Weigh 0.0143g of (S)-4,5-dihydroxy-2,3-pentanedione (AI-2) standard, dissolve it in 10mL of sterile deionized water to prepare a 10mM stock solution, and store it at -20℃ in the dark; before use, take 50μL of the stock solution and add it to 1000mL of LB medium to dilute it into a 0.5mM AI-2 inducer-LB mixture.

[0031] The temperature inside the shaker is controlled at 25°C and the rotation speed is 100~200 rpm during the oscillation.

[0032] Further, the preparation method of Bacillus subtilis suspension in step S4 is as follows: Take frozen Bacillus subtilis culture and inoculate it into LB liquid medium. Activate the strain at 37℃ and 150-200 rpm shaking. Then, transfer it to fresh LB medium at a 1% inoculation rate and continue shaking culture until OD600 = 0.8-1.0. Take the culture, centrifuge at 4℃ and 5000-6000 rpm for 10-15 min, discard the supernatant, resuspend in 0.1M PBS buffer (pH=7.2), and adjust the concentration to 10. 8 CFU / mL is sufficient.

[0033] Furthermore, the gradient adsorption described in step S4 is divided into two stages:

[0034] Stage 1: Add Bacillus subtilis suspension, shake at 4°C and 50 rpm for 2 hours;

[0035] Phase 2: Heat to 28-30℃, shake at 130 rpm for 4 hours, centrifuge at 4℃ and 4000 rpm for 10-15 minutes, discard the supernatant, and wash 2-3 times with 0.1M PBS buffer (pH=7.2) to obtain loaded biochar.

[0036] Furthermore, the biomineralization fixation described in step S4 includes the following steps:

[0037] A. Weigh sodium alginate and montmorillonite in a mass ratio of 2:1, then add them to deionized water and stir at 4°C to dissolve them to obtain a sodium alginate-montmorillonite composite sol. The mass-volume ratio of montmorillonite to deionized water is 1g:100~120mL, and the mass-volume ratio of loaded biochar to composite sol is L.

[0038] B. Disperse the loaded bacterial char evenly into the sodium alginate-montmorillonite composite sol at a mass-volume ratio of 1g:20~25mL, then drop it into the cross-linking solution with a syringe, let it stand at 30℃ for 30min, then cure it at 4℃ for 12h, wash it 3 times with 0.1M PBS buffer, and then vacuum dry it.

[0039] The preparation method of the crosslinking solution is as follows: prepare 2% CaCl2 solution and 0.1M Na2HPO4-NaH2PO4 phosphate solution, mix them at a volume ratio of 1:3, and stir evenly to obtain 2% CaCl2-0.1M phosphate crosslinking solution.

[0040] The present invention has the following advantages over the prior art:

[0041] 1. Compared to single adsorption or microbial remediation technologies, this invention combines an iron salt-chitosan composite modified biochar carrier with arsenic and antimony resistant functional bacteria. The composite modified biochar not only retains excellent pore structure and specific surface area, but also forms a micro-ecosystem with slow nutrient release and microenvironment buffering through the synergistic effect between materials. The AI-2 inducer can specifically activate the quorum sensing system of Bacillus subtilis, and the biocompatible interface formed by the polydopamine coating can maintain the activity of the bacterial community, providing suitable conditions for the colonization, reproduction, and metabolic activities of the functional bacteria, ensuring that the microorganisms can continue to play a role, and achieving long-term remediation of arsenic and antimony contaminated water. Simultaneously, through sodium alginate-montmorillonite composite sol encapsulation and Ca... 2+ Phosphate cross-linking biomineralization treatment improves the mechanical strength and resistance to long-term impact from high-concentration polluted water bodies on the loaded biochar, while the layered structure of montmorillonite can block SO4 in wastewater. 2- SiO3 3- Interfering ions reduce the breakage and loss of the carrier in high-concentration polluted environments, ensuring the stability and durability of the remediation effect, while also reducing the risk of secondary pollution.

[0042] 2. The iron salt modification of this invention endows biochar with highly active iron oxide sites, enabling the fixation of As and Sb through coordination exchange and redox adsorption. The chitosan network enhances the capture capacity of heavy metals through chelation, while its film-forming properties provide stable attachment sites for functional microorganisms. Microwave-assisted reaction promotes uniform loading of iron salts and chitosan on the biochar surface. The carbothermal reduction step regulates the iron speciation ratio, enhancing the synergistic effect of As(III) / Sb(III) oxidation adsorption. The introduction of 3-mercaptopropionic acid provides thiol active sites, forming a multi-component adsorption system with iron oxides and chitosan groups, improving the capture capacity of As and Sb. The porous structure and high specific surface area of ​​the biochar itself provide space for pollutant adsorption and microbial colonization. The immobilized functional microorganisms further degrade or transform As and Sb speciation through biotransformation, achieving synergistic effects through multiple mechanisms and significantly improving the removal efficiency of arsenic and antimony composite pollution.

[0043] 3. The raw material biochar of this invention is widely available and inexpensive. Chitosan and iron salts are both environmentally compatible materials. The overall preparation process is green and environmentally friendly. Steps such as microwave-assisted reaction and carbothermal reduction avoid the secondary pollution problems caused by the use of large amounts of reagents in traditional chemical treatment. The iron salt-chitosan composite modified microbial high-adaptability biochar of this invention can be used for the treatment of arsenic and antimony polluted wastewater with high concentration and complex composition, and has good application value. Attached Figure Description

[0044] Figure 1 This is a process flow diagram of the present invention;

[0045] Figure 2 The results of the analysis of the tolerance of Bacillus subtilis to heavy metals As, Sb and pH in this invention are as follows;

[0046] Figure 3 Scanning electron microscope image of iron salt-chitosan composite modified microbial highly adaptable biochar;

[0047] Figure 4 This is a comparison chart of specific surface area and total porosity. Detailed Implementation

[0048] To further explain the present invention, the following specific embodiments are described.

[0049] Example 1: A preparation process for iron salt-chitosan composite modified microbial highly adaptable biochar, comprising the following steps:

[0050] S1. Preparation of high-strength activated biochar:

[0051] (1) Crush the corn stalks to 100 mesh, soak them in 2M K2CO3 solution, control the solid-liquid ratio to 1:8, stir at 55℃ for 20h, and then dry them at 100℃ until the moisture content is <10%;

[0052] (2) The pretreated corn stalks were heated to 450℃ in N2 atmosphere at 10℃ / min for 2h and then activated by superheated steam at 200℃. The flow rate of superheated steam was 50mL / min and the activation time was 1h. At the same time as the activation treatment, 5% nano SiO2 sol was sprayed. The sol system contained 0.05M ZrOCl2. After natural cooling, impurities were removed by supercritical CO2. The conditions of supercritical CO2 were 40℃ and 10MPa. After acid washing with 1% HF, it was neutralized with 0.1M NaOH and then washed with distilled water until neutral. After drying at 65℃, it was passed through a 100-mesh sieve to obtain high-strength activated biochar.

[0053] S2, Iron salt-chitosan composite modification:

[0054] (1) Prepare a 0.3M FeCl3·6H2O solution according to Fe 3+ NaBH4 = 1:1, 0.1M NaBH4 solution was added dropwise under ice bath conditions, and the reaction was stirred at 400 rpm to reduce 50% of Fe. 3+ nZVI, forming Fe 0 / Fe 3+ Mixture;

[0055] (2) According to chitosan: residual Fe 3+ Weigh out chitosan at a mass ratio of 1:1, dissolve the chitosan in 0.1M citrate buffer solution with a pH of 5.0, stir to dissolve, and then add the above-mentioned Fe. 0 / Fe 3+ After mixing the mixture thoroughly, add 0.05M Ce(NO3)3·6H2O and continue stirring at room temperature for 1 hour to form a chelate solution.

[0056] (3) High-strength activated biochar was immersed in chelate solution at a mass-volume ratio of 1g:15mL. 0.1MEDTA was added and the pH was adjusted to 5.8 with 0.1M NaOH and 0.1M HCl solution. After full immersion, microwave-assisted reaction was carried out with a power of 300W, a temperature of 60℃ and a time of 40min. Then, carbothermic reduction was carried out at 370℃ for 2h. Then, it was immersed in 3-mercaptopropionic acid solution, stirred at 50℃ for 5h, centrifuged at 4000rpm for 10min, washed with water until neutral, and vacuum dried at 60℃ for 8h to obtain composite biochar.

[0057] The method for preparing the 3-mercaptopropionic acid solution is as follows: 3-mercaptopropionic acid is added to anhydrous ethanol at a solid-liquid ratio of 1:10, the pH is adjusted to 8.0 with 0.1M NaOH, and the mixture is stirred until homogeneous.

[0058] S3, Microbial affinity modification:

[0059] (1) Dissolve polydopamine powder in deionized water at a ratio of 1g:100mL. Stir until dissolved, adjust pH to 8.5 with 0.1M Tris-HCl buffer, add 0.01M ZrO2 nanoparticles with a particle size of 20nm, and magnetically stir to disperse evenly to obtain polydopamine solution.

[0060] (2) Soak the composite biochar in polydopamine solution, shake at 100 rpm for 3 h at 25 °C, wash three times with 0.1 M PBS buffer, vacuum dry at 60 °C to constant weight, then immerse in the treatment solution, stir at room temperature for 2 h, centrifuge and set aside.

[0061] The preparation method of the treatment solution is as follows: MgCl2·6H2O and CaCl2 are dissolved in deionized water at a weight ratio of 2.03:0.74 and stirred until dissolved, wherein the mass-volume ratio of CaCl2 to deionized water is 1g:140mL.

[0062] S4, Microbial load:

[0063] (1) Weigh 0.0143 g of (S)-4,5-dihydroxy-2,3-pentanedione standard, dissolve it in 10 mL of sterile deionized water to prepare a 10 mM stock solution, and store it at -20℃ in the dark; before use, take 50 μL of the stock solution and add it to 1000 mL of LB medium to dilute it into a 0.5 mM MAI-2 inducer-LB mixture;

[0064] (2) Add the composite biochar treated in step S3 to the AI-2 inducer-LB mixture at a mass-volume ratio of 1g:4mL. After shaking at 25℃ and 100rpm for 2h, add an equal volume of Bacillus subtilis suspension. After shaking at 4℃ and 50rpm for 2h, raise the temperature to 28℃ and shake at 130rpm for 4h. Centrifuge at 4℃ and 4000rpm for 10min, discard the supernatant, and wash twice with 0.1M PBS buffer to obtain loaded biochar.

[0065] The method for preparing the Bacillus subtilis suspension is as follows: Inoculate frozen Bacillus subtilis culture into LB liquid medium, activate the strain at 37°C and 150 rpm shaking, then transfer to fresh LB medium at a 1% inoculation rate. Continue shaking culture until OD600 = 0.8. Take the culture, centrifuge at 4°C and 5000 rpm for 10 min, discard the supernatant, resuspend in 0.1M PBS buffer, and adjust the concentration to 10. 8 CFU / mL is sufficient;

[0066] S5, Curing and Molding:

[0067] (1) Weigh sodium alginate and montmorillonite in a mass ratio of 2:1, then add them to deionized water and stir at 4°C to dissolve them to obtain a sodium alginate-montmorillonite composite sol, wherein the mass-volume ratio of montmorillonite to deionized water is 1g:100mL.

[0068] (2) Disperse the loaded biochar evenly into the sodium alginate-montmorillonite composite sol at a mass-volume ratio of 1g:20mL, then drop it into the cross-linking solution with a syringe, let it stand at 30℃ for 30min, then solidify at 4℃ for 12h, rinse 3 times with 0.1M PBS buffer, and then vacuum dry.

[0069] The preparation method of the crosslinking solution is as follows: prepare 3% CaCl2 solution and 0.2M Na2HPO4-NaH2PO4 phosphate solution, mix them at a volume ratio of 1:3, and stir evenly to obtain 3% CaCl2-0.2M phosphate crosslinking solution.

[0070] Example 2: A preparation process for iron salt-chitosan composite modified microbial highly adaptable biochar, comprising the following steps:

[0071] S1. Preparation of high-strength activated biochar:

[0072] (1) Crush the corn stalks to 100 mesh, soak them in 2M K2CO3 solution, control the solid-liquid ratio to 1:10, stir at 60℃ for 24h, and then dry them at 105℃ until the moisture content is <10%;

[0073] (2) The pretreated corn stalks were heated to 450℃ in N2 atmosphere at 12℃ / min for 2.5h and then activated by superheated steam at 200℃. The flow rate of superheated steam was 60mL / min and the activation time was 1.5h. At the same time as the activation treatment, 5% nano SiO2 sol was sprayed. The sol system contained 0.05M ZrOCl2. After natural cooling, impurities were removed by supercritical CO2. The conditions of supercritical CO2 were 40℃ and 10MPa. After acid washing with 1% HF, it was neutralized with 0.1M NaOH and then washed with distilled water until neutral. After drying at 70℃, it was passed through a 100-mesh sieve to obtain high-strength activated biochar.

[0074] S2, Iron salt-chitosan composite modification:

[0075] (1) Prepare a 0.3M FeCl3·6H2O solution according to Fe 3+ A 0.1M NaBH4 solution was added dropwise under ice bath conditions with a NaBH4 ratio of 1:1.5. The reaction was stirred at 450 rpm to reduce 50% of Fe. 3+ nZVI, forming Fe 0 / Fe 3+ Mixture;

[0076] (2) According to chitosan: residual Fe 3+ Weigh out chitosan at a mass ratio of 1:1, dissolve the chitosan in 0.1M citrate buffer solution with a pH of 5.0, stir to dissolve, and then add the above-mentioned Fe. 0 / Fe 3+ After mixing the mixture thoroughly, add 0.05M Ce(NO3)3·6H2O and continue stirring at room temperature for 1.5h to form a chelate solution.

[0077] (3) High-strength activated biochar was immersed in chelate solution at a mass-volume ratio of 1g:16mL. 0.1MEDTA was added and the pH was adjusted to 5.8 with 0.1M NaOH and 0.1M HCl solution. After full immersion, microwave-assisted reaction was carried out with a power of 350W, a temperature of 65℃ and a time of 45min. Then, carbothermic reduction was carried out at 380℃ for 2.5h. Then, it was immersed in 3-mercaptopropionic acid solution, stirred at 53℃ for 5.5h, centrifuged at 5000rpm for 12min, washed with water until neutral, and vacuum dried at 65℃ for 10h to obtain composite biochar.

[0078] The method for preparing the 3-mercaptopropionic acid solution is as follows: 3-mercaptopropionic acid is added to anhydrous ethanol at a solid-liquid ratio of 1:10, the pH is adjusted to 8.0 with 0.1M NaOH, and the solution is stirred evenly.

[0079] S3, Microbial affinity modification:

[0080] (1) Dissolve polydopamine powder in deionized water at a ratio of 1.2g:100mL. Stir until dissolved, adjust the pH to 8.5 with 0.1M Tris-HCl buffer, and then add 0.01M ZrO2 nanoparticles with a particle size of 40nm. Stir magnetically to disperse evenly to obtain polydopamine solution.

[0081] (2) Soak the composite biochar in polydopamine solution, shake at 150 rpm for 3.5 h at 25 °C, wash three times with 0.1 M PBS buffer, vacuum dry at 65 °C to constant weight, then immerse in the treatment solution, stir at room temperature for 2.5 h, centrifuge and set aside.

[0082] The preparation method of the treatment solution is as follows: MgCl2·6H2O and CaCl2 are dissolved in deionized water at a weight ratio of 2.03:0.74 and stirred until dissolved, wherein the mass-volume ratio of CaCl2 to deionized water is 1g:150mL.

[0083] S4, Microbial load:

[0084] (1) Weigh 0.0143 g of (S)-4,5-dihydroxy-2,3-pentanedione standard, dissolve it in 10 mL of sterile deionized water to prepare a 10 mM stock solution, and store it at -20℃ in the dark; before use, take 50 μL of the stock solution and add it to 1000 mL of LB medium to dilute it into a 0.5 mM MAI-2 inducer-LB mixture;

[0085] (2) The composite biochar treated in step S3 was added to the AI-2 inducer-LB mixture at a mass-volume ratio of 1g:5mL. After shaking at 25℃ and 150rpm for 2.5h, an equal volume of Bacillus subtilis suspension was added. After shaking at 4℃ and 50rpm for 2h, the temperature was raised to 29℃ and shaken at 130rpm for 4h. After centrifugation at 4℃ and 4000rpm for 12min, the supernatant was discarded and the mixture was washed twice with 0.1M PBS buffer to obtain the loaded biochar.

[0086] The method for preparing the Bacillus subtilis suspension is as follows: Inoculate frozen Bacillus subtilis culture into LB liquid medium, activate the strain at 37°C and 180 rpm shaking, then transfer to fresh LB medium at a 1% inoculation rate. Continue shaking culture until OD600 = 0.9. Take the culture, centrifuge at 4°C and 5500 rpm for 12 min, discard the supernatant, resuspend in 0.1M PBS buffer, and adjust the concentration to 10. 8 CFU / mL is sufficient;

[0087] S5, Curing and Molding:

[0088] (1) Weigh sodium alginate and montmorillonite in a mass ratio of 2:1, then add them to deionized water and stir at 4°C to dissolve them to obtain a sodium alginate-montmorillonite composite sol, wherein the mass-volume ratio of montmorillonite to deionized water is 1g:110mL.

[0089] (2) Disperse the loaded biochar evenly into the sodium alginate-montmorillonite composite sol at a mass-volume ratio of 1g:22mL, then drop it into the cross-linking solution with a syringe, let it stand at 30℃ for 30min, then solidify at 4℃ for 12h, rinse 3 times with 0.1M PBS buffer, and then vacuum dry.

[0090] The preparation method of the crosslinking solution is as follows: prepare 3% CaCl2 solution and 0.2M Na2HPO4-NaH2PO4 phosphate solution, mix them at a volume ratio of 1:3, and stir evenly to obtain 3% CaCl2-0.2M phosphate crosslinking solution.

[0091] Example 3: A preparation process for iron salt-chitosan composite modified microbial highly adaptable biochar, comprising the following steps:

[0092] S1. Preparation of high-strength activated biochar:

[0093] (1) Crush corn stalks to 100 mesh, soak them in 2.5M K2CO3 solution, control the solid-liquid ratio at 1:12, stir at 65℃ for 28h, and then dry at 110℃ until the moisture content is <10%;

[0094] (2) The pretreated corn stalks were heated to 450℃ in N2 atmosphere at 15℃ / min for 3h and then activated by superheated steam at 200℃. The flow rate of superheated steam was 70mL / min and the activation time was 2h. At the same time as the activation treatment, 5% nano SiO2 sol was sprayed. The sol system contained 0.05M ZrOCl2. After natural cooling, impurities were removed by supercritical CO2. The conditions of supercritical CO2 were 40℃ and 10MPa. After acid washing with 1% HF, it was neutralized with 0.1M NaOH and then washed with distilled water until neutral. After drying at 75℃, it was passed through a 100-mesh sieve to obtain high-strength activated biochar.

[0095] S2, Iron salt-chitosan composite modification:

[0096] (1) Prepare a 0.3M FeCl3·6H2O solution according to Fe 3+ NaBH4 = 1:2, 0.1M NaBH4 solution was added dropwise under ice bath conditions, and the reaction was stirred at 500 rpm to reduce 50% of Fe. 3+ nZVI, forming Fe 0 / Fe 3+ Mixture;

[0097] (2) According to chitosan: residual Fe 3+ Weigh out chitosan at a mass ratio of 1:1, dissolve the chitosan in 0.1M citrate buffer solution with a pH of 5.0, stir to dissolve, and then add the above-mentioned Fe. 0 / Fe 3+ After mixing the mixture thoroughly, add 0.05M Ce(NO3)3·6H2O and continue stirring at room temperature for 2 hours to form a chelate solution.

[0098] (3) High-strength activated biochar was immersed in chelate solution at a mass-volume ratio of 1g:20mL. 0.1M EDTA was added and the pH was adjusted to 5.8 with 0.1M NaOH and 0.1M HCl solution. After full immersion, microwave-assisted reaction was carried out with a power of 400W, a temperature of 70℃ and a time of 50min. Then, carbothermic reduction was carried out at 390℃ for 3h. Then, it was immersed in 3-mercaptopropionic acid solution, stirred at 55℃ for 6h, centrifuged at 6000rpm for 15min, washed with water until neutral, and vacuum dried at 70℃ for 12h to obtain composite biochar.

[0099] The method for preparing the 3-mercaptopropionic acid solution is as follows: 3-mercaptopropionic acid is added to anhydrous ethanol at a solid-liquid ratio of 1:10, the pH is adjusted to 8.0 with 0.1M NaOH, and the solution is stirred evenly.

[0100] S3, Microbial affinity modification:

[0101] (1) Dissolve polydopamine powder in deionized water at a ratio of 1.4g:100mL. Stir until dissolved, adjust the pH to 8.5 with 0.1M Tris-HCl buffer, and then add 0.01M ZrO2 nanoparticles with a particle size of 50nm. Stir magnetically to disperse evenly to obtain polydopamine solution.

[0102] (2) Soak the composite biochar in polydopamine solution, shake at 200 rpm for 4 h at 25 °C, wash three times with 0.1 M PBS buffer, vacuum dry at 70 °C to constant weight, then immerse in the treatment solution, stir at room temperature for 3 h, centrifuge and set aside.

[0103] The preparation method of the treatment solution is as follows: MgCl2·6H2O and CaCl2 are dissolved in deionized water at a weight ratio of 2.03:0.74 and stirred until dissolved, wherein the mass-volume ratio of CaCl2 to deionized water is 1g:160mL.

[0104] S4, Microbial load:

[0105] (1) Weigh 0.0143 g of (S)-4,5-dihydroxy-2,3-pentanedione standard, dissolve it in 10 mL of sterile deionized water to prepare a 10 mM stock solution, and store it at -20℃ in the dark; before use, take 50 μL of the stock solution and add it to 1000 mL of LB medium to dilute it into a 0.5 mM MAI-2 inducer-LB mixture;

[0106] (2) Add the composite biochar treated in step S3 to the AI-2 inducer-LB mixture at a mass-volume ratio of 1g:6mL. After shaking at 200rpm for 3h at 25℃, add an equal volume of Bacillus subtilis suspension. After shaking at 50rpm for 2h at 4℃, raise the temperature to 38℃ and shake at 130rpm for 4h. Centrifuge at 4000rpm for 15min at 4℃, discard the supernatant, and wash 3 times with 0.1M PBS buffer to obtain loaded biochar.

[0107] The method for preparing the Bacillus subtilis suspension is as follows: Inoculate frozen Bacillus subtilis culture into LB liquid medium, activate the strain at 37°C and 200 rpm shaking, then transfer to fresh LB medium at a 1% inoculation rate. Continue shaking culture until OD600 = 1.0. Take the culture, centrifuge at 4°C and 6000 rpm for 15 min, discard the supernatant, resuspend in 0.1M PBS buffer, and adjust the concentration to 10. 8 CFU / mL is sufficient;

[0108] S5, Curing and Molding:

[0109] (1) Weigh sodium alginate and montmorillonite in a mass ratio of 2:1, then add them to deionized water and stir at 4°C to dissolve them to obtain a sodium alginate-montmorillonite composite sol, wherein the mass-volume ratio of montmorillonite to deionized water is 1g:120mL.

[0110] (2) Disperse the loaded biochar evenly into the sodium alginate-montmorillonite composite sol at a mass-volume ratio of 1g:25mL, then drop it into the cross-linking solution with a syringe, let it stand at 30℃ for 30min, then cure it at 4℃ for 12h, rinse it three times with 0.1M PBS buffer, and then vacuum dry it.

[0111] The preparation method of the crosslinking solution is as follows: prepare 3% CaCl2 solution and 0.2M Na2HPO4-NaH2PO4 phosphate solution, mix them at a volume ratio of 1:3, and stir evenly to obtain 3% CaCl2-0.2M phosphate crosslinking solution.

[0112] Experimental testing and results analysis:

[0113] 1. Growth curve of the original Bacillus subtilis strain and determination of its resistance to heavy metals arsenic and antimony.

[0114] like Figure 1 As shown, the original strain of Bacillus subtilis of the present invention can maintain its growth activity under stress of 40-160 mg / L antimony (Sb) and 40-150 mg / L arsenic (As) (OD600 of the highest concentration treatment group reached 2.5 and 2.0 or higher, respectively). However, its tolerance to Sb is significantly better than that to As—the lag phase is prolonged in the 160 mg / L antimony environment but the proliferation cycle can still be completed, while the same concentration of arsenic leads to a 20% decrease in biomass. In terms of pH adaptability, the strain grows best under neutral conditions (pH=7), while the biomass is retained at approximately 73% and 83% in acidic (pH=4) and weakly alkaline (pH=9) environments, respectively. This indicates that it can synergistically exert a remediation effect with the iron salt-chitosan composite modified microbial high-adaptability biochar prepared in Examples 1-3 under a wide range of environments (pH range of 4 to 9) and medium to low concentrations of arsenic and antimony pollution (<100 mg / L).

[0115] 2. Characterization and analysis of iron salt-chitosan composite modified microbial highly adaptable biochar:

[0116] 2.1 SEM: SEM image ( Figure 2 The surface morphology characteristics of different biochars were described. SEM images clearly show that after synergistic modification with ferric chloride and chitosan, the surface structure of the biochar was significantly optimized and exhibited a gradient enhancement effect: the natural porous substrate of the original biochar provided an ideal carrier for modification, while Example 1 successfully constructed a uniform and dense mesoporous network on its surface, with significantly improved pore distribution regularity; Example 2 exhibited a highly developed interconnected pore structure, with the pore walls roughened by nitrogen-doped carbon fibers derived from chitosan pyrolysis; Example 3 formed a multi-layered pore topology network, nZVI(Fe 0 The Fe3O4 particles are dispersed in the pores under the anchoring of the chitosan flexible carbon chain.

[0117] Figure 2 In the image: (a) is a SEM image of the original biochar; (b) is a SEM image of the iron salt-chitosan composite modified microbial biochar of Example 1; (c) is a SEM image of the iron salt-chitosan composite modified microbial biochar of Example 2; and (d) is a SEM image of the iron salt-chitosan composite modified microbial biochar of Example 3.

[0118] 2.2 BET: such as Figure 3 and Figure 4 As shown, chitosan-iron modification significantly reconstructed the pore structure of biochar: the original biochar had extremely underdeveloped pores (specific surface area of ​​only 4.6 cm²). 2 / g, pore volume ≈ 0.018cm 3 / g), while the porosity of the modified material exhibits a gradient change—Example 1 yielded the optimal specific surface area (117.1 cm²). 2 / g) and maximum pore volume (0.261cm³) 3 / g), mainly forming a microporous dominant structure; Example 2: Balanced pore parameters (104.7cm) 2 / g specific surface area, 0.245 cm 3 / g pore volume), the proportion of mesopores was significantly improved; although the specific surface area of ​​Example 3 decreased to 88.4cm³. 2 / g, but the nitrogen adsorption-desorption curve showed that its adsorption capacity was the highest in the high-pressure region, indicating that the proportion of large mesopores (>10nm) increased significantly, confirming the high Fe loading (Fe 3+ The "micropore blockage-mesopore expansion" pattern caused by (NaBH4=1:2) is consistent with the dispersion characteristics of nZVI particles under high-speed stirring at 500rpm.

[0119] To further compare the technical effects of the present invention, a comparative example was designed based on Example 2 for effect comparison, specifically as follows:

[0120] Comparative Example 1: Compared with Example 2, step S2 in Comparative Example 1 is replaced with the following operation, while the other steps are the same as in Example 2.

[0121] S2, Chitosan Modification:

[0122] (1) Chitosan was dissolved in 0.1M citrate buffer solution with pH 5.0 and stirred until dissolved. Then, high-strength activated biochar was immersed in it at a mass-volume ratio of 1g:16mL. After full immersion, microwave-assisted reaction was carried out with a power of 350W, a temperature of 65℃, and a time of 45min. Then, it was carbotherm reduced at 380℃ for 2.5h. Then, it was immersed in 3-mercaptopropionic acid solution, stirred at 53℃ for 5.5h, centrifuged at 5000rpm for 12min, washed with water until neutral, and vacuum dried at 65℃ for 10h to obtain composite biochar.

[0123] The 3-mercaptopropionic acid solution is prepared by adding 3-mercaptopropionic acid to anhydrous ethanol at a solid-liquid ratio of 1:10, adjusting the pH to 8.0 with 0.1M NaOH, and stirring until homogeneous.

[0124] Comparative Example 2: Compared with Example 2, Comparative Example 2 omits the entire step S3, while the other steps are the same as in Example 2.

[0125] Comparative Example 3: Compared with Example 2, Comparative Example 3 replaces the AI-2 inducer-LB mixture in step S4 with pure LB medium, while the other steps are the same as in Example 2.

[0126] Comparative Example 4: Compared with Example 2, Comparative Example 4 omits the entire step S4, replaces the loaded microbial char in step S5 with the composite biochar obtained in step S3, and the other steps are the same as in Example 2.

[0127] Comparative Example 5: Compared with Example 2, Comparative Example 5 omits montmorillonite in step S5, while the other steps are the same as in Example 2.

[0128] 3. Pollutant Removal Rate Test: The simulated wastewater in this experiment contained As(III) 80 mg / L, Sb(III) 100 mg / L, and SO42-. 2- 500 mg / L, SiO3 2- 200 mg / L, pH=6.5, 25℃, 200 rpm dynamic oscillation (simulating water flow impact).

[0129] The test results are shown in Table 1 below.

[0130] Table 1. Comparison of pollutant removal efficiency between Example 2 and each comparative example.

[0131]

[0132] As shown in Table 1 above, the removal rates of As(III) and Sb(III) reached 98.2% and 96.8% in the short term (24h) and long term (168h, 7 days), respectively, and remained at 96.7% and 94.3%. This indicates that the iron salt-chitosan composite modified microbial high-adaptability biochar prepared in this invention not only has high initial removal efficiency in high concentrations of arsenic and antimony composite pollution (As 80mg / L, Sb 100mg / L), but also resists water flow impact and interference ions, and has excellent long-term stability.

[0133] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A preparation process for iron salt-chitosan composite modified microbial highly adaptable biochar, characterized in that, Includes the following steps: S1. The pretreated corn stalks are pyrolyzed under N2 atmosphere, then activated by superheated steam, and after natural cooling, impurities are removed, washed, dried and sieved to obtain high-strength activated biochar. S2. Under N2 protection, high-strength activated biochar is immersed in a chelate solution, 0.1M EDTA is added and the pH is adjusted to 5.

8. After full immersion, a microwave-assisted reaction is carried out, followed by carbothermic reduction. Then, it is immersed in a 3-mercaptopropionic acid solution and stirred at 50~55℃ for 5~6 hours. The mixture is then centrifuged, washed with water, and vacuum dried to obtain composite biochar. The mass-to-volume ratio of the high-strength activated biochar to the chelate solution is 1g:15~20mL; The pH adjustment is specifically achieved using a 0.1M NaOH and 0.1M HCl solution system. The preparation of the chelate solution includes the following steps: 1) Prepare a 0.3M FeCl3·6H2O solution, according to Fe 3+ The ratio of NaBH4 is 1:1~2. A 0.1M NaBH4 solution is added dropwise under ice bath conditions, and the reaction is stirred at 400~500 rpm to reduce 50% of Fe. 3+ nZVI, forming Fe 0 / Fe 3+ Mixture; 2) According to chitosan: residual Fe 3+ Weigh out chitosan at a mass ratio of 1:1, dissolve the chitosan in 0.1M citrate buffer solution with a pH of 5.0, stir to dissolve, and then add the above-mentioned Fe. 0 / Fe 3+ After mixing the mixture thoroughly, add 0.05M Ce(NO3)3·6H2O and continue stirring at room temperature for 1~2 hours to form a chelate solution. The method for preparing the 3-mercaptopropionic acid solution is as follows: 3-mercaptopropionic acid is added to anhydrous ethanol at a solid-liquid ratio of 1:10, the pH is adjusted to 8.0 with 0.1M NaOH, and the solution is stirred evenly. S3. Soak the composite biochar in polydopamine solution and shake for 3-4 hours. Rinse three times with 0.1M PBS buffer, vacuum dry to constant weight, then immerse in the treatment solution and stir at room temperature for 2-3 hours. Centrifuge and set aside. S4. Add the composite biochar treated in step S3 to the AI-2 inducer-LB mixture, shake for 2-3 hours, then add an equal volume of Bacillus subtilis suspension, perform gradient adsorption to obtain loaded biochar, and then perform biomineralization fixation. The mass-to-volume ratio of the composite biochar to the AI-2 inducer-LB mixture is 1g:4~6mL; The preparation method of the AI-2 inducer-LB mixture is as follows: Weigh 0.0143g of (S)-4,5-dihydroxy-2,3-pentanedione standard, dissolve it in 10mL of sterile deionized water to prepare a 10mM stock solution, and store it at -20℃ in the dark; before use, take 50μL of the stock solution and add it to 1000mL of LB medium to dilute it into a 0.5mM AI-2 inducer-LB mixture. The temperature inside the shaker is controlled at 25°C and the rotation speed is 100~200 rpm during the oscillation process. The aforementioned biomineralization fixation includes the following steps: A. Weigh sodium alginate and montmorillonite in a mass ratio of 2:1, then add them to deionized water and stir at 4°C to dissolve them to obtain a sodium alginate-montmorillonite composite sol, wherein the mass-volume ratio of montmorillonite to deionized water is 1g:100~120mL. B. Disperse the loaded bacterial char evenly into the sodium alginate-montmorillonite composite sol at a mass-volume ratio of 1g:20~25mL, then drop it into the cross-linking solution with a syringe, let it stand at 30℃ for 30min, then cure it at 4℃ for 12h, wash it 3 times with 0.1M PBS buffer, and then vacuum dry it. The preparation method of the crosslinking solution is as follows: prepare 3% CaCl2 solution and 0.2M Na2HPO4-NaH2PO4 phosphate solution, mix them at a volume ratio of 1:3, and stir evenly to obtain 3% CaCl2-0.2M phosphate crosslinking solution.

2. The preparation process of the iron salt-chitosan composite modified microbial highly adaptable biochar according to claim 1, characterized in that, Step S1 includes the following steps: (1) Crush corn stalks to 100 mesh, soak them in 2~2.5M K2CO3 solution, control the solid-liquid ratio to 1:8~12, stir at 55~65℃ for 20~28h, and then dry at 100~110℃ until the moisture content is <10%; (2) The pretreated corn stalks were heated to 450℃ in a N2 atmosphere at a rate of 10~15℃ / min for 2~3h, and then activated by superheated steam at 200℃. The flow rate of the superheated steam was 50~70mL / min and the activation time was 1~2h. At the same time as the activation treatment, 5% nano SiO2 sol was sprayed. The sol system contained 0.05M ZrOCl2. After natural cooling, impurities were removed by supercritical CO2 at 40℃ and 10MPa. After acid washing with 1% HF, the mixture was neutralized with 0.1M NaOH and washed with distilled water until neutral. After drying at 65~75℃, the mixture was passed through a 100-mesh sieve to obtain high-strength activated biochar.

3. The preparation process of the iron salt-chitosan composite modified microbial highly adaptable biochar according to claim 1, characterized in that, In step S2, the microwave-assisted reaction is controlled with a microwave power of 300-400W, a temperature of 60-70℃, and a time of 40-50min. The carbothermal reduction is carried out at a temperature of 370~390℃ for 2~3 hours. The centrifugation speed is 4000~6000 rpm, the centrifugation time is 10~15 min, and the mixture is washed with water until neutral. The vacuum drying temperature is 60~70℃, and the drying time is 8~12h.

4. The preparation process of the iron salt-chitosan composite modified microbial highly adaptable biochar according to claim 1, characterized in that, The preparation method of the polydopamine solution in step S3 is as follows: dissolve polydopamine powder in deionized water at a ratio of 1~1.4g:100mL, stir until dissolved, adjust the pH to 8.5 with 0.1M Tris-HCl buffer, add 0.01M ZrO2 nanoparticles with a particle size of 20~50nm, and disperse evenly by magnetic stirring. The preparation method of the treatment solution is as follows: MgCl2·6H2O and CaCl2 are dissolved in deionized water at a weight ratio of 2.03:0.74 and stirred until dissolved. The mass-volume ratio of CaCl2 to deionized water is 1g:140~160mL.

5. The preparation process of the iron salt-chitosan composite modified microbial highly adaptable biochar according to claim 1, characterized in that, During the oscillation process described in step S3, the temperature inside the shaker is controlled at 25°C and the rotation speed is 100~200 rpm. The temperature is controlled at 60~70℃ during vacuum drying.

6. The preparation process of the iron salt-chitosan composite modified microbial highly adaptable biochar according to claim 1, characterized in that, The preparation method of Bacillus subtilis suspension in step S4 is as follows: Inoculate frozen Bacillus subtilis culture into LB liquid medium, activate the strain at 37℃ and 150-200 rpm shaking, then transfer to fresh LB medium at a 1% inoculation rate. Continue shaking culture until OD600 = 0.8-1.

0. Take the culture, centrifuge at 4℃ and 5000-6000 rpm for 10-15 min, discard the supernatant, resuspend in 0.1M PBS buffer, and adjust the concentration to 10. 8 CFU / mL is sufficient.

7. The preparation process of the iron salt-chitosan composite modified microbial highly adaptable biochar according to claim 1, characterized in that, The gradient adsorption described in step S4 consists of two stages: Stage 1: Add Bacillus subtilis suspension, shake at 4°C and 50 rpm for 2 hours; Phase 2: Heat to 28-30℃, shake at 130 rpm for 4 hours, centrifuge at 4℃ and 4000 rpm for 10-15 minutes, discard the supernatant, and wash 2-3 times with 0.1M PBS buffer to obtain loaded bacterial carbon.

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