A preparation method of a composite microorganism load type citrus peel-based porous biochar for removing heavy metals in water

Abstract

The application discloses a composite microorganism load type citrus peel based porous biochar for removing heavy metals in water and a preparation method thereof, and belongs to the technical field of water treatment functional materials and agricultural waste resource utilization. The application takes citrus peels as raw materials, and through cleaning pretreatment, pectinase and alpha amylase double enzyme biological activation, cob metal doping modification and segmented temperature control inert atmosphere carbonization, porous biochar with high specific surface area and multiple active sites is prepared; then bacillus subtilis, beer yeast and lactic acid bacteria are compounded into a composite microbial population at a specific ratio, and through physical pre-adsorption and sodium alginate and gelatin gel embedding double immobilization technology, the microorganisms are stably loaded on the surface and inside the pores of the biochar to prepare a composite adsorbent. The application combines physical and chemical adsorption of biochar and microbial biosorption / complexation, and the removal rate of heavy metals such as lead, copper and chromium in water reaches more than 95%, and the application has the characteristics of cheap raw materials, green and environment-friendly process, firm microbial loading and recyclable use, realizes agricultural solid waste resource utilization and efficient treatment of water body heavy metal pollution, and is suitable for industrial heavy metal wastewater purification treatment.

Description

Technical Field

[0001] Specifically, this relates to a method for preparing a composite microbial-supported biochar adsorbent using citrus peel as a base and its application in wastewater treatment. Background Technology

[0002] With industrial development and intensified human activities, heavy metal pollution has become a prominent environmental problem threatening the ecological environment and human health. Heavy metal ions in wastewater (such as lead, copper, and chromium) are characterized by high toxicity, recalcitrant degradation, and high bioaccumulation. Their continuous discharge not only damages aquatic ecosystems but can also harm human health through the food chain. Furthermore, wastewater often contains compound pollution from recalcitrant organic pollutants (such as antibiotics and herbicides), further increasing the difficulty of remediation. Therefore, developing efficient, low-cost, and environmentally friendly adsorption and purification materials is of great significance for water remediation.

[0003] Citrus fruits are produced in large quantities, and their peels (such as orange peel, tangerine peel, and grapefruit peel), as agricultural waste, are rich in natural polymers such as cellulose and lignin. They possess a porous structure and abundant surface functional groups (such as hydroxyl and carboxyl groups), making them ideal raw materials for preparing biochar adsorbents. In existing technologies, the preparation of citrus peel-based biochar is mostly carried out through single methods such as carbonization, chemical modification (such as metal doping and coupling agent modification), or biological activation (such as enzymatic hydrolysis). Although these methods can improve the adsorption performance for pollutants, they still have significant drawbacks: 1) Single biochar adsorption easily reaches saturation, resulting in limited removal efficiency for complex pollution systems (especially those with both heavy metals and organic pollutants); 2) The adsorption process relies on physical or chemical reactions, lacking the ability to subsequently degrade pollutants, leading to short adsorbent lifespan, difficulty in regeneration, and a high risk of secondary pollution.

[0004] To address these issues, researchers have attempted to combine microorganisms with biochar, leveraging the bio-adsorption and degradation effects of microorganisms to enhance purification. However, existing microbial-biochar composite adsorbent technologies still face the following key challenges: 1) Simple microbial immobilization methods (such as direct mixing or physical adsorption) lead to easy loss of microorganisms from the biochar surface, resulting in poor stability and difficulty in maintaining long-term activity in complex aquatic environments; 2) The synergistic effect between biochar and microorganisms is not fully realized; the pore structure of biochar may be excessively blocked by microorganisms, affecting mass transfer efficiency, or microbial activity may be inhibited by residual chemical reagents from the biochar preparation process; 3) Existing preparation processes are complex (such as relying on toxic chemical cross-linking agents or high-temperature treatment), posing a risk of secondary pollution and incurring high costs, making large-scale application difficult. Furthermore, traditional biochar adsorbents suffer from limited adsorption capacity and low removal efficiency for low-concentration pollutants; while microbial methods are environmentally friendly, free microorganisms are easily inactivated in environments with high concentrations of heavy metals or toxic organic matter, and solid-liquid separation is difficult. Although existing technologies have optimized the porosity of biochar through physical or chemical methods (acid activation, microwave treatment), the disposal cost after adsorption saturation remains high; the method of combining agricultural waste with microbial inoculum has failed to effectively resolve the contradiction between carrier porosity and the maintenance of microbial activity.

[0005] Therefore, developing a citrus peel-based composite adsorbent with a green and simple process, stable microbial adhesion, significant synergistic effect between carbon and bacteria, and both high-efficiency adsorption and biodegradation capabilities has become an urgent technical problem to be solved in the current water treatment field. This material needs to achieve the following objectives: (1) Improve the adsorption capacity of heavy metals and organic pollutants by optimizing the pore structure of biochar; (2) Stabilization and immobilization technology is used to ensure the long-term activity of microorganisms and achieve in-situ degradation of pollutants; (3) Reduce preparation costs and secondary pollution risks, and promote the sustainable development of environmental governance technologies. Summary of the Invention

[0006] To achieve the above objectives, the technical problem to be solved by the present invention is to provide a method for preparing composite microbial-supported citrus peel-based porous biochar for removing heavy metals from water. The adsorbent prepared by this method can effectively remove heavy metals from water, reduce the cost of adsorbent use, and improve economic benefits.

[0007] To address the aforementioned technical problems, this invention provides a method for preparing composite microbially supported citrus peel-based porous biochar for removing heavy metals from water, comprising the following steps: A. Raw material pretreatment: Cut citrus peel into small pieces, wash with deionized water and anhydrous ethanol three times alternately to remove surface impurities, sugar and pectin floating layer, dry in a forced air at 60-100℃ for 8-12 hours until constant weight, pulverize and pass through a 40-100 mesh sieve to obtain citrus peel powder. B. Modification: Prepare a mixture of citrus peel powder and deionized water at a ratio of 1g:10-15mL. Add citrate-sodium citrate buffer, and add pectinase at a rate of 120U / g. Incubate at 50℃ for 1.5h. Then add CaCl2 to make the Ca2+ concentration of the system 0.01mol / L. Adjust the pH to 6.0 with dilute HCl, and add α-amylase at a rate of 120U / g. Incubate at 59℃ for 40min. Filter through a 200-mesh nylon screen. Wash the filter residue with deionized water until neutral and dry at 80℃ to constant weight to obtain enzymatically modified citrus peel powder. C. Enhancement and modification: Modified citrus peel powder and cobalt nitrate hexahydrate were mixed in a ratio of 1 to 3:1 (by mass), and ultrapure water was added. The mixture was ultrasonically mixed at 300 to 500 W for 3 to 10 minutes, stirred at 300 to 500 rpm at room temperature for 4 to 6 hours, filtered, and washed three times alternately with deionized water and anhydrous ethanol. The mixture was then vacuum dried at 60°C for 24 hours to obtain the cobalt-doped precursor. D. High-temperature carbonization: The modified precursor is loaded into a ceramic crucible, compacted, covered, and sealed with tin foil. It is then placed in a tube furnace, and nitrogen is introduced at a rate of 0.5-1 L / min to purge the air. The temperature is increased to 500-700℃ at a rate of 3-10℃ / min and calcined at a constant temperature for 3-5 h. The temperature is then decreased to 300-350℃ at a rate of 12℃ / min and held at a constant temperature for 30-40 min. The mixture is then allowed to cool naturally to room temperature, ground, and washed three times alternately with deionized water and anhydrous ethanol. Finally, it is vacuum dried at 60℃ for 24 h to obtain porous citrus peel-based biochar. E. Preparation of compound microbial inoculum: Bacillus subtilis inoculum, Saccharomyces cerevisiae inoculum and Lactobacillus inoculum are mixed in a volume ratio of (6-9):(3-5):(1-2), sodium bentonite is added and stirred to adsorb, and then added dropwise to sodium alginate solution to obtain microbial mixture; F. Composite Microbial Loading: A mixture of porous citrus peel-based biochar and activated composite bacterial solution (1g:20mL) was prepared and incubated at 30℃ for 1 hour with low-speed stirring at 50rpm to achieve initial microbial colonization. 2–4g of sodium alginate and 1–2g of gelatin were added to 100mL of sterile water and stirred in a 60℃ water bath until completely dissolved. The solution was then cooled to ≤35℃ (to avoid high-temperature inactivation) to obtain a 2–4wt% sodium alginate-1–2wt% gelatin composite embedding solution. After the biochar adsorbed the bacterial solution, it was added to the composite embedding solution and stirred at low speed at room temperature for 30 min to form a char-bacteria-gel mixture (biochar content 8%–12%). The mixture was then dropped into a 4–5 wt% sterile CaCl2 solution using a syringe at a rate of 1 drop / second and cross-linked and fixed at room temperature for 7–8 h to form composite particles with a diameter of 3–5 mm. The particles were washed three times with sterile physiological saline to remove residual CaCl2 and then activated in a 30°C constant temperature incubator for 2 h to promote stable colonization of microorganisms. G. Drying and storage of finished product: The obtained composite particles are placed in a vacuum drying oven at 30℃ and dried at low temperature for 4 hours, with the moisture content controlled at 10% to 20%. After sealing, they are stored at 4℃ to obtain a citrus peel-based composite adsorbent containing microbial attachment.

[0008] In step A, the citrus peel is any one or a mixture of orange peel, tangerine peel, or grapefruit peel.

[0009] In step B, the citrate-sodium citrate buffer solution has a pH of 5.

[0010] In step E, the sodium-based bentonite contains 80% montmorillonite by mass, with a particle size / mesh count ≥200 mesh, an expansion capacity of 25~50 ml / g, and a cation exchange capacity (CEC) ≥70 mmol / 100g.

[0011] In step G, the composite adsorbent is in the form of granules with a diameter of 3-5 mm, and its surface and pores are colonized with composite microorganisms (Bacillus subtilis, Saccharomyces cerevisiae, and lactic acid bacteria), with a viable count ≥ 1.0 × 10⁻⁶. 8 cfu / g; the composite adsorbent retains a microporous-mesoporous composite structure with a specific surface area of ​​500~1200m² / g, a pore size of 3.9~4.3nm, and a surface rich in cobalt active sites (metal-doped type).

[0012] This invention provides a method for removing heavy metals from water using composite microbial-supported porous biochar based on citrus peels, belonging to the technical fields of water treatment functional materials and agricultural waste resource utilization. Using citrus peels (orange peel, tangerine peel, grapefruit peel, etc.) as the core raw material, the invention sequentially processes raw material purification pretreatment, pectinase-α-amylase dual-enzyme bioactivation, cobalt metal doping modification, and segmented temperature-controlled inert atmosphere carbonization to prepare citrus peel-based porous biochar with high specific surface area, multiple active sites, and well-developed pore structure. Then, Bacillus subtilis, Saccharomyces cerevisiae, and lactic acid bacteria are compounded in a specific ratio to form a heavy metal-tolerant composite microbial community. Using a dual immobilization technology of physical pre-adsorption + sodium alginate-gelatin gel encapsulation, the composite microorganisms are stably loaded onto the surface and pores of the porous biochar, ultimately yielding a composite adsorbent with synergistic effects of biochar physicochemical adsorption and microbial bioadsorption / complexation. This invention combines the resource utilization of waste fruit peels, biochar structural modification, and microbial enhanced removal. The preparation process is green and controllable, with mild conditions. The resulting product has a high efficiency in removing heavy metal ions such as lead, copper, and chromium from water. It also has the advantages of structural stability, minimal microbial loss, and recyclability, providing an integrated solution for the treatment of heavy metal pollution in water and the high-value utilization of agricultural solid waste.

[0013] The beneficial effects of this invention are as follows: Citrus peel, as an agricultural waste, has significant advantages in its utilization: firstly, it has a large yield and wide range of sources; secondly, the raw material price is low; thirdly, the raw material sources are concentrated; and fourthly, it has social, economic, and environmental benefits, both mitigating environmental pollution and increasing the utilization value of citrus peel waste. The synergistic activation of pectinase and α-amylase effectively removes pectin and starch impurities from the peel, loosening the fibrous structure and laying the foundation for subsequent carbonization and pore formation. Combined with cobalt metal doping and segmented temperature-controlled carbonization, the resulting biochar has a large specific surface area, a rich microporous-mesoporous composite structure, and a surface rich in oxygen-containing functional groups and cobalt active sites, which not only enhances the physicochemical adsorption capacity for heavy metals but also provides sufficient carrier sites for microbial colonization.

[0014] This treatment utilizes a composite microbial community of Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus. These three microorganisms exhibit strong synergistic effects and high tolerance to heavy metals. Through extracellular polymeric complexation, cell wall adsorption, and metabolic acid production to regulate the microenvironment, they achieve the bioimmobilization and removal of heavy metals, overcoming the limitations of single-bacterial biochar adsorption, which is prone to saturation and has limited removal efficiency. The dual-loading technology results in a thin and uniform gel layer that does not clog the pores of the biochar, ensuring strong microbial colonization and preventing detachment. Even after repeated use, the microorganisms maintain high activity, demonstrating stability far superior to traditional direct mixing loading methods. Biochar rapidly enriches heavy metal ions in water, while the composite microorganisms simultaneously perform bioadsorption and complexation fixation. The synergistic effect of physicochemical adsorption and biological action results in high removal rates for common heavy metals such as lead, copper, and chromium, making it suitable for treating wastewater with varying heavy metal concentrations. The entire process uses non-toxic and environmentally friendly reagents, and the enzymatic hydrolysis, doping, carbonization, and microbial loading processes are mild, producing no toxic or harmful byproducts. The finished adsorbent is a biodegradable granular material, causing no secondary pollution after use and meeting environmentally friendly material requirements. The prepared composite adsorbent consists of regular particles of 3-5 mm with high mechanical strength and convenient solid-liquid separation. It can be reused after simple activation treatment, has a long service life, and effectively reduces the operating cost of wastewater treatment. Detailed Implementation

[0015] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are merely preferred implementations of the present invention and are not intended to limit the scope of protection of the present invention. Example 1:

[0016] A method for preparing composite microbially supported citrus peel-based porous biochar for removing heavy metals from water, comprising the following steps: S1 Raw Material Pretreatment: Select fresh orange peel, cut it into 1cm×1cm small pieces, wash it three times alternately with deionized water and anhydrous ethanol to remove surface impurities, sugar and pectin floating layer, dry it at 80℃ for 10h to constant weight, pulverize it and pass it through an 80-mesh sieve to obtain orange peel powder. S2 Bio-activation Modification: Prepare a mixture of orange peel powder and deionized water at a ratio of 1g:12mL. Add citrate-sodium citrate buffer solution at pH 5, add pectinase at 120U / g, and incubate at 50℃ for 1.5h. Then add CaCl2 to make the Ca2+ concentration of the system 0.01mol / L, adjust the pH to 6.0 with dilute HCl, add α-amylase at 120U / g, and incubate at 59℃ for 40min. Filter through a 200-mesh nylon screen, wash the filter residue until neutral, and dry at 80℃ to constant weight to obtain enzymatically modified orange peel powder. S3 Enhancement Modification: Mix enzymatically modified orange peel powder and cobalt nitrate hexahydrate in a ratio of 2:1 (mass ratio), add ultrapure water, sonicate at 400W for 5 min, stir at 400rpm at room temperature for 5 h, filter, wash with deionized water and anhydrous ethanol alternately 3 times, and vacuum dry at 60℃ for 24 h to obtain cobalt-doped precursor. S4 High-Temperature Carbonization: Cobalt-doped precursor is loaded into a ceramic crucible, sealed with tin foil, and purged with nitrogen at 0.8 L / min in a tube furnace. The temperature is raised to 600℃ at 5℃ / min and calcined for 4 hours. Then the temperature is lowered to 320℃ at 12℃ / min and calcined for 35 minutes. After natural cooling, the material is ground, cleaned, and dried to obtain porous orange peel-based biochar. Preparation of S5 composite microbial culture: Bacillus subtilis culture (4.0 × 10⁻⁶) was prepared. 8 CFU / mL), brewer's yeast culture (2.0 × 10⁻⁶) 8 cfu / mL), lactic acid bacteria culture (4.0×10⁻⁶) 8 Mix (cfu / mL) at a volume ratio of 8:4:1, add sodium bentonite and stir to adsorb, then add 2% sodium alginate solution and mix well to obtain microbial mixture; S6 composite microbial loading: Porous orange peel-based biochar: activated composite bacterial solution = 1g: 20mL, and adsorbed at 30℃ for 1h (stirred at 50rpm); prepare 3wt% sodium alginate-1.5wt% gelatin composite embedding solution, add biochar and mix well, add 4wt% sterile CaCl2 solution dropwise, crosslink at room temperature for 7.5h to obtain composite particles with a diameter of 4mm; wash with physiological saline and activate at 30℃ for 2h; S7 Finished Product Drying and Storage: The composite particles were vacuum dried at 30℃ for 4 hours, the moisture content was controlled at 15%, and the product was refrigerated at 4℃ to obtain a composite microbial-supported orange peel-based porous biochar adsorbent.

[0017] Treatment effect: For the treatment of 50 mg / L hexavalent chromium wastewater, the composite microbial-supported orange peel-based porous biochar adsorbent is superior to organic bentonite and ordinary citrus peel biochar.

[0018]

[0019] Organic bentonite 60.30% 69.10% 80.30% 89% 91.00% Common citrus peel biochar 54.80% 62.20% 68.80% 70.10% 72.20% Composite microbial-supported porous biochar based on citrus peel 65.00% 79.50% 87.60% 97.80% 98.90% Example 2:

[0020] A method for preparing composite microbially supported citrus peel-based porous biochar for removing heavy metals from water, comprising the following steps: S1 Raw material pretreatment: Select fresh grapefruit peel, cut it into 1cm×1cm small pieces, wash it 3 times with deionized water and anhydrous ethanol alternately, dry it at 60℃ for 12h until constant weight, pulverize it and pass it through a 60-mesh sieve to obtain grapefruit peel powder. S2 Bio-activation Modification: Prepare a mixture of grapefruit peel powder and deionized water at a ratio of 1g:10mL. Perform the same enzymatic hydrolysis steps as in Example 1. Filter and dry to obtain enzymatically modified grapefruit peel powder. S3 Enhancement Modification: Mix enzymatically modified grapefruit peel powder and cobalt nitrate hexahydrate in a ratio of 1:1 (mass ratio), ultrasonically mix at 300W for 3 min, stir at 300rpm at room temperature for 4 h, and wash and dry to obtain the cobalt-doped precursor; S4 High-Temperature Carbonization: Nitrogen gas is introduced into the tubular furnace at a rate of 0.5 L / min, and the temperature is raised to 500℃ at a rate of 3℃ / min and calcined at a constant temperature for 3 hours; the temperature is then lowered to 300℃ and held at a constant temperature for 30 minutes, followed by cooling, grinding, and cleaning to obtain porous grapefruit peel-based biochar. Preparation of S5 composite microbial culture: Bacillus subtilis culture (2.5 × 10⁻⁶) 8 CFU / mL), brewer's yeast culture (1.5 × 10⁻⁶) 8 CFU / mL), lactic acid bacteria culture (3.0 × 10⁻⁶) 8 Mix (cfu / mL) in a volume ratio of 6:3:1, add sodium bentonite and sodium alginate solution and mix well to obtain microbial mixture; S6 composite microbial loading: After the biochar and bacterial solution are mixed and adsorbed, a 2wt% sodium alginate-1wt% gelatin embedding solution is prepared, and 4wt% sterile CaCl2 solution is added dropwise for cross-linking for 7h to obtain composite particles with a diameter of 3mm, which are then activated. S7 Finished Product Drying and Storage: Vacuum dry at 30℃ for 4 hours, moisture content controlled at 10%, and refrigerate at 4℃.

[0021] Treatment effect: 200 mg / L Pb 2+ Cu 2+ Cr 6+ After 2 hours of treatment, the removal rates of mixed heavy metal wastewater were 96.5%, 95.8%, and 95.1%, respectively.

[0022] Example 3:

[0023] A method for preparing composite microbially supported citrus peel-based porous biochar for removing heavy metals from water, comprising the following steps: S1 Raw material pretreatment: Select fresh orange peel, cut it into 1cm×1cm small pieces, wash it 3 times alternately with deionized water and anhydrous ethanol, dry it at 100℃ for 8 hours until constant weight, pulverize it and pass it through a 100-mesh sieve to obtain orange peel powder. S2 Bio-activation Modification: Prepare a mixture of orange peel powder and deionized water at a ratio of 1g:15mL. Perform the enzymatic hydrolysis as in Example 1, and filter and dry to obtain enzymatically modified orange peel powder. S3 Enhancement Modification: Mix enzymatically modified orange peel powder and cobalt nitrate hexahydrate in a ratio of 3:1 (mass ratio), ultrasonically mix at 500W for 10 min, stir at 500rpm at room temperature for 6 h, and wash and dry to obtain the cobalt-doped precursor; S4 High-Temperature Carbonization: Nitrogen gas is introduced into the tubular furnace at a rate of 1L / min, and the temperature is raised to 700℃ at a rate of 10℃ / min and calcined at a constant temperature for 5 hours; the temperature is then lowered to 350℃ and held at a constant temperature for 40 minutes, followed by cooling, grinding and cleaning to obtain porous orange peel-based biochar. Preparation of S5 composite microbial culture: Bacillus subtilis culture (5.0 × 10⁻⁶) 8 CFU / mL), brewer's yeast culture (3.0 × 10⁻⁶) 8 CFU / mL), lactic acid bacteria culture (5.0 × 10⁻⁶) 8 Mix (cfu / mL) in a volume ratio of 9:5:2, add sodium bentonite and sodium alginate solution and mix well to obtain microbial mixture; S6 Composite Microbial Loading: After adsorption by mixing biochar and bacterial solution, a 4wt% sodium alginate-2wt% gelatin embedding solution was prepared, and 5wt% sterile CaCl2 solution was added dropwise for cross-linking for 8 hours to obtain composite particles with a diameter of 5mm, which were then activated. S7 Finished Product Drying and Storage: Vacuum drying at 30℃ for 4 hours, with the moisture content controlled at 20%, and refrigerated storage at 4℃.

[0024] Analysis of the effect of temperature on the adsorption capacity of the adsorbent in Example 3 on 100 mg / L hexavalent chromium wastewater showed that the adsorption capacity increased slowly with increasing temperature, and was positively correlated within the test temperature range.

[0025]

[0026] With the increase of the adsorbent dosage in Example 3, the adsorption rate of hexavalent chromium by the adsorbent in Example 3 significantly increased. This phenomenon is mainly due to the additional adsorption sites provided by the increased biochar dosage. When the dosage reached 4 g / L, the adsorption rate of hexavalent chromium by biochar was 98.1%.

[0027]

Claims

1. A method for preparing composite microbially supported citrus peel-based porous biochar for removing heavy metals from water, characterized in that, Includes the following steps: S1 raw material pretreatment: Citrus peels are cut into small pieces and washed three times alternately with deionized water and anhydrous ethanol to remove surface impurities, sugars and pectin layers. They are then dried at 60-100℃ for 8-12 hours until constant weight, pulverized and passed through a 40-100 mesh sieve to obtain citrus peel powder. S2 bio-activation modification: Prepare a mixture of S1 citrus peel powder and deionized water at a ratio of 1g:10-15mL. Add citrate-sodium citrate buffer solution at pH 5, and add pectinase at a rate of 120U / g. Incubate at 50℃ for 1.5h. Then add CaCl2 to make the Ca2+ concentration of the system 0.01mol / L, adjust the pH to 6.0 with dilute HCl, add α-amylase at a rate of 120U / g, and incubate at 59℃ for 40min. Filter through a 200-mesh nylon screen, wash the filter residue with deionized water until neutral, and dry at 80℃ to constant weight to obtain enzymatically modified citrus peel powder. S3 Enhancement Modification: The cobalt-doped precursor was obtained by mixing enzymatically modified citrus peel powder and cobalt nitrate hexahydrate in a ratio of 1 to 3:1 (mass ratio), adding ultrapure water, ultrasonically mixing at 300 to 500 W for 3 to 10 min, stirring at 300 to 500 rpm at room temperature for 4 to 6 h, filtering, washing with deionized water and anhydrous ethanol alternately 3 times, and vacuum drying at 60 °C for 24 h. S4 high temperature carbonization: The modified precursor obtained in step S3 was loaded into a ceramic crucible, compacted, covered, and sealed with tin foil. It was then placed in a tube furnace, and nitrogen gas was introduced at a rate of 0.5–1 L / min to purge the air. The temperature was increased to 500–700 °C at a rate of 3–10 °C / min and calcined at a constant temperature for 3–5 h. The temperature was then decreased to 300–350 °C at a rate of 12 °C / min and held at a constant temperature for 30–40 min. The mixture was then allowed to cool naturally to room temperature. After grinding, the mixture was washed three times alternately with deionized water and anhydrous ethanol. It was then vacuum dried at 60 °C for 24 h to obtain porous citrus peel-based biochar. Preparation of S5 composite microbial inoculum: Bacillus subtilis culture, Saccharomyces cerevisiae culture and Lactobacillus culture were mixed in a volume ratio of (6-9):(3-5):(1-2), sodium bentonite was added and stirred to adsorb, and then added dropwise to sodium alginate solution to obtain microbial mixture. S6 composite microbial load: A mixture of porous citrus peel-based biochar and activated composite bacterial solution was prepared at a ratio of 1g:20mL. The mixture was incubated at 30℃ for 1 hour with low-speed stirring at 50rpm to achieve initial microbial colonization. 2–4g of sodium alginate and 1–2g of gelatin were added to 100mL of sterile water and stirred in a 60℃ water bath until completely dissolved. The solution was then cooled to ≤35℃ (to avoid high-temperature inactivation) to obtain a 2–4wt% sodium alginate-1–2wt% gelatin composite embedding solution. The biochar after bacterial solution adsorption was added to the composite embedding solution and stirred at low speed at room temperature for 30 minutes to form a char-bacteria-gel mixture (biochar content 8%–12%). This mixture was then dropped into a 4–5wt% sterile CaCl2 solution using a syringe at a rate of 1 drop / second. Cross-linking and fixation were performed at room temperature for 7–8 hours to form composite particles with a diameter of 3–5mm. The particles were washed three times with sterile physiological saline to remove residual CaCl2 and then activated in a 30℃ incubator for 2 hours to promote stable microbial colonization. S7 Finished Product Drying and Storage: The obtained composite particles were placed in a vacuum drying oven at 30℃ and dried at low temperature for 4 hours, with the moisture content controlled at 10% to 20%. After sealing, they were stored at 4℃ to obtain a citrus peel-based composite adsorbent containing microbial attachment.

2. The citrus peel according to claim 1 is any one or a mixture of tangerine peel, orange peel, or grapefruit peel.

3. The effective viable count of Bacillus subtilis according to claim 1 is 2.5–5.0 × 10⁻⁶. 8 The effective viable count of brewer's yeast is 1.5–3.0 × 10⁻⁶ CFU / ml. 8 The cfu / ml concentration and the effective viable count of lactic acid bacteria are 3.0–5.0 × 10⁻⁶. 8 The concentration of the bentonite solution is 10-20% of the volume of the microbial culture solution, and the concentration of the sodium alginate solution is 2-4 wt%.

4. The sodium-based bentonite according to claim 1 contains 80% montmorillonite by mass, with a particle size / mesh count ≥ 200 mesh, an expansion volume of 25~50 ml / g, and a cation exchange capacity (CEC) ≥ 70 mmol / 100g.

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