Composite bioflocculant, preparation method and application thereof

Abstract

The application provides a composite biological flocculant and a preparation method and application thereof, the flocculant is composed of 20-40 parts of flocculation bacteria fermentation extract, 5-10 parts of modified plant-based flocculation component, 8-18 parts of cationized nanocellulose, 5-15 parts of porous mineral powder and 2-6 parts of biological surfactant. The application adopts bacillus licheniformis and candida utilis double-strain synergistic fermentation to produce flocculation, and respectively modifies the jatropha seed protein and nanocellulose, and through gradient compounding process, the biological components are uniformly loaded on the surface of inorganic carrier to form a composite system. The application solves the problems of low activity, slow settling and single function of the existing biological flocculant, the removal rates of turbidity, COD, total phosphorus and heavy metal are respectively ≥96%, 85%, 92% and 97%, the settling speed is increased by 2-3 times, there is no secondary pollution, and the application is suitable for purification treatment of municipal sewage, industrial wastewater and various water bodies.

Description

Technical Field

[0001] This invention relates to the field of water pollution control technology, and in particular to a composite bioflocculant, its preparation method, and its application. Background Technology

[0002] Water pollution is one of the major environmental problems facing the world today. Flocculation is one of the most commonly used physicochemical methods in water treatment, widely applied to industrial wastewater, urban sewage, and natural water body remediation, playing an irreplaceable role in water purification. Traditional inorganic flocculants, such as aluminum and iron salts, are inexpensive, but require large dosages, produce high sludge yields, and their residual metal ions may pose potential risks to the environment and human health. Synthetic organic polymeric flocculants, such as polyacrylamide, while having high flocculation efficiency, have monomer residues that are neurotoxic and carcinogenic, and are difficult to biodegrade, easily causing secondary pollution.

[0003] In recent years, bioflocculants, represented by microbial metabolites and natural plant extracts, have attracted attention due to their advantages such as safety and biodegradability. However, bioflocculants produced by single-strain fermentation have low flocculation activity and poor adaptability to complex water quality. The molecular chains of bioflocculants are generally short, with insufficient bridging ability, resulting in small and loose flocs with slow settling speed. They are also not very effective in removing pollutants such as heavy metal ions and total phosphorus in water, and their functions are relatively limited. At the same time, they have high production costs and poor storage stability, making it difficult to achieve large-scale industrial production and application. Summary of the Invention

[0004] In view of this, the present invention proposes a composite bio-flocculator, its preparation method and application, to solve the above problems.

[0005] The technical solution of the present invention is as follows: a composite bio-flocculator, comprising the following raw materials in parts by weight: 20-40 parts of fermentation extract of flocculent bacteria, 5-10 parts of modified plant-based flocculant component, 8-18 parts of cationic nanocellulose, 5-15 parts of porous mineral powder, and 2-6 parts of biosurfactant.

[0006] Preferably, the fermentation extract of the flocculent bacteria is prepared according to the following steps: seed cultures of Bacillus licheniformis and Candida utilis are inoculated into the fermentation medium at a volume ratio of 1:0.5-2, with an inoculation amount of 3-7% (v / v). Fermentation is carried out at a temperature of 28-32℃ and a stirring speed of 150-200 rpm for 48-72 hours. After fermentation, the supernatant is collected by centrifugation. 2-3 times the volume of ethanol is added to the supernatant for precipitation. The precipitate is collected by centrifugation and then freeze-dried to obtain the final product.

[0007] Preferably, the seed culture of Bacillus licheniformis is prepared by the following method: Bacillus licheniformis is inoculated into LB liquid medium at an inoculation rate of 1% to 5% (v / v), and cultured with shaking at a temperature of 35 to 38°C and a rotation speed of 160 to 220 rpm for 14 to 20 hours until the viable count reaches 1×10⁻⁶. 8 ~1×10 9 CFU / mL; The seed culture of *Candida utilis* was prepared as follows: *Candida utilis* was inoculated into malt extract medium at an inoculation rate of 1%–5% (v / v), and cultured with shaking at 28–32°C and 150–220 rpm for 20–40 hours until the cell count reached 1 × 10⁻⁶ cells / mL. 8 cells / mL or higher.

[0008] Preferably, the modified plant-based flocculant is a complex of quaternized moringa seed protein and tea saponin in a mass ratio of (3-5):1.

[0009] Preferably, the preparation method of the quaternized modified moringa seed protein is as follows: moringa seed protein is dissolved in a phosphate buffer solution with a pH of 8.0-9.0 to prepare a solution with a mass concentration of 3-6%, and 2,3-epoxypropyltrimethylammonium chloride is added. The mass ratio of moringa seed protein to 2,3-epoxypropyltrimethylammonium chloride is 1:1.0-1.2. The reaction is carried out at 40-50℃ for 3-5 hours. The reaction product is obtained by dialyzing and freeze-drying, and its degree of cationic substitution is 0.3-0.6.

[0010] Preferably, the cationic cellulose nanoparticles are quaternary ammonium salt-treated cellulose nanoparticles prepared by reacting cellulose nanoparticles with a cationic etherifying agent, wherein the cationic etherifying agent is 2,3-epoxypropyltrimethylammonium chloride, and the preparation steps are as follows: Nanocellulose is dispersed in water to form a suspension with a mass concentration of 1% to 5%. The pH is adjusted to 10 to 12, and then 2,3-epoxypropyltrimethylammonium chloride is added. The mixture is stirred at 60 to 80°C for 4 to 6 hours. The reaction product is neutralized, dialyzed, and dried to obtain the quaternized ammonium nanocellulose.

[0011] Preferably, the porous mineral powder is selected from at least one of attapulgite, diatomaceous earth, and zeolite powder, and all of them have undergone pretreatment. The specific method is as follows: the mineral powder is passed through a 150-250 mesh sieve, 1-2 mol / L hydrochloric acid solution is added, the solid-liquid ratio is 1g:10-15mL, the mixture is stirred and activated at 70-90℃ for 1-3 hours, washed with deionized water until neutral, dried at 100-110℃, and ground for later use.

[0012] Preferably, the biosurfactant is rhamnolipid or sophorolipid.

[0013] A method for preparing a composite bioflocculator according to the present invention includes the following steps: S1. Preparation of inorganic carrier dispersion: Cationic nanocellulose is added to deionized water and ultrasonically dispersed for 10-20 minutes at a power of 200-400W and a temperature of 25-35℃ to form a uniform nanocellulose suspension with a mass concentration of 1%-3%; then porous mineral powder is added and ultrasonically dispersed for another 15-30 minutes to obtain an inorganic carrier dispersion with a solid content of 5%-10%. S2. Preparation of bioflocculation mother liquor: Under normal temperature and pressure, the fermentation extract of flocculent bacteria and the modified plant-based flocculant are added to 100-200 parts of deionized water in a certain proportion. The mixture is stirred at 150-250 rpm for 20-30 minutes until completely dissolved to obtain a biological component solution. Then, a biosurfactant is added, and the mixture is stirred at the same speed for another 10-15 minutes. After mixing evenly, the bioflocculation mother liquor is obtained. S3. Gradient Composite and Post-treatment: The bioflocculation mother liquor obtained in step S2 is slowly added dropwise to the inorganic carrier dispersion obtained in step S1 at a flow rate of 10-20 mL / min. During the dropwise addition, the stirring speed is kept constant at 200-300 rpm throughout the process. After the dropwise addition is completed, stirring is continued for 30-60 minutes to ensure that the bioflocculation components are uniformly loaded on the surface of the inorganic carrier composite system. Finally, the mixture is subjected to vacuum freeze-drying or spray drying. The dried product is pulverized and passed through an 80-120 mesh standard sieve to obtain a powdered composite bioflocculator.

[0014] The application of the above-mentioned composite bioflocculant or the composite bioflocculant prepared by the above method in water environment pollution control.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention employs the synergistic fermentation of Bacillus licheniformis and Candida utilis, producing extracellular metabolites rich in polysaccharides, proteins, nucleic acids, and other flocculating active substances. These metabolites, in conjunction with modified plant-based flocculants, significantly enhance the flocculation effect. Cationic nanocellulose provides a three-dimensional network structure, strengthening the bridging ability and resulting in large, dense flocs with a settling velocity 2-3 times higher than that of a single bioflocculant.

[0016] 2. This invention not only efficiently removes suspended solids and COD from water, but also removes total phosphorus and heavy metal ions simultaneously through the adsorption of porous mineral powder and the complexation of biological components. The removal rates are ≥96% for turbidity, ≥85% for COD, ≥92% for total phosphorus, and ≥96% for Pb. 2+ The removal rate of heavy metal ions is ≥97%.

[0017] 3. This invention exhibits good flocculation effects within a pH range of 5.0 to 9.0, demonstrates good adaptability to different types of wastewater, and exhibits strong resistance to water quality fluctuations. All raw materials are of natural or biological origin, are biodegradable, and will not leave any toxic or harmful substances in the water body, thus avoiding secondary pollution problems caused by chemical flocculants. The preparation process is simple, energy consumption is low, and it is suitable for large-scale industrial production and application. Detailed Implementation

[0018] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0019] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0020] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0021] Microbial strains and raw materials in the embodiments of this invention: Bacillus licheniformis, with accession number CCTCC AB2014185, was purchased from the China Center for Type Culture Collection. Candida utilis, with accession number CCTCC KY 2008676, was purchased from the China Center for Type Culture Collection.

[0022] Biosurfactants: rhamnolipin purity ≥90%, sophorolipid purity ≥85%, both are commercially available industrial grade products.

[0023] 2,3-Epoxypropyltrimethylammonium chloride: Industrial grade, effective content ≥65%.

[0024] Ethanol: Industrial grade, concentration ≥95%.

[0025] Moringa seed protein: Purchased from Shanghai JueTu Biotechnology Co., Ltd., white powder, with an effective substance content of 60%, the main component is moringa seed protein peptide (molecular weight 500-1000 Daltons), product model JT-874567.

[0026] Culture medium formulation The composition of LB liquid culture medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and water as the solvent.

[0027] Malt extract culture medium: 10°Bx malt extract, pH 5.0~6.0, autoclaved at 121℃ for 20 minutes.

[0028] Fermentation medium: glucose 25g / L, corn steep liquor powder 15g / L, urea 4g / L, potassium dihydrogen phosphate 1.5g / L, magnesium sulfate 0.8g / L, manganese sulfate 0.08g / L, pH 7.0~7.2, autoclaved at 121℃ for 20 minutes. Example 1

[0029] This embodiment provides a composite bioflocculator, comprising the following raw materials in parts by weight: 30 parts of flocculating bacteria fermentation extract, 7 parts of modified plant-based flocculant, 13 parts of cationic nanocellulose, 10 parts of porous mineral powder, and 4 parts of biosurfactant.

[0030] The fermentation extract of the flocculent bacteria was prepared according to the following steps: Bacillus licheniformis and Candida utilis seed culture were inoculated into the fermentation medium at a volume ratio of 1:1, with an inoculation amount of 5% (v / v). Fermentation was carried out at 30°C, stirring speed of 180 rpm, and aeration rate of 1.0 vvm for 60 hours. After fermentation, the supernatant was collected by centrifugation at 8000 rpm for 15 minutes. 2.5 times the volume of anhydrous ethanol was added to the supernatant, and the mixture was allowed to stand at 4°C for 12 hours to precipitate. The precipitate was collected by centrifugation at 10000 rpm for 20 minutes and then freeze-dried at -50°C and vacuum degree of 10 Pa for 24 hours to obtain the extract.

[0031] The seed culture of Bacillus licheniformis was prepared as follows: Bacillus licheniformis was inoculated into LB liquid medium at an inoculation rate of 3% (v / v) and cultured with shaking at 37°C and 200 rpm for 18 hours until the viable count reached 5 × 10⁻⁶. 8 CFU / mL; The seed culture of *Candida utilis* was prepared as follows: *Candida utilis* was inoculated into malt extract medium at an inoculation rate of 3% (v / v) and cultured with shaking at 30°C and 200 rpm for 30 hours until the cell count reached 5 × 10⁻⁶ cells / mL. 8 cells / mL or higher.

[0032] The modified plant-based flocculant is a complex of quaternized moringa seed protein and tea saponin in a mass ratio of 4:1.

[0033] The preparation method of the quaternized modified moringa seed protein is as follows: Moringa seed protein is dissolved in 0.1 mol / L phosphate buffer at pH 8.5 to prepare a 5% (w / w) solution. 2,3-epoxypropyltrimethylammonium chloride is added, with a mass ratio of moringa seed protein to 2,3-epoxypropyltrimethylammonium chloride of 1:1.2. The reaction is carried out at 45°C for 4 hours. The reaction product is dialyzed for 48 hours using a dialysis bag with a molecular weight cutoff of 8000~14000 Da, with deionized water replaced every 6 hours. After dialysis, the product is freeze-dried to obtain a cation substitution degree of 0.45.

[0034] The preparation steps of the cationic nanocellulose are as follows: nanocellulose is dispersed in deionized water to form a suspension with a mass concentration of 3%. The pH is adjusted to 11 with 2 mol / L sodium hydroxide solution. Then, 2,3-epoxypropyltrimethylammonium chloride is added, with a mass ratio of nanocellulose to 2,3-epoxypropyltrimethylammonium chloride of 1:2. The mixture is stirred at 70°C for 5 hours. The reaction product is neutralized to pH 7.0 with 1 mol / L hydrochloric acid. The mixture is dialyzed for 72 hours using a dialysis bag with a molecular weight cutoff of 3500 Da. After freeze-drying, the quaternary ammonium salt nanocellulose with a cationic substitution degree of 0.52 is obtained.

[0035] The porous mineral powder is a mixture of attapulgite and diatomaceous earth in a mass ratio of 1:1. After pretreatment, the specific method is as follows: the mineral powder is passed through a 200-mesh sieve, 1.5 mol / L hydrochloric acid solution is added, the solid-liquid ratio is 1 g:12 mL, the mixture is stirred and activated at 80°C for 2 hours, washed with deionized water until neutral, dried at 105°C, and ground for later use.

[0036] The biosurfactant is rhamnolipid.

[0037] The preparation method of the above-mentioned composite bioflocculant includes the following steps: S1. Preparation of inorganic carrier dispersion: Cationic nanocellulose was added to deionized water and ultrasonically dispersed for 15 minutes at a power of 300W and a temperature of 30℃ to form a uniform nanocellulose suspension with a mass concentration of 2%; then pretreated porous mineral powder was added and ultrasonically dispersed for another 20 minutes to obtain a stable inorganic carrier dispersion. S2. Preparation of bioflocculation mother liquor: 30 parts of flocculating bacteria fermentation extract and 7 parts of modified plant-based flocculant components were added to 150 parts of deionized water and stirred at 200 rpm for 25 minutes until completely dissolved to obtain a biological component solution; then 4 parts of biosurfactant were added and stirred at the same speed for 12 minutes until evenly mixed to obtain bioflocculation mother liquor. S3. Gradient Composite and Post-treatment: The bioflocculation mother liquor obtained in step S2 is slowly added dropwise to the inorganic carrier dispersion obtained in step S1 at a flow rate of 15 mL / min. During the dropwise addition, the stirring speed is kept constant at 250 rpm and the temperature is kept constant at 30℃. After the dropwise addition is completed, stirring is continued for 45 minutes to allow the bioflocculation components to be uniformly loaded on the surface of the inorganic carrier through hydrogen bonding and electrostatic interaction to form a three-dimensional network composite system. Finally, the mixture was subjected to vacuum freeze drying under the following conditions: pre-freezing temperature -50℃, pre-freezing time 4 hours, vacuum degree 10Pa, and drying time 30 hours. The dried product was then pulverized and passed through a 100-mesh standard sieve to obtain a powdered composite bio-flocculator. Example 2

[0038] This embodiment provides a composite bio-flocculator, comprising the following raw materials in parts by weight: 20 parts of flocculating bacteria fermentation extract, 5 parts of modified plant-based flocculant, 8 parts of cationic nanocellulose, 5 parts of porous mineral powder, and 2 parts of biosurfactant.

[0039] The fermentation extract of the flocculent bacteria was prepared according to the following steps: Bacillus licheniformis and Candida utilis seed culture were inoculated into the fermentation medium at a volume ratio of 1:0.5, with a total inoculation amount of 3% (v / v). Fermentation was carried out at 28°C, stirring speed of 150 rpm, and aeration rate of 0.8 vvm for 72 hours. After fermentation, the supernatant was collected by centrifugation at 8000 rpm for 15 minutes. Two volumes of anhydrous ethanol were added to the supernatant, and the mixture was allowed to stand at 4°C for 12 hours to precipitate. The precipitate was collected by centrifugation at 10000 rpm for 20 minutes and then freeze-dried at -45°C and vacuum degree of 15 Pa for 30 hours to obtain the extract.

[0040] The seed culture of Bacillus licheniformis was prepared as follows: Bacillus licheniformis was inoculated into LB liquid medium at an inoculation rate of 1% (v / v) and cultured with shaking at 35°C and 160 rpm for 20 hours until the viable count reached 1×10⁻⁶. 8 CFU / mL; The seed culture of *Candida utilis* was prepared as follows: *Candida utilis* was inoculated into malt extract medium at an inoculation rate of 1% (v / v) and cultured with shaking at 28°C and 150 rpm for 40 hours until the cell count reached 1 × 10⁻⁶. 8 cells / mL or higher.

[0041] The modified plant-based flocculant is a complex of defatted moringa seed crude protein modified by quaternization and tea saponin in a mass ratio of 3:1.

[0042] The method for preparing the quaternized modified defatted moringa seed crude protein is as follows: Moringa seed protein is dissolved in 0.1 mol / L phosphate buffer at pH 8.0 to prepare a 5% (w / w) solution. 2,3-epoxypropyltrimethylammonium chloride is added, with a mass ratio of moringa seed protein to 2,3-epoxypropyltrimethylammonium chloride of 1:1.0. The reaction is carried out at 40°C for 5 hours. The reaction product is dialyzed for 48 hours using a dialysis bag with a molecular weight cutoff of 8000~14000 Da, with deionized water replaced every 6 hours. After dialysis, the product is freeze-dried to obtain the product, which has a cation substitution degree of 0.30.

[0043] The preparation steps of the cationic nanocellulose are as follows: nanocellulose is dispersed in deionized water to form a suspension with a mass concentration of 1%, the pH is adjusted to 10 with 2 mol / L sodium hydroxide solution, and then 2,3-epoxypropyltrimethylammonium chloride is added. The mass ratio of nanocellulose to 2,3-epoxypropyltrimethylammonium chloride is 1:1.5. The reaction is stirred at 60°C for 6 hours. The reaction product is neutralized to pH 7.0 with 1 mol / L hydrochloric acid. The product is dialyzed with a dialysis bag with a molecular weight cutoff of 3500 Da for 72 hours. After freeze-drying, the quaternary ammonium salt nanocellulose with a cationic substitution degree of 0.38 is obtained.

[0044] The porous mineral powder is zeolite powder. After pretreatment, the specific method is as follows: the mineral powder is passed through a 150-mesh sieve, added to a 1 mol / L hydrochloric acid solution (solid-liquid ratio 1 g:10 mL), stirred and activated at 70°C for 1 hour, washed with deionized water until neutral, dried at 100°C, and ground for later use. The biosurfactant is sophorolipid.

[0045] The preparation method of the above-mentioned composite bioflocculant includes the following steps: S1. Preparation of inorganic carrier dispersion: Cationic nanocellulose was added to deionized water and ultrasonically dispersed for 20 minutes at a power of 200W and a temperature of 25℃ to form a uniform nanocellulose suspension with a mass concentration of 1%; then pretreated porous mineral powder was added and ultrasonically dispersed for another 30 minutes to obtain a stable inorganic carrier dispersion. S2. Preparation of bioflocculation mother liquor: 20 parts of flocculating bacteria fermentation extract and 5 parts of modified plant-based flocculant components were added to 100 parts of deionized water and stirred at 150 rpm for 30 minutes until completely dissolved to obtain a biological component solution; then 2 parts of biosurfactant were added and stirred at the same speed for 15 minutes until evenly mixed to obtain bioflocculation mother liquor. S3. Gradient Composite and Post-treatment: The bioflocculation mother liquor obtained in step S2 is slowly added dropwise to the inorganic carrier dispersion obtained in step S1 at a flow rate of 10 mL / min. During the dropwise addition, the stirring speed is kept constant at 200 rpm and the temperature is kept constant at 25℃. After the dropwise addition is completed, stirring is continued for 60 minutes to allow the bioflocculation components to be uniformly loaded on the surface of the inorganic carrier through hydrogen bonding and electrostatic interaction to form a three-dimensional network composite system. Finally, the mixture is spray-dried under the following conditions: inlet air temperature 150℃, outlet air temperature 75℃, and feed flow rate 8mL / min. The dried product is then pulverized and passed through an 80-mesh standard sieve to obtain a powdered composite bio-flocculator. Example 3

[0046] This embodiment provides a composite bioflocculator, comprising the following raw materials in parts by weight: 40 parts of flocculating bacteria fermentation extract, 10 parts of modified plant-based flocculant, 18 parts of cationic nanocellulose, 15 parts of porous mineral powder, and 6 parts of biosurfactant.

[0047] The fermentation extract of the flocculent bacteria was prepared according to the following steps: Bacillus licheniformis and Candida utilis seed culture were inoculated into the fermentation medium at a volume ratio of 1:2, with a total inoculation amount of 7% (v / v). Fermentation was carried out at 32°C, stirring speed of 200 rpm, and aeration rate of 1.2 vvm for 48 hours. After fermentation, the supernatant was collected by centrifugation at 8000 rpm for 15 minutes. Three times the volume of anhydrous ethanol was added to the supernatant, and the mixture was allowed to stand at 4°C for 12 hours to precipitate. The precipitate was collected by centrifugation at 10000 rpm for 20 minutes and then freeze-dried at -55°C and vacuum degree of 5 Pa for 24 hours to obtain the extract.

[0048] The seed culture of Bacillus licheniformis was prepared as follows: Bacillus licheniformis was inoculated into LB liquid medium at an inoculation rate of 5% (v / v) and cultured with shaking at 38°C and 220 rpm for 14 hours until the viable count reached 1×10⁻⁶. 9 CFU / mL; The seed culture of *Candida utilis* was prepared as follows: *Candida utilis* was inoculated into malt extract medium at an inoculation rate of 5% (v / v) and cultured with shaking at 32°C and 220 rpm for 20 hours until the cell count reached 1×10⁻⁶. 9 cells / mL or higher.

[0049] The modified plant-based flocculant is a complex of defatted moringa seed crude protein modified by quaternization and tea saponin in a mass ratio of 5:1.

[0050] The method for preparing the quaternized modified defatted moringa seed crude protein is as follows: Moringa seed protein is dissolved in 0.1 mol / L phosphate buffer at pH 9.0 to prepare a 5% (w / w) solution. 2,3-epoxypropyltrimethylammonium chloride is added, with a mass ratio of moringa seed protein to 2,3-epoxypropyltrimethylammonium chloride of 1:1.5. The reaction is carried out at 50°C for 3 hours. The reaction product is dialyzed for 48 hours using a dialysis bag with a molecular weight cutoff of 8000~14000 Da, with deionized water replaced every 6 hours. After dialysis, the product is freeze-dried to obtain the product, which has a cation substitution degree of 0.60.

[0051] The preparation steps of the cationic nanocellulose are as follows: nanocellulose is dispersed in deionized water to form a suspension with a mass concentration of 5%. The pH is adjusted to 12 with 2 mol / L sodium hydroxide solution. Then, 2,3-epoxypropyltrimethylammonium chloride is added. The mass ratio of nanocellulose to 2,3-epoxypropyltrimethylammonium chloride is 1:2.5. The mixture is stirred at 80°C for 4 hours. The reaction product is neutralized to pH 7.0 with 1 mol / L hydrochloric acid. The mixture is dialyzed for 72 hours using a dialysis bag with a molecular weight cutoff of 3500 Da. After freeze-drying, the quaternary ammonium salt nanocellulose with a cationic substitution degree of 0.65 is obtained.

[0052] The porous mineral powder is attapulgite, which is pretreated by the following method: passing the mineral powder through a 250-mesh sieve, adding 2 mol / L hydrochloric acid solution at a solid-liquid ratio of 1 g: 15 mL, stirring and activating at 90°C for 3 hours, washing with deionized water until neutral, drying at 110°C, and grinding for later use.

[0053] The biosurfactant is rhamnolipid.

[0054] The preparation method of the above-mentioned composite bioflocculant includes the following steps: S1. Preparation of inorganic carrier dispersion: Cationic nanocellulose was added to deionized water and ultrasonically dispersed for 10 minutes at a power of 400W and a temperature of 35℃ to form a uniform nanocellulose suspension with a mass concentration of 3%; then pretreated porous mineral powder was added and ultrasonically dispersed for another 15 minutes to obtain a stable inorganic carrier dispersion. S2. Preparation of bioflocculation mother liquor: 40 parts of flocculating bacteria fermentation extract and 10 parts of modified plant-based flocculant were added to 200 parts of deionized water and stirred at 250 rpm for 20 minutes until completely dissolved to obtain a biological component solution; then 6 parts of biosurfactant were added and stirred at the same speed for 10 minutes until evenly mixed to obtain bioflocculation mother liquor. S3. Gradient Composite and Post-treatment: The bioflocculation mother liquor obtained in step S2 is slowly added dropwise to the inorganic carrier dispersion obtained in step S1 at a flow rate of 20 mL / min. During the dropwise addition, the stirring speed is kept constant at 300 rpm and the temperature is kept constant at 35℃. After the dropwise addition is completed, stirring is continued for 30 minutes to allow the bioflocculation components to be uniformly loaded on the surface of the inorganic carrier through hydrogen bonding and electrostatic interaction to form a three-dimensional network composite system. Finally, the mixture was subjected to vacuum freeze drying under the following conditions: pre-freezing temperature -55℃, pre-freezing time 6 hours, vacuum degree 5Pa, and drying time 24 hours. The dried product was then pulverized and passed through a 120-mesh standard sieve to obtain a powdered composite bio-flocculator. Comparative Example 1

[0055] The difference between this comparative example and Example 1 is that cationized nanocellulose is not added, while the other components and preparation methods are the same. Comparative Example 2

[0056] The difference between this comparative example and Example 1 is that unmodified moringa seed protein was used instead of quaternized moringa seed protein, while the other components and preparation methods were the same. Comparative Example 3

[0057] The difference between this comparative example and Example 1 is that only a single strain of Bacillus licheniformis was used to ferment and prepare the flocculent bacteria fermentation extract, while the other components and preparation methods are the same. Comparative Example 4

[0058] This comparative example uses commercially available polyaluminum chloride (PAC) with an active ingredient content of ≥28% Al2O3. Comparative Example 5

[0059] The difference between this comparative example and Example 1 is that: no cationic nanocellulose is added, the amount of modified plant-based flocculant is increased to 20 parts, and only cationic moringa seed protein is used as the cationic flocculant. The other components and preparation methods are the same. Comparative Example 6

[0060] The difference between this comparative example and Example 1 is that no modified plant-based flocculant is added, the amount of cationic nanocellulose is increased to 20 parts, and only cationic nanocellulose is used as the cationic flocculant. The other components and preparation methods are the same. Performance testing

[0061] The influent from the secondary sedimentation tank of a municipal wastewater treatment plant was analyzed. The water quality parameters were: turbidity 285 NTU, COD 320 mg / L, total phosphorus 4.2 mg / L, and Pb... 2+ Concentration 0.8 mg / L, pH 7.2.

[0062] Take 1000 mL of the above wastewater and place it in a beaker. Adjust the pH to 7.0. Add the flocculants prepared in Examples 1-3 and Comparative Examples 1-6 at a dosage of 100 mg / L. Stir rapidly for 2 minutes at 300 rpm, then stir slowly for 8 minutes at 80 rpm. Let it stand for 20 minutes to settle. Take the supernatant and measure various water quality indicators. Test method: 1. Turbidity (based on HJ 1075-2019 Determination of Turbidity in Water) Calibrate the turbidity meter to zero and full scale using standard turbidity solution. Take the supernatant after settling and inject it directly into the turbidity meter cuvette. Read and record the turbidity value. Perform three parallel measurements and take the average value. Calculation formula: Turbidity removal rate = (Initial turbidity - Residual turbidity) / Initial turbidity × 100% 2. COD (based on HJ828-2017) Take 20.0 mL of water sample into a digestion tube, add 10.0 mL of potassium dichromate standard solution and a few anti-bounce glass beads, slowly add 30 mL of sulfuric acid-silver sulfate solution, shake well, and heat in a digester at 165℃ for 15 minutes. After cooling to room temperature, add 3 drops of ferroin indicator, and titrate with ferrous ammonium sulfate standard solution until the solution changes from yellow to blue-green to reddish-brown. At the same time, perform a blank test. Calculate the COD value based on the titration volume difference. Calculation formula: COD removal rate = (initial COD - remaining COD) / initial COD × 100% 3. Total phosphorus (according to GB / T11893-1989 Ammonium molybdate spectrophotometric method) Take 25.0 mL of water sample into a colorimetric tube, add 4 mL of potassium persulfate solution, seal the tube tightly, and place it in an autoclave at 121℃ for 30 minutes to digest. After cooling to room temperature, add 1 mL of ascorbic acid solution and shake well. After 30 seconds, add 2 mL of ammonium molybdate solution and mix thoroughly. Develop the color at room temperature for 15 minutes. Using distilled water as a reference, measure the absorbance at 700 nm. Calculate the total phosphorus concentration based on the phosphorus standard curve. Calculation formula: Total phosphorus removal rate = (Initial total phosphorus - Residual total phosphorus) / Initial total phosphorus × 100% 4. Pb 2+ Aluminum residue (based on GB / T7475-1987 Atomic absorption spectrophotometry) Take 50.0 mL of supernatant, add 1 mL of concentrated nitric acid to acidify to pH < 2, prepare a series of Pb²⁺ / aluminum standard solutions, plot a standard curve, turn on the atomic absorption spectrophotometer, set the Pb²⁺ wavelength to 283.3 nm and the aluminum wavelength to 309.3 nm respectively, adjust the instrument to the optimal working state, and measure the absorbance of the blank solution, standard solution and sample solution in sequence. Calculate the Pb concentration in the sample based on the standard curve. 2+ / Concentration of aluminum; Calculation formula: Pb 2+ Removal rate = (initial Pb) 2+ Concentration - Residual Pb 2+ Concentration) / Initial Pb 2+ Concentration × 100% 5. Settlement velocity After slow stirring, immediately transfer the mixture to a 1000mL graduated cylinder and record the time t (h) required for the floc interface to settle from the 1000mL mark to the 100mL mark. Perform three parallel measurements and take the average value. Calculation formula: Settlement velocity (m / h) = 0.9 m / t The results are shown in the table below: Example 1 97.8 89.7 95.6 98.5 3 <0.01 Example 2 96.1 85.2 92.1 97.3 2.5 <0.01 Example 3 97.2 88.5 94.3 98.1 2.8 <0.01 Comparative Example 1 82.3 71.4 76.5 83.2 1.1 <0.01 Comparative Example 2 85.6 74.8 78.9 85.7 1.3 <0.01 Comparative Example 3 88.7 78.2 81.3 87.5 1.1 <0.01 Comparative Example 4 92.5 76.3 85.7 62.4 2.1 0.32 Comparative Example 5 78.5 72.1 65.2 76.3 1 <0.01 Comparative Example 6 72.3 68.5 58.7 71.2 1.2 <0.01 The data in the table show that the composite bioflocculants prepared in Examples 1-3 of this invention have effects on turbidity, COD, total phosphorus, and Pb. 2+ All exhibit excellent removal effects, rapid settling speed, and no aluminum residue. Compared with Comparative Examples 1-3, this invention significantly improves the performance of the flocculant by adding cationic nanocellulose, using quaternized modified moringa seed protein, and employing dual-strain synergistic fermentation. Compared with Comparative Examples 5 and 6, this invention simultaneously uses GTA-modified moringa seed protein and nanocellulose, producing a significant synergistic effect, with all indicators far exceeding those of single modified components. Compared with the commercially available chemical flocculant PAC in Comparative Example 4, the composite bio-flocculator of this invention has significant advantages in the removal of COD, total phosphorus, and heavy metals, and produces no secondary pollution, demonstrating the superiority of the technical solution of this invention.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A composite bioflocculator, characterized in that, It includes the following raw materials in parts by weight: 20-40 parts of flocculating bacteria fermentation extract, 5-10 parts of modified plant-based flocculant, 8-18 parts of cationic nanocellulose, 5-15 parts of porous mineral powder, and 2-6 parts of biosurfactant.

2. The composite bioflocculator as described in claim 1, characterized in that, The fermentation extract of the flocculent bacteria is prepared according to the following steps: seed cultures of Bacillus licheniformis and Candida utilis are inoculated into the fermentation medium at a volume ratio of 1:0.5-2, with an inoculation amount of 3-7% (v / v). Fermentation is carried out at a temperature of 28-32℃ and a stirring speed of 150-200 rpm for 48-72 hours. After fermentation, the supernatant is collected by centrifugation. 2-3 times the volume of ethanol is added to the supernatant for precipitation. The precipitate is collected by centrifugation and then freeze-dried to obtain the extract.

3. The composite bioflocculator as described in claim 2, characterized in that, The seed culture of Bacillus licheniformis was prepared as follows: Bacillus licheniformis was inoculated into LB liquid medium at an inoculation rate of 1%~5% (v / v), and cultured with shaking at 35~38℃ and 160~220 rpm for 14~20 hours until the viable count reached 1×10⁻⁶. 8 ~1×10 9 CFU / mL; The seed culture of *Candida utilis* was prepared as follows: *Candida utilis* was inoculated into malt extract medium at an inoculation rate of 1%–5% (v / v), and cultured with shaking at 28–32°C and 150–220 rpm for 20–40 hours until the cell count reached 1 × 10⁻⁶ cells / mL. 8 cells / mL or higher.

4. The composite bioflocculator as described in claim 1, characterized in that, The modified plant-based flocculant is a complex of quaternized moringa seed protein and tea saponin in a mass ratio of (3-5):

1.

5. The composite bioflocculator as described in claim 4, characterized in that, The preparation method of the quaternized modified moringa seed protein is as follows: Moringa seed protein is dissolved in phosphate buffer solution with pH 8.0-9.0 to prepare a solution with a mass concentration of 3-6%, and 2,3-epoxypropyltrimethylammonium chloride is added. The mass ratio of moringa seed protein to 2,3-epoxypropyltrimethylammonium chloride is 1:1.0-1.

2. The reaction is carried out at 40-50℃ for 3-5 hours, and the reaction product is obtained by dialyzing and freeze drying.

6. The composite bioflocculator as described in claim 1, characterized in that, The cationic cellulose nanoparticles are quaternary ammonium salt-treated cellulose nanoparticles prepared by reacting cellulose nanoparticles with a cationic etherifying agent, wherein the cationic etherifying agent is 2,3-epoxypropyltrimethylammonium chloride, and the preparation steps are as follows: Nanocellulose is dispersed in water to form a suspension with a mass concentration of 1% to 5%. The pH is adjusted to 10 to 12, and then 2,3-epoxypropyltrimethylammonium chloride is added. The mixture is stirred and reacted at a temperature of 60 to 80°C for 4 to 6 hours. The reaction product is dried to obtain the quaternized ammonium nanocellulose.

7. The composite bioflocculator as described in claim 1, characterized in that, The porous mineral powder is selected from at least one of attapulgite, diatomite, and zeolite powder, and all of them have undergone pretreatment. The specific method is as follows: the mineral powder is passed through a 150-250 mesh sieve, 1-2 mol / L hydrochloric acid solution is added, the solid-liquid ratio is 1g:10-15mL, and the powder is stirred and activated at 70-90℃ for 1-3 hours. The powder is then washed with deionized water until neutral, dried at 100-110℃, and ground for later use.

8. The composite bioflocculator as described in claim 1, characterized in that, The biosurfactant is rhamnolipin or sophorolipid.

9. A method for preparing a composite bioflocculator according to any one of claims 1-8, characterized in that, Includes the following steps: S1. Preparation of inorganic carrier dispersion: Cationic nanocellulose is added to deionized water and ultrasonically dispersed for 10-20 minutes at a power of 200-400W and a temperature of 25-35℃ to form a uniform nanocellulose suspension with a mass concentration of 1%-3%; then porous mineral powder is added and ultrasonically dispersed for another 15-30 minutes to obtain an inorganic carrier dispersion with a solid content of 5%-10%. S2. Preparation of bioflocculation mother liquor: Under normal temperature and pressure, the fermentation extract of flocculent bacteria and the modified plant-based flocculant are added to 100-200 parts of deionized water in a certain proportion. The mixture is stirred at 150-250 rpm for 20-30 minutes until completely dissolved to obtain a biological component solution. Then, a biosurfactant is added, and the mixture is stirred at the same speed for another 10-15 minutes. After mixing evenly, the bioflocculation mother liquor is obtained. S3. Gradient Composite and Post-treatment: The bioflocculation mother liquor obtained in step S2 is slowly added dropwise to the inorganic carrier dispersion obtained in step S1 at a flow rate of 10-20 mL / min. During the dropwise addition, the stirring speed is kept constant at 200-300 rpm throughout the process. After the dropwise addition is completed, stirring is continued for 30-60 minutes. Finally, the mixture is subjected to vacuum freeze-drying or spray drying. The dried product is pulverized and passed through an 80-120 mesh standard sieve to obtain a powdered composite bioflocculator.

10. The application of the composite bioflocculant according to any one of claims 1 to 8 or the composite bioflocculant prepared by the method of claim 9 in the treatment of water pollution.