Fermentation inoculant for sewage treatment and application of fermentation inoculant

Abstract

The invention relates to the technical field of environmental microorganisms and wastewater treatment, and particularly discloses a fermentation inoculant for sewage treatment and application. The fermentation inoculant is prepared by immobilizing a multifunctional composite inoculant on chitosan magnetic microspheres, the multifunctional complex microbial inoculant is prepared by compounding fermentation broth of pseudomonas koreensis, lactobacillus acidophilus, bacillus brevis, massinia pemophila and bacillus licheniformis according to a specific viable count ratio, wherein the fermentation broth of the pseudomonas koreensis, the lactobacillus acidophilus, the bacillus brevis, the massinia pemophila and the bacillus licheniformis are used as raw materials. Fe3O4 is used as a magnetic core of the chitosan magnetic microsphere, and a cross-linked chitosan layer is coated outside the magnetic core. Through combination of specific flora compounding and a magnetic immobilization technology, a microbial remediation system which has complementary functions and is easy to recover is successfully constructed. The microbial agent can efficiently and synchronously remove nitrate, tetrabromobisphenol A and heavy metal multi-component composite pollutants in underground water or polluted water, can be quickly recycled and reused through an external magnetic field, and has the advantages of high treatment efficiency, environment friendliness and low cost.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology and wastewater treatment technology, and in particular to a fermentation agent for wastewater treatment and its application. Background Technology

[0002] Physical, chemical, and biological methods are commonly used wastewater treatment technologies. Compared with physical and chemical methods, biological methods, which rely on the metabolic processes of microorganisms to decompose and transform pollutants, offer advantages such as stable treatment results, low cost, and simple and easy-to-implement technology, making them one of the most widely adopted wastewater treatment methods.

[0003] Tetrabromobisphenol A (TBBPA), a typical brominated flame retardant, exhibits environmental persistence, bioaccumulation, and endocrine-disrupting toxicity; excessive nitrates can lead to methemoglobinemia and increase the risk of cancer; heavy metals such as copper and lead have clear toxic effects on the human body. The distinct properties of these three pollutants make their simultaneous treatment a significant challenge in the field of water treatment.

[0004] Currently, biological treatment technologies for this type of combined pollution have attracted widespread attention due to their environmental friendliness and low cost. Existing technology CN120485067A provides a composite bacterial agent for treating groundwater, its preparation method, and its application. Its purpose is to address the difficulty of existing biological treatment methods in simultaneously removing TBBPA and nitrates from groundwater. This technology uses a composite bacterial agent comprising *Pseudomonas kwangsiensis* Y5-11, *Acinetobacter pietabs* LYS-5, *Pseudomonas extremaustralis* Y39-6, and psychrophilic bacteria F5-6. The main technical approach involves separately fermenting and culturing the four strains, then mixing the four fermentation broths to prepare the composite bacterial agent. This composite bacterial agent is used to simultaneously remove TBBPA and nitrates from groundwater. The composite bacterial agent exhibits biodiversity, and the treatment results are consistent with the characteristics of groundwater quality. However, this technology still has significant shortcomings: First, its microbial agent mainly targets organic pollutants and nitrates, lacking the ability to remove heavy metals and thus unable to cope with more common ternary complex pollution scenarios; second, the activity and adaptability of the microbial agent used may be insufficient in the low-temperature, oligotrophic environment of groundwater, leading to unstable remediation effects; third, this technology does not solve the problem of microbial agent loss in open environments, making it impossible to recycle and reuse, resulting in cost waste and potential ecological risks.

[0005] Furthermore, other existing microbial remediation technologies generally suffer from problems such as limited microbial agent function, poor environmental targeting, and incomplete denitrification due to insufficient carbon sources. Therefore, there is an urgent need to develop a novel composite microbial remediation technology that can efficiently, stably, and economically remove nitrates, TBBPA, and heavy metals from groundwater simultaneously, and to solve the challenges of microbial agent colonization, efficacy, and recycling in complex environments. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide a fermentation agent for wastewater treatment and its application. This agent can efficiently and stably remove the combined pollution of nitrates, tetrabromobisphenol A, and heavy metals from groundwater simultaneously, and solves the problems of traditional biological agents being easily lost and difficult to recycle in the environment.

[0007] To achieve the above-mentioned technical effects, the present invention adopts the following technical solution: In a first aspect, the present invention provides a fermentation agent for wastewater treatment, which is made by immobilizing a multifunctional composite agent on chitosan magnetic microspheres.

[0008] Preferably, the multifunctional compound microbial agent is prepared by combining fermentation broths of *Pseudomonas koreensis*, *Lactobacillus acidophilus*, *Brevibacillus brevis*, *Massilia timonae*, and *Bacillus licheniformis* in a viable cell ratio of (2-3):(1-2):(1-2):(0.5-1.5):(1-2); this specific ratio combination can produce a significant synergistic degradation effect. Furthermore, all the strains are from publicly known cultural relics. Among them, *Pseudomonas koreanum* has the accession number CCTCC S2016806; *Lactobacillus acidophilus* has the accession number CCTCC CB 20081799; *Microsporum brevis* has the accession number CCTCC AB 94218; *Masseudomonas timonene* has the accession number CCTCC AB 205314; and *Bacillus licheniformis* has the accession number CCTCC CB 20082367. These strains together constitute a highly efficient microbial system capable of simultaneously treating multiple contaminants.

[0009] Secondly, the present invention provides a core carrier for the fermentation agent used in wastewater treatment—chitosan magnetic microspheres. The chitosan magnetic microspheres consist of a magnetic Fe3O4 core and a chitosan cross-linked layer covering it.

[0010] Preferably, the preparation method of the chitosan magnetic microspheres includes the following six steps: S1. Preparation of magnetic Fe3O4 nanoparticles; S2. The magnetic Fe3O4 nanoparticles obtained in S1 are dispersed in a dilute acid solution to obtain a magnetic suspension; S3. Dissolve chitosan in a dilute acid solution to obtain a chitosan solution with a concentration of 1.5% to 2.0% (w / v); S4. Under stirring conditions, the chitosan solution and the magnetic suspension are mixed to carry out a coating reaction, thereby obtaining a mixed reaction system; S5. Add a crosslinking agent to the mixed reaction system to perform crosslinking and curing, to obtain the final system; S6. The final system is subjected to magnetic separation, washing, and drying to obtain the chitosan magnetic microspheres.

[0011] In the foregoing description, "% (w / v)" refers to a mass-volume percentage concentration, indicating the mass (in grams) of solute contained in every 100 mL of solution. For example, preparing a "5% (w / v) chitosan solution" means dissolving 5 grams of chitosan in water and bringing the volume to 100 mL.

[0012] As a further preferred technical solution, the preparation method of the chitosan magnetic microspheres includes the following six steps: S1. Magnetic Fe3O4 nanoparticles were prepared using the classic co-precipitation method as magnetic response cores; S2. The magnetic Fe3O4 nanoparticles obtained in S1 are dispersed in a dilute acid solution and a uniform magnetic suspension is obtained by ultrasonic treatment. S3. Dissolve chitosan powder in a dilute acid solution to prepare a clear and transparent chitosan solution, wherein the concentration of the chitosan solution is 1.5% to 2.0% (w / v); S4. Under mechanical stirring conditions, the chitosan solution is slowly added dropwise to the magnetic suspension to carry out the coating reaction and obtain a mixed reaction system; S5. Add an appropriate amount of crosslinking agent to the mixed reaction system for crosslinking and curing to enhance the mechanical strength and stability of the microspheres. The final system is obtained after the reaction is completed. S6. The product is collected from the final system using magnetic separation technology, and after washing and freeze-drying, the final chitosan magnetic microspheres that can be stored for a long time are obtained.

[0013] More specifically, in step S1, the preparation of the Fe3O4 nanoparticles preferably adopts the following process parameters: FeCl2·4H2O and FeCl3·6H2O are mixed according to Fe... 2+ with Fe 3+ Salt was dissolved in deionized water at a molar ratio of 1:1.5–2.2. Under nitrogen protection, the temperature was raised to 70–80°C, and 25%–28% ammonia was slowly added dropwise to adjust the pH of the reaction system to 9–10. After stirring continuously for 0.5–2 hours, hydrophilic Fe3O4 nanoparticles were obtained by magnetic separation and washing with water and ethanol alternately until neutral.

[0014] More specifically, in steps S2 and S3, the dilute acid solution is preferably an acetic acid solution with a concentration of 0.5% to 2.0% (v / v). This concentration range ensures complete dissolution of chitosan without causing excessive corrosion to the magnetic particles. In the foregoing description, "% (v / v)" is a volume percentage concentration, indicating the volume (milliliters) of solute contained in 100 mL of solution. For example, "1% (v / v) acetic acid solution" means that 100 mL of solution contains 1 mL of glacial acetic acid.

[0015] More specifically, in step S4, the mass ratio of Fe3O4 nanoparticles to chitosan is controlled within the range of 1:(0.5-3); the coating reaction is carried out under mild conditions, with the temperature maintained at 20-40°C and the reaction time controlled within 2-6 hours, to ensure the formation of a complete and uniform coating layer.

[0016] More specifically, in step S5, the crosslinking agent is preferably glutaraldehyde, and its addition amount is controlled between 0.5% and 5% of the chitosan mass; the crosslinking reaction time is controlled between 1 and 3 hours. This crosslinking process can significantly improve the stability of the carrier, making it less prone to disintegration during wastewater treatment.

[0017] Thirdly, this invention provides a complete preparation method for the aforementioned fermentation agent for wastewater treatment. The method includes the following key steps: First, chitosan magnetic microspheres are prepared as described above; then, the chitosan magnetic microspheres are mixed with the multifunctional composite microbial agent at a volume-to-mass ratio of (5-15):1 (mL:g), and cultured for 24-48 hours under shaking conditions at 28-32℃ and 120-180 r / min, allowing the microbial agent to fully colonize the carrier surface and form a biofilm, ultimately completing the immobilization of the microbial agent and obtaining a directly usable fermentation agent product. In the foregoing description, "mL" refers to the volume of the multifunctional composite microbial agent fermentation broth, and "g" refers to the mass of the chitosan magnetic microsphere carrier.

[0018] Fourthly, the present invention provides the application of the aforementioned fermentation agent for wastewater treatment. This agent is particularly suitable for the simultaneous removal of nitrates, tetrabromobisphenol A (TBBPA), and heavy metals (such as Cu) from groundwater or polluted water bodies. 2+ Pb 2+ Complex pollution (etc.).

[0019] Fifthly, the present invention provides a specific method for remediating water bodies using the aforementioned wastewater treatment fermentation agent. The method includes: adding the fermentation agent to the polluted water body at a dosage of 0.5–3 g / L, reacting for 12–72 hours, and then efficiently recovering it using an external magnetic field.

[0020] Preferably, the recovered fermentation agent can be reused after a simple activation and regeneration treatment, which greatly reduces operating costs. The activation and regeneration conditions are as follows: in a regeneration solution containing carbon and nitrogen sources, shake culture at 25-30°C for 12-24 hours to restore its biological activity.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: First, the wastewater treatment fermentation agent provided by this invention, by combining five specific strains with complementary functions in an optimized ratio and immobilizing them on a chitosan magnetic microsphere carrier, produces a significant synergistic effect. Experimental results show that this agent achieves a simultaneous removal rate of over 90% for nitrates, TBBPA, and heavy metal copper ions, demonstrating excellent comprehensive purification capabilities and effectively solving the challenge of simultaneous treatment of multiple complex pollutants.

[0022] Secondly, the chitosan magnetic microsphere carrier provided by this invention plays multiple key roles. Its chitosan shell not only provides excellent biocompatible fixation sites for the bacterial agent, promoting biofilm formation, but its functional groups can also efficiently adsorb heavy metal ions. The internal magnetic Fe3O4 core endows the bacterial agent with strong magnetic responsiveness, enabling it to be rapidly separated and recovered by an external magnetic field after water remediation, with a recovery rate exceeding 98%. This achieves the recycling of the bacterial agent, significantly reduces operating costs, and avoids secondary pollution.

[0023] Furthermore, carrier immobilization technology provides crucial microenvironment protection for the microbial agent. Comparative experiments confirmed that immobilized microbial agents exhibit significantly higher tolerance and removal efficiency for heavy metal ions than free microbial agents, indicating that the carrier effectively mitigates the direct impact of toxic substances on microorganisms. The coupling of carrier adsorption and microbial biodegradation forms a dynamic synergistic purification mechanism, whose comprehensive treatment effect far exceeds the simple superposition of single processes, ensuring the treatment efficiency and stability of the microbial agent in complex environments. Detailed Implementation

[0024] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. Those skilled in the art should understand that the present invention can be implemented even without certain specific details. In some other embodiments, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the spirit of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and the numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.

[0025] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, or instruments used, unless otherwise specified by the manufacturer, are all commercially available; and the conditions not specified in the examples are all performed under conventional conditions or conditions recommended by the manufacturer. Furthermore, this invention does not limit the source of the raw materials used; unless otherwise specified, the raw materials used in this invention are all commercially available products commonly found in this technical field. Unless otherwise specified, the "ratio" referred to in the following examples refers to a ratio of parts by mass.

[0026] It should be noted that all strains used in this invention were legally obtained from public depository institutions. Specifically, the original strains of *Pseudomonas koreensis* (CCTCC S2016806), *Lactobacillus acidophilus* (CCTCC CB 20081799), *Brevibacillus brevis* (CCTCC AB 94218), *Massilia timonae* (CCTCC AB 205314), and *Bacillus licheniformis* (CCTCC CB 20082367) used in the specific embodiments were all purchased from the China Center for Type Culture Collection (CCTCC).

[0027] Example 1 The purpose of this embodiment is to provide a method for preparing a fermentation agent for wastewater treatment, the specific steps of which are as follows: (1) Preparation of chitosan magnetic microspheres, including the following steps: S1. Preparation of Magnetic Fe3O4 Nanoparticles: 1.99 g of ferrous chloride hexahydrate (FeCl2·4H2O) and 5.41 g of ferric chloride hexahydrate (FeCl3·6H2O) were accurately weighed and dissolved together in 100 mL of nitrogen-deoxygenated deionized water. The solution was placed in a three-necked flask and heated to 75 °C in a water bath under continuous nitrogen purging. Subsequently, a 28% (w / w) ammonia solution was slowly added dropwise using a constant-pressure dropping funnel to precisely adjust the pH of the reaction system to 9.5. At this temperature, the reaction was mechanically stirred continuously at 500 rpm for 1 hour. After the reaction, solid-liquid separation was performed using a strong neodymium magnet. The resulting black magnetic precipitate was washed three times each with deionized water and anhydrous ethanol, respectively, until the washing solution was neutral, finally yielding hydrophilic Fe3O4 nanoparticles.

[0028] S2. Preparation of magnetic suspension: All the Fe3O4 nanoparticles prepared above were transferred to a 250 mL beaker, and 100 mL of 1% (volume ratio) acetic acid solution was added. Then the beaker was placed in an ultrasonic cell disruptor and ultrasonically dispersed for 30 minutes at a power of 300 W to obtain a uniform and stable black magnetic suspension.

[0029] S3. Preparation of chitosan solution: In another beaker, slowly add 2.0 g of chitosan powder (degree of deacetylation of 95%, molecular weight of 20 kDa) to 100 mL of 1% acetic acid solution. Stir continuously on a magnetic stirrer at room temperature for about 2 hours until the chitosan is completely dissolved, resulting in a clear and transparent viscous chitosan solution.

[0030] S4. Coating Reaction: The magnetic suspension obtained in S2 was placed on a mechanical stirrer and stirred at a rate of 300 rpm. Subsequently, the chitosan solution from S3 was slowly and evenly added dropwise to the magnetic suspension using a dropper. After the addition was complete, stirring was maintained, and the reaction system was transferred to a 30°C constant temperature water bath and the reaction was continued for 4 hours to allow the chitosan to fully coat the surface of the Fe3O4 nanoparticles.

[0031] S5. Crosslinking and curing: Add 0.01 mL of glutaraldehyde solution (equivalent to 2% of the chitosan mass) to the above reaction solution, and continue to stir at the same speed at 30°C for 2 hours to complete the crosslinking and curing of the chitosan layer, so as to improve the mechanical strength and chemical stability of the microspheres.

[0032] S6. Post-processing: After the reaction is complete, the product is separated again with a neodymium magnet and washed repeatedly with deionized water until there is no aldehyde odor. The obtained chitosan magnetic microspheres are placed in a freeze dryer and freeze-dried at -50℃ and 10Pa for 24 hours to obtain dry, long-term stored brown powder chitosan magnetic microspheres.

[0033] (2) Compound preparation and immobilization of compound microbial agents: Purchase the corresponding bacterial strains, and carry out high-density fermentation of the strains with preservation numbers CCTCC S2016806, CCTCC CB 20081799, CCTCC AB94218, CCTCC AB 205314, and CCTCC CB 20082367 according to the standard fermentation process. Collect the fermentation broth and determine the viable cell count.

[0034] Fermentation broths of *Pseudomonas kwangsiensis*, *Lactobacillus acidophilus*, *Microsporum brevis*, *Masséella timonale*, and *Bacillus licheniformis* were mixed according to a live bacteria ratio of 2.5:1.5:1.5:1:1.5, and the final concentration of the compound bacterial agent was adjusted to 5 × 10⁻⁶ with sterile physiological saline. 8 CFU / mL.

[0035] Measure 100 ml of the above-mentioned compound bacterial agent and place it together with 10 g of the chitosan magnetic microspheres prepared in step (1) into a 500 ml Erlenmeyer flask, and add 100 ml of LB liquid culture medium. Place the Erlenmeyer flask in a constant temperature shaker and culture it at 30°C and 150 rpm for 36 hours to allow the bacterial agent to fully colonize on the carrier surface and form a biofilm, thus obtaining the fermentation bacterial agent for wastewater treatment described in this invention.

[0036] Example 2 The purpose of this embodiment is to provide a preparation method for a fermentation agent for wastewater treatment. The specific steps are as follows: (1) Preparation of chitosan magnetic microspheres: S1: Weigh 2.30 g of FeCl2·4H2O and 5.41 g of FeCl3·6H2O (molar ratio 1:2) and dissolve them in 100 mL of deionized water. Under nitrogen protection, heat to 80 °C, add ammonia dropwise to adjust the pH to 10, and react for 1.5 hours. Subsequent magnetic separation and washing procedures are the same as in Example 1.

[0037] S2 and S3: Same as in Example 1.

[0038] S4: The Fe3O4 nanoparticles obtained in S1 were fed with chitosan at a mass ratio of 1:2, and the coating reaction was carried out at 25°C for 6 hours.

[0039] S5: The amount of glutaraldehyde added is 1% of the mass of chitosan, and the cross-linking time is 1.5 hours.

[0040] S6: Same as Example 1.

[0041] (2) Compound preparation and immobilization of compound microbial agents: The compound ratio of the compound bacterial agent was adjusted to 2:1:2:0.5:2; the volume-to-mass ratio of bacterial solution to carrier was 8:1 (mL:g); the immobilization culture time was 48 hours; and the remaining steps were the same as in Example 1.

[0042] Example 3 The preparation steps of a fermentation agent for wastewater treatment are as follows: (1) Preparation of chitosan magnetic microspheres: S1: Weigh 1.99 g of FeCl2·4H2O and 4.87 g of FeCl3·6H2O (molar ratio 1:1.8) and dissolve them in 100 mL of deionized water. Under nitrogen protection, heat to 70 °C, add ammonia dropwise to adjust the pH to 9, and react for 2 hours. Subsequent magnetic separation and washing procedures are the same as in Example 1.

[0043] S2 and S3: Same as in Example 1.

[0044] S4: The Fe3O4 nanoparticles obtained in S1 were fed with chitosan at a mass ratio of 1:1, and the coating reaction was carried out at 35°C for 2.5 hours.

[0045] S5: The amount of glutaraldehyde added is 4% of the mass of chitosan, and the cross-linking time is 3 hours.

[0046] S6: Same as Example 1.

[0047] (2) Compound preparation and immobilization of compound microbial agents: The compound ratio of the compound bacterial agent was adjusted to 3:2:1:1.5:1, the volume-to-mass ratio of bacterial solution to carrier was 12:1 (mL:g), the immobilization culture time was 24 hours, and the remaining steps were the same as in Example 1.

[0048] Experimental Example 1 This experiment aims to systematically verify the synergistic degradation mechanism of the five-strain compound system and to explore the role of chitosan magnetic carriers in the directional adsorption of pollutants, protection of bacterial agents, and recovery processes, as detailed below: 1.1 Group Settings To accurately evaluate the function of each component, this experiment included multiple control groups. In all groups involving microbial agents, the total viable bacterial count was controlled at 5 × 10⁻⁶. 8 CFU were used to ensure the comparability of biomass among groups. Equal amounts of chitosan magnetic microspheres were used as carriers in all immobilized groups. The specific design is shown in Table 1 below:

[0049] Table 1. Experimental Example 1: Group Design and Inoculum Composition for Each Group

[0050] 1.2 Experimental Methods Preparation of nitrate (NO3) - -N 50 mg / L), TBBPA (2 mg / L) and Cu 2+ Simulated groundwater at a concentration of 10 mg / L was used. Eight experimental groups were set up: Group E1 (complete five bacteria + carrier) served as the baseline; Groups 1-A (lactobacterium acidophilus missing), 1-B (lactobacter brevis missing), and 1-C (lactobacter licheniformis missing) were used to analyze the function of single bacteria; Group 1-D (free five bacteria) was used to investigate the necessity of immobilization; Group 1-E (blank carrier) was used to evaluate the adsorption performance of the carrier itself; Group 1-F (immobilized single bacteria) was used to verify the advantages of the combination; and Group CK served as a blank control. Each group was reacted at 25℃ and 120 rpm for 24 hours, and the changes in pollutant concentration and carrier recovery rate were detected.

[0051] 1.3 Experimental Results and Discussion The experimental results are shown in Table 2:

[0052] Table 2. Pollutant removal rate and carrier recovery rate for each group (n=3)

[0053] The above experimental results show that the complete system (Group E1) consisting of five specific bacterial strains immobilized on chitosan magnetic microspheres effectively inhibits NO3-. - -N, TBBPA and Cu 2+ The simultaneous removal rates all exceeded 90%, and the carrier recovery rate reached 98.2%. This result confirms that the technical approach of combining functionally complementary microbial communities with recyclable carriers is effective and can address the challenge of simultaneous removal of diverse and complex pollutants.

[0054] Meanwhile, the experiments above, by setting up control groups lacking specific strains, revealed the main functions of each strain in the composite system. Data showed that *Lactobacillus acidophilus* and *Bacillus brevis* are key functional units for efficient TBBPA degradation (removal rate significantly decreased after deletion); *Bacillus licheniformis* plays a central role in heavy metal removal (Cu levels decreased significantly after deletion). 2+ The removal rate decreased to 68.3%; while *Pseudomonas kwangsiensis* and *Masseudomonas timonene* made significant contributions to the overall stability of the system and nitrogen transformation. Single strains or combinations of strains (such as group 1-I) could not achieve the same level of comprehensive removal effect, demonstrating the necessity of setting up functional strains and combining them for different pollutants.

[0055] Furthermore, the carrier plays multiple roles in this system, and its function needs to be comprehensively analyzed in conjunction with the function of the supported microorganisms. Specifically, this experiment shows that only the carrier group (group 1-H) has an effect on Cu 2+ The removal rate of 88.9% confirmed the rapid adsorption capacity of chitosan materials for heavy metals. This was compared to free bacteria (groups 1-G, Cu...). 2+ The significant difference between the removal rate (18.9%) and the immobilized intact bacterial community (E1 group) indicates that the carrier effectively isolated some toxic substances, creating a more suitable microenvironment for the survival and function of microorganisms.

[0056] A carrier is a platform for functional expression, but not a sufficient condition. Data shows that groups with carriers but incomplete bacterial communities (such as groups 1-E and 1-F) still exhibit significant shortcomings in treatment efficiency. This indicates that the protective and enrichment effects of the carrier must be combined with the specific biodegradation and transformation capabilities of the loaded bacterial community to achieve efficient and simultaneous removal of various pollutants. Group E1 showed improvement in Cu... 2+The final removal rate (94.8%) surpassed that of the pure carrier (88.9%). This is likely due to the following reasons: the carrier first rapidly adsorbs pollutants and mitigates environmental toxicity; the protected, fully functional microbial community then deeply biodegrades or transforms the enriched pollutants; and the activity of the microorganisms may also maintain or regenerate the system's treatment capacity to some extent. This dynamic coupling mechanism of "adsorption-biological treatment" makes its overall performance superior to simple physical adsorption or unprotected biological treatment.

[0057] Experimental Example 2 This experimental example aims to explore the reusability of the immobilized fermentation agent (E1 system) described in this invention and its ability to continuously treat pollutants, and to compare it with a simple adsorption carrier to observe whether the experimental group has a sustainable treatment advantage.

[0058] 2.1 Material Preparation Experimental group: Take the complete immobilized fermentation agent prepared in Example 1 (E1 group sample).

[0059] Control group: Pure chitosan magnetic microspheres prepared in Experiment 1 without loading any bacterial agents (corresponding to the carriers of group 1-H).

[0060] Simulated contaminated water: Freshly prepared for each cycle, with the same pollutant composition and concentration as in Experiment 1 (NO3). - -N 50 mg / L, TBBPA 2 mg / L, Cu 2+ 10 mg / L).

[0061] 2.2 Experimental Methods Add 0.5 g (based on dry carrier mass) of the experimental or control group material to a conical flask containing 200 mL of fresh simulated contaminated water (i.e., the dosage is 2.5 g / L). Place the conical flask in a constant temperature shaker and react for 24 hours at 25℃ and 120 rpm, which is considered as one treatment cycle.

[0062] After each cycle, an external neodymium magnet is used to quickly separate the material from the reaction system, and the supernatant is collected to determine the residual concentration of the three pollutants and calculate the removal rate of the cycle.

[0063] Subsequently, the magnetically recovered material was transferred directly to a conical flask containing 200 mL of fresh simulated contaminated water without any washing or activation treatment, to begin the next 24-hour reaction cycle. This process was repeated for a total of four consecutive cycles.

[0064] 2.3 Test Results The results of repeated experiments are shown in Table 3.

[0065] Table 3 Comparison of reusability performance between immobilized bacterial agents and pure carriers (n=3)

[0066] The above experimental results show that the removal capacity of pure chitosan magnetic microspheres (control group) exhibits a precipitous drop after the first cycle. Its removal capacity for Cu... 2+ The removal rate plummeted from 87.6% in the first cycle to 22.3% in the second cycle, and remained low in subsequent cycles; the adsorption of TBBPA showed a similar trend. These experimental results further demonstrate the capacity limitation of simple physicochemical adsorption; once the adsorption sites are saturated in the first round, its treatment efficiency is essentially lost.

[0067] The immobilized fermentation agent of this invention (experimental group) showed a slight decrease in the removal rate of the three pollutants over four consecutive use cycles, but it remained at a high level (still above 80% in the fourth cycle). This indicates that the system has strong continuous treatment capabilities and good reusability.

[0068] Based on the above experimental results, the core advantage of the fermentation agent for wastewater treatment provided by this experimental group lies in the sustainable biological treatment capacity formed by carrier immobilization and the combination of functional microbial communities. This system fundamentally overcomes the shortcomings of simple adsorption materials, such as easy saturation and single-use, and achieves the goal of high efficiency, stability, and repeated use in the treatment of multiple complex pollutants, demonstrating significant practical value and economic benefits.

[0069] Experimental Example 3 The purpose of this experiment is to investigate the Fe3O4 preparation process. 2+ / Fe 3+ The effects of molar ratios (1:1.6–1:2.5) on the magnetic properties, microstructure, and adsorption function of the carrier are as follows: 3.1 Experimental Methods This experimental example is for Fe 2+ / Fe 3+ Seven molar ratio gradients were set, including: 2-A (1:1.6), 2-B (1:1.7), 2-C (1:1.8), 2-D (1:1.9), 2-E (1:2.0), 2-F (1:2.2), and 2-G (1:2.5). Chitosan magnetic microspheres were prepared according to the method in Example 1. The saturation magnetization was measured using a vibrating sample magnetometer, the specific surface area was analyzed by the BET method, and Cu content was determined. 2+ Adsorption experiment.

[0070] 3.2 Test Results The experimental results are shown in Table 4: Table 4 Different Fe 2+ / Fe3+ The effect of molar ratio on carrier performance (n=3)

[0071] As shown in Table 4, the experimental results indicate that the carrier performance does not reach its optimal value at the classical theoretical molar ratio (1:2). When Fe... 2+ / Fe 3+ The supports prepared in the molar ratio range of 1:1.6 to 1:1.9 exhibited the highest overall performance. Among them, the 2-C group (1:1.8) showed the highest saturation magnetization (68.5 emu / g), specific surface area (89.7 m² / g), and Cu... 2+ The adsorption capacity (95.3 mg / g) reached its peak value. Specifically: The classic reaction principle for preparing Fe3O4 by coprecipitation (Fe 2+ + 2Fe 3+ + 8OH - → Fe3O4 + 4H2O) clearly shows that a 1:2 molar ratio is the theoretically optimal stoichiometric ratio. However, the results of this experiment reveal that strictly adhering to the theoretical ratio of 1:2 (group 2-E) in the actual engineering preparation process does not yield the best overall performance. Its saturation magnetization (65.2 emu / g) and specific surface area (75.4 m²) are also shown. 2 / g) and Cu 2+ The adsorption capacity (82.1 mg / g) was significantly lower than that of the groups within the preferred range of this invention (such as group 2-C). It is speculated that this phenomenon in actual processes may be due to the fact that classical theory assumes an ideal closed reaction system, while in actual open reaction environments with an aqueous phase, heating, and a protective gas atmosphere, some Fe... 2+ It is highly susceptible to oxidation by dissolved oxygen, leading to a decrease in the actual Fe content in the system. 2+ / Fe 3+ The effective proportion is lower than the feed ratio; therefore, using a molar ratio of 1:1.6 to 1:1.9 is essentially a method for controlling the Fe content. 2+ The potential loss compensation strategy ensures that the actual ratio of ions participating in the reaction in the system is closer to the theoretically optimal 1:2 when the reaction occurs, thereby generating Fe3O4 nanocrystals with more complete crystal structure, stronger magnetism, and more uniform and fine particle size. When the molar ratio deviates further to 1:2.2 and 1:2.5 (2-F, 2-G groups), all key properties show a systematic decline.

[0072] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A fermentation agent for wastewater treatment, characterized in that, It is made by immobilizing a multifunctional composite microbial agent on chitosan magnetic microspheres; wherein the multifunctional composite microbial agent is composed of fermentation broth of Pseudomonas koreensis, Lactobacillus acidophilus, Brevibacillus brevis, Massilia timonae and Bacillus licheniformis in a viable cell ratio of (2-3):(1-2):(1-2):(0.5-1.5):(1-2).

2. The fermentation agent for wastewater treatment as described in claim 1, characterized in that, The preservation number of *Pseudomonas koreensis* is CCTCC S2016806; the preservation number of *Lactobacillus acidophilus* is CCTCC CB 20081799; the preservation number of *Brevibacillus brevis* is CCTCC AB 94218; the preservation number of *Massilia timonae* is CCTCC AB 205314; and the preservation number of *Bacillus licheniformis* is CCTCCCB 20082367.

3. The fermentation agent for wastewater treatment as described in claim 1, characterized in that, The chitosan magnetic microspheres consist of a magnetic Fe3O4 core and a chitosan cross-linked layer covering it.

4. The fermentation agent for wastewater treatment as described in claim 3, characterized in that, The preparation method of the chitosan magnetic microspheres includes the following steps: S1. Preparation of magnetic Fe3O4 nanoparticles; S2. The magnetic Fe3O4 nanoparticles obtained in S1 are dispersed in a dilute acid solution to obtain a magnetic suspension; S3. Dissolve chitosan in a dilute acid solution to obtain a chitosan solution; S4. Under stirring conditions, the chitosan solution and the magnetic suspension are mixed to carry out a coating reaction, thereby obtaining a mixed reaction system; S5. Add a crosslinking agent to the mixed reaction system to perform crosslinking and curing, to obtain the final system; S6. The final system is subjected to magnetic separation, washing, and drying to obtain the chitosan magnetic microspheres.

5. The fermentation agent for wastewater treatment as described in claim 4, characterized in that, In step S1, the Fe3O4 nanoparticles are prepared by a co-precipitation method, specifically: Fe... 2+ with Fe 3+ Dissolve the salt in deionized water at a molar ratio of 1:1.5 to 2.

2. Under nitrogen protection, heat to 70 to 80°C, then add ammonia dropwise to adjust the pH to 9 to 10. After reacting for 0.5 to 2 hours, perform magnetic separation and washing until neutral.

6. The fermentation agent for wastewater treatment as described in claim 4, characterized in that, In steps S2 and S3, the dilute acid solution is an acetic acid solution with a concentration of 0.5% to 2.0% by volume percentage.

7. The fermentation agent for wastewater treatment as described in claim 4, characterized in that, The mass ratio of Fe3O4 nanoparticles to chitosan is 1:(0.5-3); the coating reaction temperature is 20-40℃ and the time is 2-6 hours.

8. The fermentation agent for wastewater treatment as described in claim 4, characterized in that, In step S5, the crosslinking agent is glutaraldehyde, and its addition amount is 0.5% to 5% of the chitosan mass; the crosslinking reaction time is 1 to 3 hours.

9. The method for preparing the fermentation agent for wastewater treatment according to any one of claims 1-8, characterized in that, Includes the following steps: Chitosan magnetic microspheres were prepared; and the chitosan magnetic microspheres were mixed with a multifunctional composite bacterial agent at a volume-to-mass ratio of (5-15):1, and cultured under shaking conditions at 28-32℃ and 120-180 r / min for 24-48 hours to complete the immobilization of the bacterial agent.

10. The application of the fermentation agent for wastewater treatment as described in any one of claims 1-8 in the simultaneous removal of nitrates, TBBPA and heavy metals from groundwater.

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