A microbial composite hydrogel, its preparation method and application

Abstract

This invention belongs to the field of complex pollutant removal technology in water bodies, and specifically relates to a microbial composite hydrogel, its preparation method, and its application. The microbial composite hydrogel, by weight, comprises: 1-3 parts polyvinyl alcohol, 3-8 parts polyethylene glycol, 2-6 parts sodium alginate, 0.05-0.08 parts nano-titanium dioxide, 10-25 parts CaCl2, 10-25 parts boric acid, 5-15 parts potassium persulfate, and *Bacillus velezensis*, wherein the concentration of *Bacillus velezensis* is 0.35 × 10⁻⁶. 8 -0.6*10 8 Cells / mL. The microbial composite hydrogel of this invention can effectively treat multiple pollutants in wastewater simultaneously, has a more efficient pollutant degradation capacity and a wider range of applications, and realizes comprehensive integrated treatment of food wastewater.

Description

Technical Field

[0001] This invention belongs to the field of water pollution removal technology, and specifically relates to a microbial composite hydrogel, its preparation method and application. Background Technology

[0002] Food wastewater mainly includes high concentrations of organic pollutants, suspended solids and grease, chemicals such as phosphorus and chlorine used for cleaning and disinfection, antibiotics or food additives lost into the water, and nutrient residues such as carbohydrates, proteins and lipids. Direct discharge will seriously pollute the surrounding land and surface water.

[0003] Bacillus velezensis can effectively degrade pollutants in wastewater through microbial decomposition. Immobilized microbial technology can effectively protect microbial cells, increase the contact opportunities between microorganisms and pollutants, and improve the biodegradation capacity of microorganisms for organic pollutants. Immobilized carriers can provide a microenvironment for microorganisms, enabling high-density fermentation, promoting microbial metabolism, and thus improving wastewater treatment efficiency.

[0004] For example, existing technologies for COD degradation involve immobilizing Bacillus belye on the surface of sodium alginate (SA) / polyvinyl alcohol (PVA) / nano-zinc oxide (Nano-ZnO) microspheres to prepare SA / PVA / ZnO microspheres. However, this existing technology has a limited effect on the degradation of organic matter in wastewater; it can only degrade COD and cannot degrade recalcitrant substances such as tetracycline in wastewater.

[0005] Another existing technology prepares a magnetically immobilized microbial composite material using Bacillus belye, sodium alginate, nano-Fe3S4, and CaCl2. This magnetically immobilized microbial composite material has the ability to degrade tetracycline; however, the magnetic material may aggregate and be oxidized in an aqueous environment, reducing its dispersibility and adsorption capacity. Furthermore, the magnetic material may result in high costs for large-scale applications. Summary of the Invention

[0006] The purpose of this invention is to provide a microbial composite hydrogel that can effectively treat multiple pollutants in food wastewater without using magnetic materials, as well as its preparation method and its application in treating food wastewater.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A microbial composite hydrogel, by weight, comprises: 1-3 parts polyvinyl alcohol, 3-8 parts polyethylene glycol, 2-6 parts sodium alginate, 0.05-0.08 parts nano-titanium dioxide, 10-25 parts CaCl2, 10-25 parts boric acid, 5-15 parts potassium persulfate, and Bacillus velezensis, wherein the concentration of Bacillus velezensis is 0.35*10. 8 -0.6*10 8 Cells / mL.

[0009] In one preferred embodiment, the polyethylene glycol has a molecular weight of 2000-6000.

[0010] In one preferred embodiment, the polyvinyl alcohol has a molecular weight of 20,000-40,000.

[0011] In one preferred embodiment, the nano-titanium dioxide is 0.07-0.08 parts.

[0012] In one preferred embodiment, the nano-titanium dioxide has a particle size of 60-100 nm and is anatase type.

[0013] In one preferred embodiment, the nano-titanium dioxide has a particle size of 70-80 nm.

[0014] Bacillus velezensis is the Bacillus velezensis disclosed in the prior art (AJD, AMJ, AYQZ, et al. Enhanced treatment of organic matter in slaughter wastewater through live Bacillus velezensis strain using nano zinc oxide microsphere[J]. 2021).

[0015] Based on the same inventive concept, this invention also claims protection for a method for preparing the microbial composite hydrogel, comprising the following steps:

[0016] S1. Mix polyvinyl alcohol, polyethylene glycol, sodium alginate, nano titanium dioxide, and deionized water, stir evenly at 900-100℃, and cool to obtain mixed solution 1.

[0017] S2. Mix CaCl2, boric acid and potassium persulfate evenly to obtain mixed solution 2;

[0018] S3. Chemical cross-linking is carried out by adding mixed solution 1 dropwise into mixed solution 2. After addition, the mixture is allowed to stand for 6-18 hours, then washed with water and freeze-dried to prepare hydrogel microspheres.

[0019] S4. Mix the hydrogel microspheres and Bacillus belye bacterial suspension, and incubate at 35-38℃ and 150-220rpm for 3-6h to obtain the microbial composite hydrogel.

[0020] In one preferred embodiment, the concentration of the bacterial suspension is 0.8*10⁻⁶. 8 -1.2*10 8 Cells / mL.

[0021] In one preferred embodiment, the hydrogel microspheres have a particle size of 2-5 mm.

[0022] Large-sized hydrogel microspheres have a relatively small specific surface area, reducing the contact area with pollutants and lowering adsorption efficiency. Conversely, small-sized hydrogel microspheres may cause blockages in wastewater treatment systems, affecting water flow and increasing the difficulty of recycling and reuse.

[0023] In one preferred embodiment, the microbial composite hydrogel contains hydrogel microspheres loaded with 35-60% of Bacillus belye suspension.

[0024] Based on the same inventive concept, the present invention also claims protection for the use of the microbial composite hydrogel in the removal of COD, TP and / or tetracycline from wastewater.

[0025] In one preferred embodiment, the wastewater is food wastewater.

[0026] Based on the same inventive concept, the present invention also claims protection for the application of the microbial composite hydrogel in inhibiting microorganisms in wastewater.

[0027] In one preferred embodiment, the microorganism is Escherichia coli and / or Staphylococcus aureus.

[0028] The following attempts to further explain the present invention:

[0029] This invention constructs a novel PEG-PVA / SA / nano-TiO2 hydrogel and immobilizes it with Bacillus velezensis isolated from wastewater, forming a hydrogel microsphere-organic degrading bacteria system. Hydrogels are three-dimensional flexible polymer network structures and polymeric materials with excellent hydrophilicity. Hydrogel adsorbents possess superior adsorption capacity, broad pH independence, low cost, and sustainability. Pollutants can be adsorbed on the outer surface of the hydrogel or within the expanded three-dimensional hydrogel network, and then separated from the water along with the hydrogel. The composition of the hydrogel is crucial to its function. Sodium alginate (SA) is one of the most commonly used matrices, but it is generally fragile and not conducive to adsorption treatment. Mixing SA with polyvinyl alcohol (PVA) can effectively improve the mechanical strength, durability, and chemical stability of the hydrogel. Polyethylene glycol (PEG) is a pore-forming agent that can form a porous structure in the hydrogel. The high specific surface area and porous structure of the hydrogel are beneficial for absorbing large amounts of pollutants. Nano-titanium dioxide (nano-TiO2) is a low-toxicity, inert chemical material containing many hydroxyl groups. Nano-TiO2, due to its high surface area, porous structure, excellent stability under acidic conditions, environmental friendliness, and the presence of numerous surface absorption sites, can be used as an effective absorbent. Adding nanoparticles to the hydrogel framework can improve the mechanical strength, expansion behavior, thermal properties, and chemical properties of hydrogel composites, thereby increasing the practicality and lifespan of the absorbent material and increasing the available absorption sites in the hydrogel system. Simultaneously, nano-TiO2 can generate electron / hole pairs under natural light conditions, producing reactive oxygen species (ROS) through light absorption. ROS can disrupt bacterial cell walls and viral membranes, thus producing a certain antibacterial effect. However, *Bacillus belyceae* also faces the risk of being killed. Therefore, this invention, by adjusting the particle size and dosage of nano-TiO2, effectively inhibits *Staphylococcus aureus* and *Escherichia coli* without killing *Bacillus belyceae*. The nano-TiO2 powder separated as a hydrogel material after water treatment can be more easily recycled, avoiding secondary pollution. 2+ It cross-reacts with the wall material PEG-PVA / SA, reducing the solubility of the wall material and promoting the enhancement of the mechanical properties of the hydrogel microspheres. It also accelerates the formation of bacterial extracellular polymers and enhances the aggregation of microbial surfaces through c-di-GMP in the signaling molecule.

[0030] Due to the synergistic effect of the adsorption of the hydrogel carrier, the biodegradation of microorganisms, and the adsorption and antibacterial activity of titanium nanoparticles, the microbial composite hydrogel of the present invention can treat a variety of pollutants in food wastewater and maintain a long-term mechanism.

[0031] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0032] 1. The microbial composite hydrogel of the present invention can comprehensively treat multiple pollutants in wastewater, has a more efficient pollutant degradation capacity and a wider range of applications, realizes the comprehensive integrated treatment of food wastewater, and can be flexibly adjusted according to changes in pollutant type and concentration to achieve the best treatment effect.

[0033] 2. The microbial composite hydrogel of the present invention has more stable performance, higher integration and longer life cycle, and can more fully leverage the advantages of immobilization technology.

[0034] 3. The nano-TiO2 powder separated by the microbial composite hydrogel of the present invention can be more easily recovered, avoiding secondary pollution. Attached Figure Description

[0035] Figure 1 Morphological structure diagram of PEG-PVA / SA / nano-TiO2 microspheres;

[0036] Figure 2 The images show SEM and FT-IR spectra of PEG-PVA / SA / nano-TiO2 microspheres. In the figures, (a) is the SEM of nano-TiO2; (b) is the SEM of PEG-PVA / SA microspheres; (c) is the SEM of PEG-PVA / SA / nano-TiO2 microspheres; and (d) is the FT-IR spectrum.

[0037] Figure 3 A bar chart showing the ability of microspheres with different nano-TiO2 contents to immobilize Bacillus velezensis;

[0038] Figure 4 A bar chart showing the physical stability of hydrogel microspheres treated at different temperature and pH levels;

[0039] Figure 5 The graph shows the degradation efficiency of microbial composite hydrogel microspheres on organic compounds, phosphorus-containing components, and antibiotics in simulated food wastewater. In the graph, a represents the biodegradation efficiency of COD, b represents the fitting equation of COD biodegradation efficiency over time, c represents the biodegradation efficiency of TP, d represents the fitting equation of TP biodegradation efficiency over time, e represents the biodegradation efficiency of tetracycline, and f represents the fitting equation of tetracycline biodegradation efficiency over time. Detailed Implementation

[0040] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other. The polyethylene glycol used in the embodiments of this invention has a molecular weight of 2000. The polyvinyl alcohol has a molecular weight of 20,000.

[0041] Example 1

[0042] Preparation of microspheres

[0043] Solution 1 was prepared by dissolving 1.0 wt% PVA (polyvinyl alcohol), 3.0 wt% PEG polyethylene glycol 2000, and 2.0 wt% SA sodium alginate in deionized water and stirring continuously at 95°C for 15 minutes. Then, 0-2.0 g / L of nano-TiO2 (80 nm particle size, anatase type) was added and the mixture was thoroughly dispersed until a milky white, gel-like homogeneous system of 10 mL was formed. Solution 2 was prepared by dissolving 2.0 wt% CaCl2, 2.0 wt% H3BO3 boric acid, and 1.0 wt% K2S2O8 potassium persulfate in deionized water to obtain 50 mL of solution 2. After solution 1 was cooled to room temperature, it was added dropwise to solution 2 using a syringe. After immobilization for 12 hours, hydrogel microspheres containing different concentrations of nano-TiO2 were formed. The microspheres were rinsed three times with sterile water and stored at 4°C.

[0044] The results of visual observation of PEG-PVA / SA / nano-TiO2 microspheres are as follows: Figure 1 As shown in the figure. The results indicate that the hydrogel microspheres containing 0.5 g / L nano-TiO2 are elastic, with uniform nano-TiO2 distribution, minimal tailing, better morphology, and easier handling. With increasing nano-TiO2 concentration, the viscosity of solution 1 increases, leading to more pronounced tailing during preparation. The prepared material exhibits poor molding, a hard texture, and uneven nano-TiO2 distribution. Poor microsphere morphology affects bacterial immobilization and the carrier's absorption capacity, further impacting pollutant degradation. Simultaneously, the Ca in solution 2... 2+ Cross-reaction with the wall material PEG-PVA / SA (solution 1) reduced the solubility of the wall material and promoted the enhancement of the mechanical properties of the hydrogel microspheres.

[0045] PEG-PVA / SA / nano-TiO2 microspheres were freeze-dried, fixed, and sputter-coated with gold. The microstructure of the microspheres was observed using a scanning electron microscope (SEM, Zeiss Zeiss Sigma 300, Germany) at an accelerating voltage of 3 kV and a magnification of 2000x.

[0046] Lyophilized PEG-PVA / SA microspheres (with zero nano-TiO2 added), PEG-PVA / SA / nano-TiO2 microspheres, and nano-TiO2 powder were ground in an agate mortar and then passed through a 200-mesh sieve. 40 mg of the powder was taken, fixed with KBr as a background, and measured using Fourier transform infrared spectroscopy (FT-IR, Thermo Scientific, Germany). The spectral recording range was 4000 to 500 cm⁻¹. -1 The resolution is 4cm. -1 .

[0047] The results obtained are as follows Figure 2 As shown in the figure, (a) is the SEM image of nano-TiO2; (b) is the SEM image of PEG-PVA / SA microspheres; (c) is the SEM image of PEG-PVA / SA / nano-TiO2 microspheres; and (d) is the FT-IR spectrum. The figure shows that a large number of uniformly enlarged pores are formed on the surface of the PEG-PVA / SA microspheres, which is beneficial for bacterial strain loading, substance exchange, and pollutant absorption. The particle size of the PEG-PVA / SA microspheres is 5 mm (e.g., ...). Figure 2 (as shown in b). Figure 2 a and 2c indicate that nano-TiO2 in PEG-PVA / SA / nano-TiO2 microspheres is effectively aggregated in the voids on the surface of the microspheres, occupying fewer sites and not affecting the immobilization effect and absorption efficiency.

[0048] FT-IR was performed on nano-TiO2 and hydrogel microspheres to investigate the functional groups and interactions present in the microspheres. The results are as follows: Figure 2 As shown in d. PEG-PVA / SA microspheres at 3473 cm⁻¹ -1 The broad peak at 2926 cm⁻¹ is attributed to the stretching vibration of -OH. -1 1627cm -1 1402cm -1 and 1096cm -1 The absorption peaks generated at 647 cm⁻¹ correspond to the stretching vibrations of CH, -COO, -CH₂-, and CO, respectively. PEG-PVA / SA / nano-TiO₂ microspheres at 647 cm⁻¹... -1 The observed broadband is a result of adding nano-TiO2. The increase in peak width and the lower wavenumber from 3300 cm⁻¹... -1 Slightly moved to 3400cm -1 This indicates that hydrogen bonds have formed between nano-TiO2 and the support. The nano-TiO2 is bound at 400-700 cm⁻¹. -1The characteristic peaks and stretching vibrations of -OH can indicate that nano-TiO2 has been successfully integrated into the hydrogel.

[0049] Example 2

[0050] Preparation of microbial composite hydrogels

[0051] The *Bacillus velezensis* strain used in the experiment is a COD-degrading strain isolated from slaughterhouse wastewater. 100 μL of *Bacillus velezensis* was inoculated into 50 mL of LB broth and cultured at 37°C and 180 rpm for 24 hours. Subsequently, the culture was transferred to sterile centrifuge tubes and centrifuged at 6000 rpm for 5 minutes. The supernatant was discarded, and the centrifuge tubes were rinsed with sterile water; this process was repeated three times. Sterile water was added and vortexed to completely suspend the bacteria in the sterile water. The concentration of the bacterial suspension was adjusted to achieve the desired COD. 600 =1.0 (The concentration of the bacterial suspension at this absorbance is approximately 1.0 * 10⁻⁶) 8 (cells / mL). 6 g of each microsphere containing different concentrations of nano-TiO2 was transferred to an Erlenmeyer flask containing 50 mL of the above bacterial suspension. The flasks were incubated at 37°C and 180 rpm for 4 hours to immobilize the hydrogel microspheres and allow Bacillus velezensis to adsorb. This constructed a microbial composite hydrogel containing different concentrations of nano-TiO2, namely a hydrogel microsphere-organic degrading bacteria system.

[0052] Four hours after microsphere immobilization, the absorbance of the bacterial suspension was measured at a 600 nm ultraviolet wavelength. A bacterial suspension without microspheres served as a control. The absorbance was calculated using OD... 600 The degree of reduction in [something] determines the ability of microspheres to immobilize bacteria. The bacterial immobilization efficiency of microspheres is calculated using the following formula:

[0053]

[0054] In the formula, OD0 is the OD of the initial bacterial suspension. 600 OD1 is the OD of the immobilized bacterial suspension. 600 .

[0055] The bacterial carrying capacity of the microspheres for Bacillus velezensis was determined by measuring the change in absorbance at 600 nm after co-culturing the microspheres with bacteria, thus reflecting the immobilization efficiency. Effective immobilization of Bacillus velezensis by the microspheres led to a decrease in the OD value, as shown in the results. Figure 3As shown in the figure, the hydrogel microspheres exhibited a significant carrying capacity for this organically degradable bacterium. The ability of the microspheres to immobilize *Bacillus velezensis* gradually increased as the nano-TiO2 concentration increased from 0 g / L to 0.5 g / L, with the highest bacterial loading rate reaching 57.5% for the 0.5 g / L nano-TiO2 microspheres. With further increases in nano-TiO2 concentration, the bacterial carrying capacity of the microspheres decreased or showed no significant difference.

[0056] Different concentrations of microbial composite hydrogels were placed on culture media coated with Escherichia coli, Staphylococcus aureus, and Bacillus bellsii, and incubated at 37°C for 24 hours. The antibacterial properties of the microbial composite hydrogel microspheres with different concentrations were determined by measuring the diameter of the inhibition zone. The results are shown in Table 1.

[0057] Table 1. Diameters of the inhibition zones of different microspheres

[0058]

[0059]

[0060] Note: N / D indicates that no inhibition zone was detected.

[0061] The results are shown in Table 1. No inhibition zone was detected when the concentration of nano-TiO2 in the microspheres was 0-0.2 g / L. When the concentration was 0.5-0.8 g / L, the microspheres showed inhibition zones against *Escherichia coli* and *Staphylococcus aureus*, but no inhibition zone was detected against *Bacillus velezensis*. When the concentration increased to 1 g / L and above, the microspheres inhibited all three bacteria, and the diameter of the inhibition zone gradually increased.

[0062] Nano-TiO2 microspheres containing 0.5-0.8 g / L showed significant inhibitory effects on Escherichia coli and moderate inhibitory effects on Staphylococcus aureus. At this concentration, the inhibitory effect on Escherichia coli was significantly stronger than that on Staphylococcus aureus, while having little effect on the growth and reproduction of degradable bacteria.

[0063] The results show that at a nano-TiO2 concentration of 0.5-0.8 g / L, the microspheres exhibit better morphology, more uniform nano-TiO2 distribution, and easier operation. The microspheres demonstrate the strongest ability to immobilize Bacillus velezensis, and this concentration inhibits the growth of Escherichia coli and Staphylococcus aureus without affecting the growth and reproduction of biodegradable bacteria, *Veilles velezensis*. Furthermore, a nano-TiO2 concentration of 0.7-0.8 g / L shows optimal activity against Escherichia coli and Staphylococcus aureus while not inhibiting Bacillus velezensis.

[0064] Furthermore, based on Example 1, this invention adjusted the particle size of nano-TiO2 to 40, 60, 100, and 120 nm, with an addition amount of 0.5 g / L, to prepare a series of microbial composite hydrogels. The results showed that the microbial composite hydrogels prepared with nano-TiO2 particle sizes of 60-100 nm exhibited better inhibitory effects against *Escherichia coli* and *Staphylococcus aureus*. Simultaneously, the microbial composite hydrogels prepared within this range showed relatively good immobilization ability against *Bacillus velezensis*. Excessively large nano-TiO2 particle sizes reduced the specific surface area, decreasing the contact area with bacteria and thus reducing the ability to penetrate bacterial cell walls. Furthermore, excessively large nano-TiO2 particle sizes were difficult to distribute uniformly in the hydrogel, leading to gel structure instability, affecting the gel's mechanical properties, and consequently impacting overall performance and application effectiveness. Conversely, excessively small nano-TiO2 particle sizes had a large specific surface area, increasing van der Waals forces and surface energy between particles, resulting in aggregation. Agglomeration reduces the effective specific surface area of ​​the material, leading to decreased system stability and adversely affecting photocatalytic and antibacterial properties.

[0065] The physical stability of the prepared microbial composite hydrogel (nano-titanium dioxide particles with a diameter of 80 nm, added at a concentration of 0.5 g / L) was tested: the physical stability of the microspheres at different temperatures and pH values ​​was studied by detecting the weight loss rate. A certain mass (m0) of microspheres was weighed and placed in solutions with different pH values ​​(4, 5, 6, 7, 8, 9, 10), and stored at different temperatures (25℃ and 4℃). Every 5 days, the microspheres were taken out, and their mass (m0) was accurately measured after absorbing surface moisture. i The weightlessness rate is calculated using the following formula:

[0066]

[0067] In the formula, m0 is the mass (g) of the microspheres on day 0; mi is the mass (g) of the microspheres stored on day i.

[0068] The results are as follows Figure 4 As shown. Figure 4 The changes in weight loss rate of hydrogel microspheres at 4℃ and 25℃ with pH and time were illustrated. The weight loss rate of hydrogel microspheres in each treatment showed an increasing trend with increasing storage time. The weight loss rate of hydrogel microspheres at 4℃ was generally lower than that at 25℃ (p<0.01), indicating that PEG-VA / SA / nano-TiO2 microspheres are more stable at 4℃. After 20 days of storage at 4℃, significantly lower weight loss was observed under neutral conditions.

[0069] Under the same conditions, the particle size and amount of nano-TiO2 also affect the stability. The microbial composite hydrogel prepared with a nano-TiO2 particle size of 60-100 nm and an addition amount of 0.5-0.8 g / L exhibits the best stability.

[0070] Example 3

[0071] Comprehensive degradation of food wastewater

[0072] The biodegradability of the hydrogel microsphere-organic degrading bacteria system was investigated using simulated wastewater. Ammonium sulfate (300 mg / L) was added to LB medium to examine the degradation effect of the microbial composite hydrogel (80 nm nano-titanium dioxide particles, 0.5 g / L) on COD. Potassium dihydrogen phosphate (100 mg / L) was used to simulate phosphorus pollutants to examine the degradation effect of the microbial composite hydrogel (80 nm nano-titanium dioxide particles, 0.5 g / L) on TP. Tetracycline was used as an alternative to antibiotic pollutants to determine the degradation effect of the microbial composite hydrogel (80 nm nano-titanium dioxide particles, 0.5 g / L) on antibiotics. The hydrogel microsphere-organic degrading bacteria system was added to simulated wastewater (6 g / 50 mL) and cultured at 37℃, 180 rpm, and under white light. Samples were collected every 24 hours. COD and TP in simulated wastewater were measured using a water quality analyzer (GL-800, Shandong Greluy) and matching reagents, and the degradation rate was calculated. The degradation rate of COD and TP by the system was calculated using the following formula:

[0073]

[0074]

[0075] Where COD0 and TP0 are the initial COD or TP values ​​of the simulated wastewater, and COD1 and TP1 are the COD or TP values ​​after a period of degradation.

[0076] The hydrogel microsphere-organic degrading bacteria system demonstrates high degradation efficiency for organic compounds, phosphorus-containing components, and antibiotics in simulated food wastewater, such as... Figure 5 As shown in the figure, figures 5a-5d demonstrate that the system has a significant degradation effect on COD and TP, with the degradation rate reaching its peak on day 7, at 71.85% and 36.84%, respectively. In contrast, the hydrogel without nano-TiO2 showed degradation rates of only 35.04% and 19.87% for COD and TP, respectively.

[0077] Introducing tetracycline into bacterial cells inhibits bacterial peptide chain elongation. Therefore, the critical inhibitory concentration of tetracycline was first investigated. Simulated wastewater containing 0, 1.0, 5.0, and 10.0 mg / L tetracycline was prepared. 50 mL of bacterial suspension was added to the simulated wastewater, and the initial OD was measured. 600 Absorbance. After 24 hours of incubation, observe the turbidity of the culture medium and measure the OD. 600 The inhibitory effects of different concentrations of tetracycline on the growth of Bacillus velezensis were compared. A 50 mL simulated wastewater was prepared using tetracycline, and the initial amount of the mycotoxin was determined based on the UV absorbance at 357 nm. The system (6 g / 50 mL simulated wastewater) was added to the culture medium and incubated. The UV absorbance at 357 nm was measured every 24 hours, and the tetracycline degradation rate was calculated.

[0078]

[0079] Where OD0 is the initial OD of the simulated wastewater. 357 Value, OD1 is the OD value after a period of degradation. 357 value.

[0080] The results are shown in Table 2.

[0081] Table 2. Inhibitory effects of different concentrations of tetracycline on the growth of Bacillus velezensis.

[0082]

[0083] A solution containing 5 g / L tetracycline was selected as simulated antibiotic wastewater to investigate the degradation effect of the system. The results are as follows: Figure 5 As shown in e and 5f, the system exhibited similar degradation effects and trends for tetracycline, with a degradation rate of 40.52% on day 5. In contrast, the hydrogel without nano-TiO2 showed a degradation rate of only 17.33% for tetracycline on day 5. Furthermore, the sodium alginate (SA) / polyvinyl alcohol (PVA) / nano-zinc oxide (Nano-ZnO) microspheres prepared under optimal conditions (SA 6%, PVA 9%, ZnO 0.015%) according to existing technology (AJD, AMJ, AYQZ, et al. Enhanced treatment of organic matter in slaughter wastewater through live Bacillus velezensis strain using nano zinc oxide microsphere[J]. 2021) showed a degradation rate of only 20.50% for tetracycline on day 5.

[0084] Through the Figure 5 Matching the results shown in a, 5b, and 5e yields the degradation rates y of COD, TP, and TC as a function of time x. The COD degradation rate equation is y = -1.763x. 2 +20.915x+6.274. The equation for TP is y=0.432x 2 +6.858x + 8.565. The equation for TC is y = -0.772x. 2 +9.612x+8.875. The coefficients of determination (R²) of the three equations. 2 The values ​​were 0.9590, 0.9960, and 0.9898, respectively, indicating that the model explained 95.90%, 99.60%, and 98.98% of the degradation rate index changes, showing good agreement and helping to predict the subsequent degradation rate changes over time.

[0085] Due to the combined effects of adsorption by the hydrogel carrier, biodegradation by microorganisms, and adsorption and antibacterial activity of nano-TiO2, the microbial composite hydrogel of this invention can treat multiple pollutants in food wastewater and maintain a long-lasting mechanism. *Bacillus velezensis* exhibits strong resistance, can grow and reproduce in wastewater environments, and is highly safe. In particular, *Bacillus velezensis* demonstrates highly efficient biodegradation of wastewater. The Ca in the microspheres... 2+ This may accelerate the formation of bacterial extracellular polymers and enhance microbial surface aggregation through c-di-GMP in signaling molecules. Furthermore, Ca... 2+ Hydrogels can improve sedimentation efficiency in wastewater, regulate biofilm formation, and promote attachment and growth processes. Immobilization of hydrogels promotes microbial growth and enhances the secretion of a range of bioactive substances. *Bacillus velezensis* can biologically remove phosphorus, especially in eutrophic water environments, effectively reducing phosphorus content and improving water quality. PEG-PVA / SA microspheres exhibit good pore size variation and biofriendliness, effectively absorbing pollutants from the aquatic environment. *Bacillus velezensis* cells can grow and biodegrade more continuously on the microspheres. PVA improves the mechanical strength, durability, and chemical stability of SA hydrogel microspheres. PEG, as a porogen, forms the porous structure of the hydrogel and can be used for immobilization to obtain excellent mechanical and mass transfer properties. The addition of H3BO3 and K2S2O8 helps extend the durability and stability of polyvinyl alcohol hydrogel beads in the aquatic environment. The high specific surface area and porous structure of the hydrogel are beneficial for absorbing large amounts of pollutants. Adding nanomaterials to hydrogels can improve the mechanical strength, swelling behavior, thermal properties, and pollutant removal capacity of synthesized hydrogel composites.

[0086] Nano-TiO2 is a low-toxicity, inert chemical material containing numerous hydroxyl groups. Its high specific surface area and surface activity make it an effective adsorbent, increasing the available absorption sites in hydrogel systems and showing great application potential in wastewater treatment. Under ultraviolet irradiation, the cavities generated on the surface of nano-TiO2 can oxidize water molecules or hydroxide ions, producing highly oxidizing hydroxyl radicals (-OH) and reactive oxygen species. These radicals can oxidize and degrade organic pollutants in water into harmless carbon dioxide and water, while reactive oxygen species can disrupt bacterial cell structure, leading to cell death and thus producing an antibacterial effect. Furthermore, recovering nano-TiO2 in powder form after water treatment is challenging. Encapsulating nano-TiO2 in a biopolymer matrix facilitates easier recovery.

[0087] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A microbial composite hydrogel, characterized in that, By weight, it includes: 1-3 parts polyvinyl alcohol, 3-8 parts polyethylene glycol, 2-6 parts sodium alginate, 0.05-0.08 parts nano titanium dioxide, 10-25 parts CaCl2, 10-25 parts boric acid, 5-15 parts potassium persulfate and Bacillus belysin. Bacillus velezensis Bacillus belesi Bacillus velezensis The concentration is 0.35*10 8 -0.6*10 8 cells / mL; The nano-titanium dioxide has a particle size of 60-100 nm and is anatase type. The preparation method of the microbial composite hydrogel includes the following steps: S1. Mix polyvinyl alcohol, polyethylene glycol, sodium alginate, nano titanium dioxide, and deionized water, stir evenly at 900-100℃, and cool to obtain mixed solution 1. S2. Mix CaCl2, boric acid and potassium persulfate evenly to obtain mixed solution 2; S3. Chemical cross-linking is carried out by adding mixed solution 1 dropwise into mixed solution 2. After addition, the mixture is allowed to stand for 6-18 hours, then washed with water and freeze-dried to prepare hydrogel microspheres. S4. Mix the hydrogel microspheres and Bacillus belye bacterial suspension, and incubate at 35-38℃ and 150-220 rpm for 3-6 hours to obtain the microbial composite hydrogel.

2. The microbial composite hydrogel according to claim 1, characterized in that, The polyethylene glycol has a molecular weight of 2,000-6,000; the polyvinyl alcohol has a molecular weight of 20,000-40,000.

3. The microbial composite hydrogel according to claim 1, characterized in that, The nano-titanium dioxide content is 0.07-0.08 parts.

4. The microbial composite hydrogel according to claim 1, characterized in that, The nano-titanium dioxide has a particle size of 70-80 nm.

5. A method for preparing the microbial composite hydrogel according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Mix polyvinyl alcohol, polyethylene glycol, sodium alginate, nano titanium dioxide, and deionized water, stir evenly at 900-100℃, and cool to obtain mixed solution 1. S2. Mix CaCl2, boric acid and potassium persulfate evenly to obtain mixed solution 2; S3. Chemical cross-linking is carried out by adding mixed solution 1 dropwise into mixed solution 2. After addition, the mixture is allowed to stand for 6-18 hours, then washed with water and freeze-dried to prepare hydrogel microspheres. S4. Mix the hydrogel microspheres and Bacillus belye bacterial suspension, and incubate at 35-38℃ and 150-220 rpm for 3-6 hours to obtain the microbial composite hydrogel.

6. The method according to claim 5, characterized in that, The particle size of the hydrogel microspheres is 2-5 mm.

7. The method according to claim 5, characterized in that, In the microbial composite hydrogel, the hydrogel microspheres are loaded with 35-60% of Bacillus belye bacterial suspension.

8. The application of the microbial composite hydrogel according to any one of claims 1-4 in the removal of COD, TP and / or tetracycline from wastewater.

9. The application according to claim 8, characterized in that, The wastewater is food wastewater.

10. The application of the microbial composite hydrogel according to any one of claims 1-4 in inhibiting microorganisms in wastewater.

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