Preparation method and application of microbial flocculant for efficiently removing antibiotics

Abstract

The invention provides a preparation method of a microbial flocculant for efficiently removing antibiotics, which comprises the following steps: inoculating a Vilnelande azotobacter AZ6 strain into fermentation liquor, carrying out shake culture, and mixing the obtained fermentation liquor with bacterial liquid with glycerol to obtain preserved bacterial liquid; inoculating the preserved bacteria solution into a new fermentation solution, carrying out shake culture, centrifuging, and collecting a supernatant; adding absolute ethyl alcohol into the supernate, uniformly mixing, standing, centrifuging and collecting precipitate; and freeze-drying the precipitate to obtain the microbial flocculant. The invention also provides application of the microbial flocculant for removing antibiotics in water. The microbial flocculant is used for removing antibiotics in water. The microbial flocculant disclosed by the invention is applied to a mechanism of removing antibiotics through a synergistic effect of surface adsorption, charge neutralization, adsorption bridging and sweeping and netting.

Description

Technical Field

[0001] This invention belongs to the field of water pollution control technology, specifically relating to a method for preparing and applying a highly efficient microbial flocculant for removing antibiotics. Background Technology

[0002] New pollutants such as antibiotics are biotoxic, environmentally persistent, and bioaccumulative. They are widely present in rivers, lakes, groundwater, and even drinking water. After accumulating along the food chain, they pose a serious threat to ecosystem stability and human health. They are included in the "List of Key Controlled New Pollutants (2023 Edition)" and the "Standards for Drinking Water Quality" (GB 5749-2022).

[0003] Conventional water treatment processes (such as coagulation, sedimentation, filtration, and disinfection) are mainly effective against conventional pollutants such as suspended particulate matter and organic matter. They have extremely low removal efficiency (usually less than 30%) for new pollutants like antibiotics, which are structurally stable and highly soluble. Furthermore, antibiotics are prone to migration and transformation during water treatment, further increasing the environmental risk of resistance gene transmission, and thus failing to meet the technical requirements for the treatment of new pollutants.

[0004] Traditional flocculants have drawbacks: inorganic flocculants (iron salts, aluminum salts, etc.) require large dosages, produce a lot of sludge, and residual metal ions can easily cause secondary pollution; organic flocculants (polyacrylamide, etc.) have degradation products that may be biotoxic, and long-term use poses environmental accumulation and ecological risks.

[0005] There is a shortage of existing microbial flocculants: Some microbial flocculants have problems such as limited flocculation activity, narrow applicability, unclear culture conditions, and insufficient verification of their removal efficiency for specific new pollutants such as antibiotics, making it difficult to meet the actual needs of new water pollution control.

[0006] Therefore, it is necessary to prepare a flocculant with high flocculant removal capacity to overcome the shortcomings of existing technologies. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a method for preparing and applying a highly efficient microbial flocculant for removing antibiotics, addressing the shortcomings of the prior art. The microbial flocculant produced by this invention using Azotobacter vinelandii strain AZ6 is mainly composed of biomacromolecules, which are biodegradable, non-toxic, and do not cause secondary pollution. It has good biocompatibility, overcoming the environmental risks of traditional inorganic / organic flocculants; and it has a good removal effect on antibiotics.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing a highly efficient microbial flocculant for removing antibiotics, the preparation method comprising the following steps:

[0009] S1. Inoculate the fermentation broth with strain AZ6 of *Venelandia diffusa* and culture it with shaking to obtain a fermentation broth containing bacteria.

[0010] S2. Mix the fermentation broth containing bacteria obtained in S1 with glycerol to obtain the preservation broth;

[0011] S3. Take the preserved bacterial culture obtained in S2 and inoculate it into the new fermentation broth. Shake and culture it, then centrifuge and collect the supernatant.

[0012] S4. Add anhydrous ethanol to the supernatant obtained in S3, mix well, let stand, and then centrifuge to collect the precipitate.

[0013] S5. The precipitate obtained in step 4 is freeze-dried to obtain a dry powder, which is the microbial flocculant.

[0014] Preferably, the fermentation broth in S1 has the following formula: 15g glucose, 10g sucrose, 0.7g yeast extract, 0.1g urea, 0.5g ammonium sulfate, 1g KH2PO4, 2.5g K2HPO4, 0.3g MgSO4, 0.2g NaCl, and 1L H2O; the shaking culture is carried out at a temperature of 36℃, a rotation speed of 198r / min, and a time of 39h; the amount of *Venerendaeca AZ6* strain added is 100μL / L.

[0015] Preferably, the volume ratio of the fermentation broth containing bacteria to glycerol in S2 is 8:2.

[0016] Preferably, the amount of the preservative bacterial solution used in S3 is 120 μl / L; the temperature of the shaking culture is 36℃, the rotation speed is 198 r / min, and the time is 39 h; the rotation speed of the centrifugation is 6000 rpm, and the time is 10 min.

[0017] Preferably, the volume ratio of the supernatant to anhydrous ethanol in step S4 is 1:2; the settling temperature is 4°C and the settling time is 18 hours; the centrifugation speed is 6000 rpm and the centrifugation time is 10 minutes.

[0018] Preferably, the freeze-drying temperature in S5 is -55°C and the time is 24 hours.

[0019] The present invention also provides an application of a highly efficient microbial flocculant for removing antibiotics, the microbial flocculant being used to remove antibiotics from water.

[0020] Preferably, when the antibiotic is oxytetracycline, the amount of microbial flocculant added is 300 mg / L.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] 1. The *Azotobacter vinelandii* strain AZ6 used in this invention is specifically designed to target novel pollutants such as oxytetracycline antibiotics in water bodies. It exhibits high flocculation activity and strong stability, unlike existing broad-spectrum strains. Using the microbial flocculant of this invention, the flocculation rate of kaolin suspension reached 44.01%, significantly higher than that of conventional microbial flocculants (the flocculation rates of Bacillus subtilis spore-type microbial flocculants, common strains of *Pseudomonas*, and *Saccharomyces cerevisiae* extracellular flocculants are typically 30-35%).

[0023] 2. This invention establishes and verifies a mathematical model by combining single-factor experiments with response surface methodology, thereby obtaining the optimal combination of culture parameters for two strains and achieving a synergistic improvement in flocculant yield and activity, overcoming the problems of ambiguous culture conditions and unstable efficacy in existing methods.

[0024] 3. This invention simplifies the flocculant extraction and purification process by using an ethanol precipitation-freeze-drying process, which is simple to operate and can retain the activity of biological macromolecules, resulting in high product purity.

[0025] 4. The mechanism of action of this invention is clear: the principle of synergistic action of multiple mechanisms is elucidated through multi-method characterization, providing theoretical support for the targeted optimization of antibiotic removal, which is different from the current situation where the mechanism research is not in-depth.

[0026] 5. The microbial flocculant prepared by this invention has strong application targeting, focusing on new pollutants such as antibiotics, optimizing specific adsorption conditions, and forming a complete technology chain of "screening-preparation-optimization-application", which is compatible with the control requirements of the "List of Key Controlled New Pollutants".

[0027] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0028] Figure 1 Standard deviation and R0 of the regression equation for Azotobacter vinelandii strain AZ6 2 ;

[0029] Figure 2 Fitting curves for Azotobacter vinelandii strain AZ6;

[0030] Figure 3 Contour lines (a) and response surface plot (b) showing the interaction between culture time and culture temperature of Azotobacter vinelandii strain AZ6.

[0031] Figure 4Contour lines (a) and response surface plot (b) illustrating the interaction between culture time and shaker speed of Azotobacter vinelandii strain AZ6.

[0032] Figure 5 Contour lines (a) and response surface plot (b) showing the interaction between culture temperature and shaking speed for Azotobacter vinelandii strain AZ6.

[0033] Figure 6 This is the Fourier transform infrared spectrum of the microbial flocculant of the present invention;

[0034] Figure 7 This is a SEM image of the microbial flocculant of the present invention;

[0035] Figure 8 The effect of adsorption time on the adsorption of oxytetracycline by microbial flocculants;

[0036] Figure 9 The effect of microbial flocculant concentration on the adsorption of oxytetracycline by microbial flocculant;

[0037] Figure 10 The effect of initial antibiotic concentration on the adsorption of oxytetracycline by microbial flocculants. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1

[0040] This embodiment provides a method for preparing a highly efficient microbial flocculant for removing antibiotics, the method comprising the following steps:

[0041] S1. Inoculate the fermentation broth with Azotobacter vinelandii strain AZ6 and culture it with shaking at 36℃ and 198 r / min for 39 h to obtain the fermentation broth with bacteria.

[0042] The fermentation broth formula is as follows: 15g glucose, 10g sucrose, 0.7g yeast extract, 0.1g urea, 0.5g ammonium sulfate, 1g KH2PO4, 2.5g K2HPO4, 0.3g MgSO4, 0.2g NaCl, and 1L H2O; the amount of Venetian violaceum strain AZ6 added is 100μL / L.

[0043] S2. Mix the fermentation broth containing bacteria obtained in S1 at a volume ratio of 8:2 with glycerol to obtain the preservation broth.

[0044] S3. Inoculate the preserved bacterial culture obtained in S2 into a new fermentation broth, and culture it with shaking at 36℃ and 198 r / min for 39 h. Then, centrifuge at 6000 rpm for 10 min and collect the supernatant.

[0045] The amount of preservative culture solution used was 120 μl / L; the new fermentation broth formula was the same as that in S1.

[0046] S4. Add anhydrous ethanol to the supernatant obtained in S3, mix well, let stand at 4℃ for 18h, then centrifuge at 6000rpm for 10min and collect the precipitate.

[0047] The volume ratio of the supernatant to anhydrous ethanol is 1:2;

[0048] S5. The precipitate obtained in S4 is freeze-dried at -55℃ for 24 hours to obtain a dry powder, which is the microbial flocculant.

[0049] The infrared spectral analysis (FTIR) of the microbial flocculant prepared in this embodiment is as follows: Figure 6 As shown in the figure, the microbial flocculant prepared by Azotobacter vinelandii strain AZ6 was analyzed based on the absorption peaks in the figure as follows: The absorption peak at 3406.57 cm⁻¹ belongs to the stretching vibration absorption peak of hydroxyl (OH), which is superimposed with the stretching vibration of amino (NH). This indicates that the microbial flocculant contains polysaccharides such as starch, cellulose or protein / peptide components. The hydroxyl group is a typical characteristic group of polysaccharides, while the amino group indicates the presence of amino acids or proteins.

[0050] The absorption peaks at 2929.12 cm⁻¹ and 2859.34 cm⁻¹ are stretching vibrations of saturated alkyl groups (CH2, CH3), which are characteristic peaks of hydrocarbon chains in organic matter. This indicates that the microbial flocculant contains aliphatic carbon chain structures, which are commonly found in the sugar chain backbone of polysaccharides, fatty acids, or hydrocarbon side chains of proteins.

[0051] The absorption peak at 1638.6 cm⁻¹ belongs to the stretching vibration of carbonyl (C=O), representing the amide I band, and is superimposed with the imine (C=N) vibration. Combined with the NH peak at 3406 cm⁻¹, it confirms the presence of protein / peptide (amide bond), or possibly carbonyl group of carboxylic acid (-COOH) or ester, suggesting that the microbial flocculant contains amino acid residues or organic acid components.

[0052] The absorption peaks at 1541.11 cm⁻¹ and 1455.17 cm⁻¹ are the bending vibrations of amino groups (NH) (representing the amide II band) and CH bending vibrations, which further confirm the presence of proteins or peptides. Amino groups are also important charged groups in microbial flocculants and participate in charge neutralization in the microbial flocculation process.

[0053] The absorption peak at 1123.34 cm⁻¹ belongs to the stretching vibration of the ether bond (COC), which represents the presence of glycosidic bonds. It is the core characteristic peak of polysaccharides, indicating that polysaccharides are one of the main components in microbial flocculants, and glycosidic bonds constitute the backbone structure of sugar chains.

[0054] The absorption peak at 619.63 cm⁻¹ is due to the bending vibration of aromatic / heterocyclic (CH) or the vibration of SO or PO. If phosphorus and sulfur elements are present, it may be a component of nucleic acid, phospholipid or sulfate, or it may be a characteristic peak of heterocyclic compound, indicating a small amount of polar groups containing phosphorus and sulfur.

[0055] Based on the above analysis, it can be concluded that the microbial flocculant is mainly composed of polysaccharide-protein complex, with hydroxyl, amino, and carbonyl groups as the main polar functional groups, glycosidic bonds and amide bonds forming the molecular skeleton, and also contains a small amount of aliphatic hydrocarbon chains and possible phosphorus / sulfur polar groups.

[0056] The morphological characteristics of the microbial flocculant prepared in this embodiment were analyzed, and the SEM images are shown below. Figure 7 As shown in the SEM images, this microbial flocculant is a typical micron-nano composite system, composed of irregular polyhedral crystalline particles of 1-5 μm and spherical nanoparticles of 20-50 nm. The micron-sized crystalline particles serve as the skeletal support structure, with clear edges and no obvious etching damage, indicating a stable crystal structure that provides rigid support during flocculation. The nanospherical particles, in clusters, are tightly attached to the surface and grain boundaries of the micron-sized crystals, forming a core-shell structure of "nano-coated micron." The absence of cracks or separation at the interface indicates a strong physical adsorption between the two types of particles.

[0057] From the distribution and aggregation characteristics, the sample exhibits regional aggregation under low magnification, with nanoparticle clusters forming aggregates of 5-10 μm in size. Micrometer-level gaps exist between these aggregates, and no large-area agglomeration occurs. This characteristic stems from the dispersion and self-assembly behavior of the particles during sample preparation. Under high magnification, the non-directional attachment of the nanoparticle clusters further confirms that their bonding with the micron-sized crystals is primarily physical adsorption through non-covalent interactions, without chemical bonding or the formation of new phases.

[0058] In terms of pore structure, the microbial flocculant forms a hierarchical pore system consisting of mesopores (2-50 nm), macropores (50-500 nm), and micron-sized fissures. This structure is determined by the packing pattern of the nanoparticle clusters and the spatial distribution of the aggregates. Mesopores and macropores are mainly distributed within the nanoclusters, while micron-sized fissures exist between the aggregates. The formation of this hierarchical pore system significantly increases the specific surface area of ​​the sample, providing channels for the adsorption and mass transfer of pollutants.

[0059] Correlation between morphological characteristics and mechanism of action

[0060] Based on the above microscopic morphological characteristics, it can be inferred that surface roughening and hierarchical pore structure are the core factors for improving the adsorption performance of microbial flocculants. The attachment of nanoparticles significantly increases the surface roughness of the sample, forming a large number of surface protrusions and adsorption sites, which can make multi-point contact with suspended particles in the water through van der Waals forces, electrostatic attraction, etc. The hierarchical pore structure further expands the specific surface area, enabling the adsorption capacity and adsorption rate of pollutants by microbial flocculants to be improved simultaneously. At the same time, the "bridging effect" of nanoparticle clusters and the "sweeping effect" of micron crystals form a synergistic effect: nanoparticle clusters connect fine suspended particles through intermolecular forces to form micro flocs; micron crystals, with their larger particle size and rigid structure, sweep away micro flocs and unadsorbed particles in the water, ultimately forming large-sized flocs, which greatly improves flocculation efficiency.

[0061] In addition, the composite structure of "nanoparticles coated with micron-sized particles" endows the flocs with excellent stability. The tight adhesion of nanoparticles to the surface of micron-sized crystals enhances the binding force between particles, resulting in a dense floc structure that is less prone to breakage in the water flow field. At the same time, the skeletal support of the micron-sized crystals reduces the porosity of the flocs, increases their density, and thus accelerates their settling velocity. Furthermore, the sample exhibits no large-area agglomeration, allowing it to disperse rapidly in water, shortening the contact time with pollutants, and further optimizing the kinetic characteristics of the flocculation process.

[0062] Based on the above analysis, it can be concluded that the microscopic morphology of this microbial flocculant exhibits the core characteristics of dual-size composite, multi-level pores, and surface roughening. This type of structure enhances adsorption, bridging, and sweeping effects, synergistically improving flocculation efficiency, floc stability, and dispersibility, which is the core reason for its excellent flocculation performance.

[0063] Screening and identification of Venetian AZ6 strain in Example S1

[0064] 1. Sample collection:

[0065] Samples were collected from environments rich in organic matter and with a high risk of antibiotic contamination, such as activated sludge from the aerobic tank of a sewage treatment plant in Zhengzhou, river sediment, and farmland soil. The samples were placed in sterile containers and transported at low temperatures.

[0066] 2. Strain strain isolation and purification:

[0067] The samples were serially diluted 10-fold (dilution range 10⁻). 4 After being coated with beef extract peptone medium, potato dextrose agar medium and Gao's No. 1 medium respectively, the mixture was incubated at 37℃ for 48 h.

[0068] The formulations for each culture medium are as follows:

[0069] Beef extract peptone medium: 5g beef extract, 10g peptone, 5g NaCl, 15-20g agar, 1L H2O, pH 7.2-8.0;

[0070] Potato glucose agar medium: 200g potato, 20g glucose, 15-20g agar, 1L H2O;

[0071] Gao's No. 1 culture medium: 20g soluble starch, 3g KNO3, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 0.01g FeSO4, 0.5g NaCl, 1L H2O.

[0072] After preparing the culture medium, adjust the pH to 7.0 and sterilize at 120℃ for 20-30 minutes.

[0073] Single colonies were picked and purified using the streak plating method. They were then inoculated into fermentation medium (15g glucose, 10g sucrose, 0.7g yeast extract, 0.1g urea, 0.5g ammonium sulfate, 1g KH2PO4, 2.5g K2HPO4, 0.3g MgSO4, 0.2g NaCl, with water added to 1L, pH 7.0) and cultured at 30-37℃ with shaking at 120r / min for 48h. After centrifugation, the fermentation supernatant was collected.

[0074] 3. Highly efficient strain screening:

[0075] Preliminary screening: Using 4 g / L kaolin suspension (pH 7.0) as a simulated system, 2 mL of fermentation supernatant was added, and the mixture was stirred slowly at 80 r / min for 3 min, then stirred rapidly at 120 r / min for 3 min, and then allowed to stand for 3 min. The absorbance was measured at 600 nm to screen strains with a flocculation rate ≥30%.

[0076] Flocculation rate / % = (AB) / A × 100%

[0077] In the formula: A—OD of the control group of kaolin suspension 600 ;

[0078] B—Kaolin suspension OD after adding flocculant 600 .

[0079] Secondary screening: Based on the actual wastewater simulation system, the Azotobacter vinelandii strain AZ6 with high and stable flocculation performance was screened out.

[0080] Table 1 Record of Flocculation Calculation Results

[0081]

[0082] 4. Strain identification:

[0083] By combining morphological observation (colony morphology, cell morphology and Gram staining), physiological and biochemical characteristic analysis, and 16S rRNA sequence sequencing and evolutionary analysis, the taxonomic position of the strain was clarified.

[0084] The sequencing results are as follows:

[0085]

[0086] Based on the above results, the strain was identified as *Venerendica AZ6*.

[0087] Culture medium and culture conditions optimization experiment

[0088] 1. Culture medium optimization:

[0089] Carbon source: Glucose was selected as the optimal carbon source (flocculation rate > 35%), balancing activity and cost;

[0090] Nitrogen source: Yeast extract was determined to be the optimal nitrogen source for cell growth and flocculant synthesis;

[0091] Inorganic salts: Mg²⁺ is the key promoting factor, and the optimal mass concentration of MgSO₄ was determined to be 0.3 g / L. Na⁺ maintains cell osmotic pressure, and Cu²⁺ is the inhibiting factor.

[0092] 2. Optimization of single-factor culture medium conditions and response surface methodology:

[0093] Temperature: The optimal temperature for the strain is 30℃ (flocculation rate 40.3%).

[0094] Air flow rate: The optimal air speed for strain 2 is 160 r / min (flocculation rate 41.6%).

[0095] Incubation time: The optimal incubation time is 36 hours (flocculation rate 42.3%).

[0096] With incubation time (A), temperature (B), and rotation speed (C) as independent variables and flocculation rate as the response value, CCD experiments were designed using Design-Expert software.

[0097] The optimal combination of Venetian azeotropic bacteria strain AZ6 was optimized by single-factor culture medium conditions: 36 h, 30 ℃, 160 r / min (predicted flocculation rate 43.57%, actual 44.01%). After optimization by Design-Expert response surface model, the final optimized conditions were determined to be: 39 h, 36 ℃, 198 r / min.

[0098] Table 2. CCD factor levels of *Venelandia diffusa* strain AZ6

[0099]

[0100] Table 3. Experimental design and response results of *Venelandia diffusa* strain AZ6.

[0101]

[0102] Seventeen calculation results were obtained using Design-Expert software. The flocculation rate of *Venerenella AZ6* strain under different experimental factors was used as the response value. Regression analysis was performed on these results using Design-Expert software, yielding the following quadratic regression equation:

[0103] Y=43.57+3.77A+1.37B+4.94C−5.77A 2 -7.54B 2 -4.13C 2 +1.47AB−0.73AC−0.5825BC

[0104] Standard deviation and R of the regression equation 2 like Figure 1 As shown, the fitted curve is as follows Figure 2 As shown, by Figure 2 The experimental data showed no outliers and were basically linearly distributed. This indicates that the actual experimental values ​​were similar to the model predictions, further demonstrating the model's good predictive ability for the flocculation rate of *Venerendaeonia AZ6* strain under different conditions.

[0105] Table 4. Analysis of variance of regression equations for Vinerland nitrogen-fixing bacterium strain AZ6

[0106]

[0107] Figure 3 Contour lines (a) and response surface plots (b) illustrating the interaction between culture time and culture temperature of Azotobacter vinelandii strain AZ6. Figure 3 It was found that the surface inclination was high in the interaction between time A and temperature B, indicating that the interaction between the two was significant. In the other two groups, the strain had poor flocculation efficiency when the rotation speed C was too low, and the strain's flocculation efficiency decreased slightly when the rotation speed C was too high, reaching a maximum value at 160 r / min.

[0108] Figure 4 Contour lines (a) and response surface plots (b) illustrating the interaction between culture time and shaker speed of Azotobacter vinelandii strain AZ6. Figure 4 It was found that the surface inclination was high in the interaction between culture time A and shaker speed C, indicating that the interaction between the two was obvious. In the other two groups, the strain flocculation efficiency was poor when the culture time A was too short, and the strain flocculation efficiency decreased slightly when the culture time A was too long, reaching the maximum value at the middle level within the experimental range.

[0109] Figure 5Contour lines (a) and response surface plots (b) illustrating the interaction between culture temperature and shaker speed for Azotobacter vinelandii strain AZ6. Figure 5 The study revealed a high surface inclination in the interaction between temperature B and shaking speed C, indicating a significant interaction between the two factors. In the single-factor effect of temperature B, the strain's flocculation efficiency was poor at low temperatures and slightly decreased at high temperatures, reaching its maximum value within the experimental range. Compared to the interaction between time A and shaking speed C, both showed significant interaction effects, and the influence of shaking speed C followed the same pattern: poor flocculation efficiency at low temperatures and slightly decreased at high temperatures, peaking at approximately 160 r / min. This indicates that shaking speed is a key regulatory factor affecting the flocculation efficiency of *Azotobacter vinelandii* strain AZ6.

[0110] In this embodiment S1, the *Venelandia diffusa* strain AZ6 can also be a commercially available product.

[0111] Example 2

[0112] This embodiment provides an application of a highly efficient microbial flocculant for removing antibiotics. The microbial flocculant is used to remove antibiotics from water. When the antibiotic is oxytetracycline, the optimal addition amount of the microbial flocculant is 300 mg / L.

[0113] The specific antibiotic removal application process is as follows:

[0114] 1. Preparation of simulated wastewater: Accurately weigh oxytetracycline antibiotic, dissolve it in high-purity deionized water, and prepare stock solutions and simulated wastewater with gradient concentrations of 0.1 mg / L, 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, 1.0 mg / L, 1.2 mg / L, 1.4 mg / L, and 1.6 mg / L.

[0115] 2. Optimization of adsorption conditions:

[0116] Reaction system: 30℃, 100mL system, initial antibiotic concentration 1mg / L;

[0117] Optimization sequence: First, determine the optimal adsorption time by adding 400 mg / L of microbial flocculant, then screen the optimal microbial flocculant concentration based on this time, and finally verify the adsorption effect of different initial concentrations.

[0118] (1) Optimization of adsorption time: With the initial concentration of oxytetracycline fixed at 1 mg / L and the flocculant concentration at 400 mg / L, adsorption time gradients of 0 min, 10 min, 20 min, 30 min, 45 min, 60 min, 75 min, 90 min, 105 min, and 120 min were set. The results are as follows: Figure 8 As shown, the adsorption rate first increases rapidly with time and then tends to level off. The rapid adsorption period is from 0 to 75 min (the removal rate increases from 0 to 40.92%). Adsorption equilibrium is reached at 90 min, with a removal rate of 46.85%. The optimal adsorption time is determined to be 90 min.

[0119] (2) Optimization of flocculant concentration: With the initial concentration of oxytetracycline fixed at 1 mg / L and the adsorption time at 90 min, concentration gradients of 0 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L were set. The results are as follows: Figure 9 As shown, when the dosage concentration is 0-300 mg / L, the removal rate increases significantly with increasing concentration. When the dosage concentration is 300-500 mg / L, the rate of increase slows down. The removal rate reaches a peak of 49.47% at 300 mg / L, and the optimal dosage concentration is determined to be 300 mg / L.

[0120] (3) Optimization of initial antibiotic concentration: With a fixed flocculant concentration of 300 mg / L and an adsorption time of 90 min, initial concentration gradients of 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, 1.0 mg / L, 1.2 mg / L, and 1.4 mg / L were set. The results are as follows: Figure 10 As shown, when the initial concentration is 0.2-1.2 mg / L, the removal rate remains above 50% (54.86% at 1.0 mg / L), and the removal rate decreases slowly when the concentration is 1.0-1.4 mg / L. The optimal treatment concentration is ≤1.0 mg / L.

[0121] 3. Optimal combination of conditions for oxytetracycline antibiotic removal

[0122] Combination Figures 8-10 Based on the data from the three charts, the optimal combination of conditions for oxytetracycline removal was determined: adsorption time: 90 min, microbial flocculant concentration: 300 mg / L, and initial antibiotic concentration range: optimal treatment concentration ≤ 1.0 mg / L.

[0123] 4. Determination of removal efficiency: The concentration of antibiotic residues in the filtrate was detected by a UV-Vis spectrophotometer, and the removal rate and unit adsorption capacity were calculated.

[0124] Removal rate (%) = (AB) / A × 100%

[0125] In the formula: A—initial concentration of oxytetracycline antibiotic;

[0126] B—Concentration of oxytetracycline antibiotic after the addition of flocculant.

[0127] Based on the above formula, the removal rate of the microbial flocculant is 53.86%.

[0128] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a highly efficient microbial flocculant for removing antibiotics, characterized in that, The preparation method includes the following steps: S1. Inoculate the fermentation broth with strain AZ6 of *Venelandia diffusa* and culture it with shaking to obtain a fermentation broth containing bacteria. S2. Mix the fermentation broth containing bacteria obtained in S1 with glycerol to obtain the preservation broth; S3. Take the preserved bacterial culture obtained in S2 and inoculate it into the new fermentation broth. Shake and culture it, then centrifuge it and collect the supernatant. S4. Add anhydrous ethanol to the supernatant obtained in S3, mix well, let stand, and then centrifuge to collect the precipitate. S5. The precipitate obtained in step 4 is freeze-dried to obtain a dry powder, which is the microbial flocculant.

2. The method for preparing a highly efficient antibiotic-removing microbial flocculant according to claim 1, characterized in that, The fermentation broth formulation in S1 is as follows: 15g glucose, 10g sucrose, 0.7g yeast extract, 0.1g urea, 0.5g ammonium sulfate, 1g KH2PO4, 2.5g K2HPO4, 0.3g MgSO4, 0.2g NaCl, and 1L H2O; the shaking culture temperature is 36℃, the rotation speed is 198r / min, and the time is 39h; the amount of *Venerendaenia AZ6* strain added is 100μL / L.

3. The method for preparing a highly efficient antibiotic-removing microbial flocculant according to claim 1, characterized in that, The volume ratio of the fermentation broth containing bacteria to glycerol in S2 is 8:

2.

4. The method for preparing a highly efficient antibiotic-removing microbial flocculant according to claim 1, characterized in that, The amount of the preservation bacterial solution used in S3 is 120 μl / L; the temperature of the shaking culture is 36℃, the rotation speed is 198 r / min, and the time is 39 h; the centrifugation speed is 6000 rpm, and the time is 10 min.

5. The method for preparing a highly efficient antibiotic-removing microbial flocculant according to claim 1, characterized in that, The volume ratio of the supernatant to anhydrous ethanol in S4 is 1:2; the settling temperature is 4°C and the settling time is 18 hours; the centrifugation speed is 6000 rpm and the settling time is 10 minutes.

6. The method for preparing a highly efficient antibiotic-removing microbial flocculant according to claim 1, characterized in that, The freeze-drying temperature described in S5 is -55℃, and the time is 24 hours.

7. The application of a highly efficient microbial flocculant for removing antibiotics, characterized in that, The microbial flocculant is used to remove antibiotics from water.

8. The application of the highly efficient antibiotic removal microbial flocculant according to claim 7, characterized in that, When the antibiotic is oxytetracycline, the amount of microbial flocculant added is 300 mg / L.

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