Norfloxacin degrading bacterium as well as gel ball and application thereof
By screening and immobilizing the norfloxacin-degrading strain Pedobacter sp. KHR607 to form gel spheres, the problem of microbial inactivation in norfloxacin wastewater treatment was solved, achieving efficient and stable wastewater degradation.
Patent Information
- Application Number
- CN202511040660.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-28
- Publication Date
- 2025-11-04
AI Technical Summary
Existing technologies are difficult to effectively treat norfloxacin wastewater, especially high-concentration norfloxacin wastewater, and microorganisms are easily deactivated in the wastewater environment, affecting the treatment effect.
The highly efficient norfloxacin-degrading strain Pedobacter sp. KHR607 was screened and immobilized into gel spheres. These spheres were then encapsulated using a suspension of polyvinyl alcohol, sodium alginate, and Fe3O4 to enhance their stability and degradation ability in harsh environments.
It achieves efficient and rapid degradation of norfloxacin wastewater concentration. The gel balls maintain stability and degradation efficiency during multiple cycles of use, improve the adaptability to acidity and high concentrations of norfloxacin, and enhance the environmental adaptability of microorganisms.
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Figure CN120888442A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of water treatment, and particularly relates to a norfloxacin-degrading bacterium, a gel ball thereof and application thereof, which are used for the biodegradation of norfloxacin in wastewater. BACKGROUND
[0002] Antibiotics are a kind of substances with anti-pathogen or other activities produced by bacteria, fungi or other microorganisms in the life process, and are widely used in medical industry, livestock and aquaculture, etc. Antibiotics cannot be completely absorbed or metabolized, and are finally discharged into the surrounding environment in the form of themselves or metabolites through urine and feces. Antibiotics themselves are difficult to degrade, and wastewater containing antibiotics has high concentration, low biodegradability, large impact load and many toxic and harmful substances. The entry of antibiotic wastewater into the environment will induce the generation of drug-resistant microorganisms, interfere with the composition of microbial populations in the environment, and affect the operation and treatment effect of the biochemical treatment process of municipal wastewater, which has a very great impact on human health, ecological balance and environmental pollution.
[0003] Fluoroquinolone drugs are a kind of antibiotics commonly used by humans and animals, which are mainly discharged into the natural environment through pharmaceutical enterprise wastewater, medical industry waste, livestock manure and aquaculture water, etc. In recent years, fluoroquinolone antibiotics have been frequently detected in different environments, including water environment, soil and aquatic organisms, and the detection rate and concentration are both high. Therefore, there are a large number of residues of fluoroquinolone antibiotics in the environment, which poses a great security risk to the environment and human health, and it is particularly important to effectively remove such drug residues. Norfloxacin is a common fluoroquinolone antibiotic, and the methods for treating norfloxacin wastewater include physical method, chemical method and biological method. Compared with physical and chemical methods, biological treatment has the advantages of green, high efficiency and low cost, and is usually used as the main process of antibiotic wastewater treatment technology. The biological degradation of norfloxacin is mainly based on microbial metabolism, and free microorganisms are easily stressed by the wastewater environment and easily inactivated. Therefore, it is particularly important to explore microorganisms with high and stable degradation efficiency and improve the adaptability of microorganisms to the wastewater environment. SUMMARY
[0004] To solve the problems in the prior art, the present application provides a norfloxacin-degrading bacterium, a gel ball thereof and application thereof. Specifically, the present application screens a strain with high degradation efficiency for norfloxacin. The strain exhibits strong resistance to norfloxacin and can effectively convert norfloxacin, which is difficult to degrade and has biological toxicity in wastewater, by using its biochemical action. This process quickly and economically converts norfloxacin-containing wastewater that cannot be directly treated by microorganisms into low-concentration wastewater, greatly improving the biodegradability.
[0005] The specific technical scheme of the present application is as follows:
[0006] The application provides a norfloxacin-degrading bacterium, which is classified as Pedobacter sp. KHR607 and is preserved in the China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC M2025139.
[0007] Further, the 16S rRNA gene sequence of the norfloxacin-degrading bacterium is shown in SEQ ID No. 1.
[0008] The application also provides application of the norfloxacin-degrading bacterium in degrading norfloxacin in water.
[0009] The application also provides a gel ball containing the norfloxacin-degrading bacterium, and a preparation method of the gel ball.
[0010] 1) mixing a polyvinyl alcohol solution, a sodium alginate solution and a Fe3O4 suspension; placing the mixed solution on a magnetic stirrer, stirring at room temperature to fully mix and uniformly distribute the components; and sterilizing the mixed embedding material solution by autoclaving and cooling to room temperature;
[0011] 2) adding the norfloxacin-degrading bacterium of claim 1 to the cooled embedding material mixed solution and uniformly stirring to uniformly distribute the bacterium in the embedding material;
[0012] 3) transferring the embedding material mixed solution containing the bacterium to a syringe, and dropping the mixed solution into a saturated boric acid CaCl2 solution drop by drop by using the syringe to form uniformly spherical gel beads; solidifying the gel beads in a crosslinking solution; and washing the solidified material with sterile water to remove residual CaCl2 solution on the surface, thereby obtaining the gel ball containing the norfloxacin-degrading bacterium.
[0013] According to the preferred scheme of the application, in step 1), the mass percentage concentration of the polyvinyl alcohol solution is 8%-16%, the mass percentage concentration of the sodium alginate solution is 5%, and the mass percentage concentration of the Fe3O4 suspension is 5%; and the volume ratio of the polyvinyl alcohol solution, the sodium alginate solution and the Fe3O4 suspension is 5:4:1.
[0014] According to the preferred scheme of the application, in step 2), the inoculation amount of the norfloxacin-degrading bacterium in the embedding material mixed solution is 10 (V / V) %.
[0015] According to the preferred scheme of the application, in step 3), the saturated boric acid CaCl2 solution is prepared by adding boric acid into deionized water until there is solid residue, then adding CaCl2 to make the mass fraction 2.5%, and adjusting the pH to 6-7 with a Na2HCO3 solution.
[0016] The application also provides application of the gel ball in degrading norfloxacin in water.
[0017] According to a preferred embodiment of the present invention, after the degradation of norfloxacin in the current batch of water is completed, the gel balls are reused to degrade norfloxacin in the next batch of water.
[0018] According to a preferred embodiment of the invention, the concentration of norfloxacin in the water body ranges from 20 mg / L to 100 mg / L, and the pH ranges from 5 to 9.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] 1) This invention provides a strain Pedobacter p. KHR607 that can efficiently degrade norfloxacin in water. This strain is a new species in the genus Pedobacter. It has a certain tolerance to high concentrations of norfloxacin and can achieve efficient degradation of norfloxacin (it can completely degrade 20 mg / L of norfloxacin within 5 days).
[0021] 2) The present invention prepared an immobilized material (a gel ball containing norfloxacin-degrading bacteria) using the obtained strain. The obtained immobilized material has a stable structure (high mechanical strength and wear resistance), which can prevent the embedded microorganisms from leaking or breaking under external pressure (such as water flow impact or reactor stirring), and the immobilized material can adsorb norfloxacin to improve the degradation efficiency.
[0022] 3) The immobilized material exhibits excellent reusability in terms of recycling. The immobilized material can maintain its complete spherical shape for more than ten days and can still rapidly degrade antibiotics after the wastewater is replaced. Due to the large number of active bacteria inside the gel spheres, the degradation rate is faster than the first time.
[0023] After immobilizing the 4 strains, the system's resistance to acidic environments was significantly improved. Furthermore, the immobilized material provided some protection to the strain cells, resulting in a relatively low NOR concentration within the material, allowing the strains to proliferate normally and exhibiting a better degradation rate than free-floxacin. Therefore, the immobilized material of this invention has a wider pH adaptability and adaptability to high concentrations of norfloxacin, showing promising application prospects in the treatment of norfloxacin-containing wastewater. Attached Figure Description
[0024] Figure 1 Phylogenetic tree (NJ tree) constructed for strain KHR607 and similar standard strains;
[0025] Figure 2 A phylogenetic tree (ML tree) was constructed for strain KHR607 and similar standard strains;
[0026] Figure 3 Phylogenetic tree (MP tree) constructed for strain KHR607 and similar standard strains;
[0027] Figure 4 The growth curve of strain KHR607 in wastewater;
[0028] Figure 5 The removal effect of strain KHR607 on norfloxacin;
[0029] Figure 6 This is a photograph of the immobilized material (gel ball) obtained by embedding bacterial cells in PVA / SA / Fe3O4 according to the present invention.
[0030] Figure 7 The degradation effect of immobilized materials (gel spheres) under different PVA and SA ratios;
[0031] Figure 8 The recycling performance of the immobilized material (gel spheres) of this invention is shown (solid line for the first time, dashed line for the second time);
[0032] Figure 9 The degradation effects of single bacteria at different pH values are shown in (a), where (a) is the degradation effect of free bacteria at pH=5, and (b) is the degradation effect of encapsulated bacteria.
[0033] Figure 10 The degradation effects of free bacteria and hydrogel-embedded bacteria with different initial NOR concentrations were investigated. Detailed Implementation
[0034] The present invention will be further described and illustrated below with reference to specific embodiments. The embodiments described are merely examples of the content of this disclosure and do not limit the scope of the invention. The technical features of each embodiment in the present invention can be combined accordingly, provided that there is no mutual conflict.
[0035] Example 1. Strain Screening
[0036] 1.1 Simulated wastewater
[0037] Given the complexity of real wastewater substrates and the presence of various organic interferences, this embodiment employs a component-controlled simulated wastewater system for subsequent experiments to accurately analyze the effects of different culture conditions on the metabolic activity of the strain and the degradation efficiency of norfloxacin. The composition of the simulated wastewater is shown in Table 1. The vitamin solution composition (mg / L) is as follows: Vitamin B12 0.1, Biotin 2, Folic acid 2, Riboflavin 5, Niacin 5, Pantothenic acid 5, Ammonium sulfate 5, Para-aminobenzoic acid 5, Lipoic acid 5, Pyridoxine hydrochloride 10; the mineral solution composition (g / L) is as follows: H3BO3 0.01, CuSO4·5H2O 0.01, AlK(SO4)2·12H2O 0.01, NiCl2·6H2O 0.024, NaMoO4 0.025, NaWO4·2H2O 0.025, FeSO4·7H2O 0.1, CaCl2·2H2O 0.1, CoCl2·6H2O 0.1, ZnCl2 0.13, MnSO4·H2O 0.5, NaCl 1, C6H9NO6 1.5, MgSO4 3.
[0038] Table 1 Composition of Simulated Wastewater
[0039]
[0040] 1.2 The components of the culture medium involved in this invention are as follows:
[0041] LB medium: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, pH 7.
[0042] LB-Fe medium: ferric citrate 1 g / L, yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, pH 7.
[0043] 1.3 Source of strains
[0044] The Extremophile Microbiology Laboratory of the College of Life Sciences at Zhejiang University has accumulated a large number of microbial strains from extreme environments such as oceans, salt lakes, salt mines, chemical wastewater, and soil through long-term basic research, providing the source of strains for this study. The strains were preserved using a vacuum freeze-drying method.
[0045] 1.4 Strain screening
[0046] Strains with a growth cycle of less than 3 days, culture temperatures of 25–35℃, and simple culture media were selected from the strain library of the Extremophile Microbiology Laboratory, College of Life Sciences, Zhejiang University, as candidate strains. These strains were activated in sterile 96-well plates using LB and LB-Fe media. Simulated wastewater was filtered through a 0.22 μm membrane for sterilization before being dispensed into sterile 96-well plates. The activated candidate strains were inoculated into the wastewater at a concentration of 1% (V / V) and cultured at 30℃ for 3 days. During the culture process, the OD of the strains in the 96-well plates was monitored using a microbial growth analyzer. 600 Value, select OD 600 >0.5 was used as the target strain. After multiple subculturings, strains that could still grow stably were selected as the test strains.
[0047] Example 2: Single-strain degradation experiment of norfloxacin
[0048] 2.1 Seed liquid preparation
[0049] The strains screened in Example 1 were amplified and cultured in LB medium to OD. 600 When the bacterial concentration reaches approximately 1.5, centrifuge at 8000 rpm for 3 minutes, discard the supernatant, and collect the bottom bacterial cells. Resuspend the cells in sterile distilled water to prepare the seed culture.
[0050] 2.2 Degradation performance
[0051] After preparing the simulated wastewater, it was filtered through a 0.22 μm filter membrane for sterilization. 100 mL of the filtered water was then added to a 250 mL sterilized Erlenmeyer flask. Seed culture was added to adjust the initial OD value in the Erlenmeyer flask. 600 =1. Three replicates were set up for each strain. The experimental system was carried out at a constant temperature of 30℃ and a shaking speed of 150 rpm. Samples were taken every 24 hours to determine the residual norfloxacin concentration, total organic carbon content, and total nitrogen content of the experimental groups.
[0052] Example 3: Strain Identification
[0053] This study screened a strain capable of eliminating 100% of norfloxacin within 5 days and performed 16S rRNA gene sequencing on it. PCR amplification was performed using universal primers 27F (5'-GAGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGAC-3'), and the sequencing results were submitted to the EzBioCloud database (http: / / www.ezbiocloud.net / eztaxon / identify) for sequence alignment. The 16S rRNA gene alignment information is shown in Table 2.
[0054] Table 2 Sequencing alignment information of 16S rRNA gene of single strains
[0055]
[0056] The full-length 16S rRNA gene sequence of strain KHR607 (1397 bp) showed 98.79% similarity to its closest relative, *Pedobacter montanisoli*. Phylogenetic trees were constructed using the Neighbor-Joining, Maximum Likelihood, and Maximum Parsimony algorithms. The results showed that KHR607 consistently remained within the *Pedobacter* genus in all three phylogenetic trees. Figure 1 -to Figure 3 It belongs to the same branch as the standard strain Pedobactermontanisoli CYS-01, but there is a stable 42bp evolutionary difference between the two.
[0057] The characteristics of Pedobacter sp. KHR607 are as follows: the colonies are pale yellow, smooth, with irregular edges, slightly raised in the center, 3 mm in diameter, easy to pick up, and have a uniform texture; they grow well on conventional media such as R2A, LB, or nutrient agar; catalase activity was observed using 3% hydrogen peroxide and the colonies were positive; PNPG (4-nitrophenyl-β-galactopyranoside) was weakly positive.
[0058] In summary, strain KHR607 has been identified as a new species within the genus *Pedobacter*, tentatively named *Pedobacter* sp. KHR607. This new species was deposited with the China Center for Type Culture Collection (CCTCC) in January 2025, at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. The accession number is CCTCC M 2025139, and the deposit date is January 15, 2025.
[0059] Example 4 Immobilization of strains
[0060] 4.1 Solution Preparation
[0061] Polyvinyl alcohol (PVA) solution: Accurately weigh a certain amount of PVA (type 1799), add an appropriate amount of deionized water, heat and stir in a constant temperature water bath at 90-95℃ until the PVA is completely dissolved, and prepare a PVA solution with a mass fraction of 8%-16%.
[0062] Sodium alginate (SA) solution: Weigh an appropriate amount of SA, add deionized water, stir to dissolve, and sonicate to remove air bubbles to obtain a 5% (w / w) SA solution.
[0063] Fe3O4 suspension: A certain amount of Fe3O4 nanoparticles are dispersed in deionized water and ultrasonically treated to make them uniformly dispersed, thus preparing a suspension with a mass percentage concentration of 5%.
[0064] Saturated boric acid CaCl2 solution: Add boric acid to a certain volume of deionized water until solid remains, then add an appropriate amount of CaCl2 to make the mass fraction 2.5%. Adjust the pH to 6-7 using Na2HCO3 solution.
[0065] 4.2 Material Mixing
[0066] Mix 10 wt% PVA solution, 5 wt% SA solution, and 5 wt% Fe3O4 suspension at a volume ratio of 5:4:1. Place the mixture on a magnetic stirrer and stir for 30-60 minutes at room temperature to ensure thorough mixing. Autoclave the mixed embedding material solution (121°C, 20 minutes) and cool to room temperature.
[0067] 4.3 Strains encapsulation
[0068] The collected and washed bacterial strain KHR607 was added to 10 mL of cooled embedding material mixture at an inoculum size of 10 (V / V), and gently stirred to ensure uniform dispersion of the bacteria in the embedding material. The bacterial-containing embedding material mixture was transferred to a syringe, and the mixture was dripped dropwise into a saturated boric acid CaCl2 solution, forming uniform spherical gel beads. The gel beads were cured in the cross-linking solution for 24 hours. The cured embedding beads were washed 3-5 times with sterile water to remove residual CaCl2 solution from the surface. The washed embedding beads were then transferred to a sterile container and stored at 4°C for later use.
[0069] 4.4 Degradation experiment of immobilized strains
[0070] Gel beads containing bacteria served as the experimental group, while gel beads without bacteria served as the control group. Both groups of gel beads were added to simulated norfloxacin wastewater. 100 mL of simulated wastewater was added to each 250 mL Erlenmeyer flask, and the flasks were placed in a shaker and cultured under suitable conditions. Samples were taken every 24 hours to determine the residual amount of norfloxacin. After one degradation experiment, the embedded gel beads were washed 3–5 times with sterile water and then added to fresh simulated norfloxacin wastewater. The above degradation experiment was repeated to assess the reusability of the embedded bacteria, for 3–5 cycles.
[0071] Figure 4 The growth curve of strain Pedobacter sp. KHR607 in simulated wastewater is shown. C-9 / C-10 / C-11 represent three parallel experimental groups. Dynamic monitoring data from a biological replication experiment (n=3) over 72 hours showed that the OD... 600 A 25-30% decrease in OD value indicates that the bacteria have entered a metabolic stress phase; subsequently, they maintain steady-state growth (OD) for 60 hours. 600=0.31±0.04), indicating that this single bacterium has a certain tolerance to an environment of 20 mg / L norfloxacin.
[0072] Figure 5 To investigate the removal effect of free strains on norfloxacin, experimental data showed that the strains had a good removal effect on norfloxacin, and could completely degrade 20 mg / L of norfloxacin within 5 days.
[0073] Figure 6 This image shows a physical representation of the PVA / SA / Fe3O4 encapsulation material (hydrogel beads) prepared in this invention. The hydrogel beads are 3-4 mm in diameter, with smooth, undamaged surfaces, and maintain a relatively intact spherical shape throughout the experiment. The high mechanical strength and abrasion resistance of PVA provide structural stability to the composite material, preventing leakage or breakage of the encapsulated microorganisms under external pressure (such as water flow impact or reactor agitation). Simultaneously, the flexibility of sodium alginate alleviates the brittleness of PVA, forming a "rigid-flexible" matrix and reducing the risk of breakage during long-term use. This work optimized the ratio of PVA to SA (…). Figure 7 The control group consisted of a single material and showed only weak adsorption of norfloxacin. High-concentration PVA (8 wt%) exhibited high mechanical properties but poor mass transfer performance, resulting in a lower degradation effect compared to free bacteria. Considering both mechanical strength and degradation performance, 5 wt% PVA and 2 wt% SA were the optimal immobilization ratio.
[0074] In terms of recycling, hydrogel spheres exhibit excellent reusability. Figure 8 The gel balls can maintain their complete spherical shape for more than ten days and can still rapidly degrade antibiotics after the wastewater is replaced. Because there are a large number of active bacteria inside the gel balls, the degradation rate is faster than the first time.
[0075] This single bacterium exhibits excellent adaptability to alkaline conditions, surviving in simulated wastewater ranging from neutral to alkaline and efficiently degrading norfloxacin. In contrast, the individual strain is not acid-tolerant, showing poor degradation performance in simulated wastewater at pH 5, with a degradation rate of only 37% within 5 days. After immobilizing the strain by embedding it, the system's resistance to acidic environments significantly improved, achieving a degradation rate of 86% within 5 days.
[0076] When the NOR concentration is high, NOR not only exists as a carbon source, but also forms an environmental stress factor, which slows down the proliferation rate of free cells. Hydrogel spheres provide a certain degree of protection for cells, resulting in a relatively low NOR concentration inside the hydrogel spheres, allowing the strains to proliferate normally and exhibiting a better degradation rate of free bacteria. Figure 9 The degradation effects of free bacteria and hydrogel-embedded bacteria with different initial NOR concentrations were investigated.
[0077] This invention uses a composite material of polyvinyl alcohol and sodium alginate, doped with Fe3O4 nanoparticles, to achieve the immobilization and encapsulation of bacterial strains. The encapsulated system has a wider pH adaptability and adaptability to high concentrations of norfloxacin, and has good application prospects in the treatment of wastewater containing norfloxacin.
[0078] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.
Claims
1. A norfloxacin-degrading bacterium, characterized in that, The norfloxacin-degrading bacterium is classified and named Pedobactersp.KHR607, and is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M2025139 on January 15, 2025.
2. The norfloxacin-degrading bacteria according to claim 1, characterized in that, Its 16S rRNA gene sequence is shown in SEQ ID No.
1.
3. The application of the norfloxacin-degrading bacteria according to claim 1 in the degradation of norfloxacin in water.
4. A gel ball comprising the norfloxacin-degrading bacteria of claim 1, characterized in that, Its preparation method includes the following steps: 1) Mix the polyvinyl alcohol solution, sodium alginate solution, and Fe3O4 suspension; place the mixture on a magnetic stirrer and stir at room temperature to ensure that all components are thoroughly mixed; autoclave the mixed embedding material solution and cool it to room temperature; 2) Add the norfloxacin-degrading bacteria as described in claim 1 to the cooled embedding material mixture, stir evenly, so that the bacteria are evenly dispersed in the embedding material; 3) Transfer the embedding material mixture containing bacteria into a syringe, and use the syringe to drop the mixture dropwise into a CaCl2 solution saturated with boric acid, so that the droplets form uniform spherical gel beads; the gel beads are solidified in the cross-linking solution; wash the solidified material with sterile water to remove the residual CaCl2 solution on the surface, and obtain gel beads containing norfloxacin-degrading bacteria.
5. The gel sphere according to claim 4, characterized in that, In step 1), the mass percentage concentration of the polyvinyl alcohol solution is 8%-16%; the mass percentage concentration of the sodium alginate solution is 5%; the mass percentage concentration of the Fe3O4 suspension is 5%; and the volume ratio of the polyvinyl alcohol solution, sodium alginate solution, and Fe3O4 suspension is 5:4:
1.
6. The gel sphere according to claim 4, characterized in that, In step 2), the inoculum amount of norfloxacin-degrading bacteria in the embedding material mixture is 10%.
7. The gel sphere according to claim 4, characterized in that, In step 3), the method for preparing the CaCl2 solution of saturated boric acid is as follows: add boric acid to deionized water until there is solid residue, then add CaCl2 to make its mass fraction 2.5%, and adjust the pH to 6-7 with Na2HCO3 solution.
8. The application of the gel spheres according to any one of claims 4-7 in the degradation of norfloxacin in water.
9. The application according to claim 8, characterized in that, After the degradation of norfloxacin in the current batch of water is completed, the gel balls are reused to degrade norfloxacin in the next batch of water.
10. The application according to claim 9, characterized in that, In the water body, the concentration of norfloxacin ranges from 20 mg / L to 100 mg / L, and the pH ranges from 5 to 9.