Preparation method and application of artificially synthesized charcoal immobilized degrading enzyme material based on tylosin degrading bacteria

Abstract

The invention discloses a preparation method and application of an artificially synthesized charcoal immobilized degrading enzyme material based on tylosin degrading bacteria, and relates to the technical field of material synthesis and modification. The method comprises the following steps: adding the biochar into a tylosin degradation intracellular enzyme solution, fully mixing, putting into a constant-temperature oscillation shaking table, and adsorbing at the temperature of 29-31 DEG C; after the adsorption is finished, centrifuging and collecting the precipitate, washing with a phosphate buffer solution, centrifuging again, and washing and centrifuging twice again to obtain the artificially synthesized charcoal immobilized degrading enzyme material based on the tylosin degrading bacteria. The artificially synthesized charcoal immobilized degrading enzyme material based on the tylosin degrading bacteria is applied to synchronous removal of antibiotics and resistance genes in soil. The invention can obtain the preparation method and the application of the artificially synthesized charcoal immobilized degrading enzyme material based on the tylosin degrading bacteria.

Description

Technical Field

[0001] The present invention relates to the technical field of material synthesis and modification, and in particular to a preparation method and application of an artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria. Background Art

[0002] Veterinary antibiotics, such as tylosin, are widely used in livestock and poultry farming to prevent disease, treat infection, and promote growth. Of these, 60–90% of oral and intramuscular antibiotics are excreted in the form of parent substances and metabolites in livestock urine and feces, entering the ecosystem through soil infiltration, air movement, and soil erosion. Furthermore, residual antibiotics exert selective pressure on indigenous soil bacteria, altering bacterial community structure and triggering the proliferation of resistant bacteria. They can also enhance horizontal gene transfer (HGT) among soil bacteria, significantly promoting the spread and dissemination of soil antibiotic resistance genes (ARGs). This not only increases the risk of agricultural soil contamination but also impacts public health through enrichment in the food chain. Therefore, from the perspectives of food safety and human health, developing a broadly applicable method that can simultaneously and efficiently degrade antibiotics in livestock and poultry soils and effectively control the spread of resistance genes is an unmet need. This approach is crucial for controlling and mitigating the environmental risks of antibiotic resistance in soil.

[0003] For antibiotic-contaminated soil, the addition of highly efficient microbial agents is a widely used bioremediation method both domestically and internationally. However, the use of microbial agents carrying resistance genes also increases the risk of soil-resistant bacteria and the spread of resistance gene contamination. The main principle of bioremediation technology is to utilize enzymes secreted by microorganisms to catalyze the degradation of pollutants. Therefore, the use of enzyme remediation technology to repair contaminated soil can achieve the goal of highly efficient degradation of antibiotics without introducing resistance risks. However, free enzymes are highly susceptible to the soil environment, have poor stability in the soil, and have low mass transfer efficiency, so their use alone has certain limitations.

[0004] Antibiotic residues in the soil not only pollute the soil, but the spread of resistance genes they cause also poses potential risks to soil ecosystems, public health and food security. Summary of the Invention

[0005] The purpose of the present invention is to solve the above technical problems and to provide a preparation method and application of an artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria.

[0006] A tylosin-degrading bacterium is disclosed. The tylosin-degrading bacterium is Klebsiella sp. TN-T1, which is deposited in the China Center for Type Culture Collection on October 28, 2024, and has a deposit number of CCTCCNO: M 20242349.

[0007] An artificially synthesized biochar-immobilized degradation enzyme material based on the tylosin-degrading bacteria.

[0008] The method for preparing the artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria is carried out according to the following steps:

[0009] Step S1, extraction and preparation of tylosin degrading enzyme:

[0010] The tylosin degrading bacteria were inoculated into the inorganic salt liquid culture medium at a volume fraction of 1%, and then placed in a constant temperature shaking shaker, and cultured at a temperature of 29-31 ° C and a speed of 125-135 r / min until the bacterial liquid OD 600 The method comprises the steps of: the step of: the tylosin-degrading bacterial solution having a pH value of 0.4 to 0.6, obtaining a tylosin-degrading bacterial solution in a logarithmic phase; centrifuging the tylosin-degrading bacterial solution once at a temperature of 3.9 to 4.1° C.; collecting the supernatant, continuously adding ammonium sulfate powder while stirring until the saturation reaches 9.9 to 10.1%, and standing at a temperature of 3.9 to 4.1° C. for salt precipitation; collecting the precipitate by centrifugation to obtain an extracellular enzyme solution; washing the extracellular enzyme solution with a phosphate buffer solution, resuspending the solution in a phosphate buffer solution, performing ultrasonic disruption in an ice bath, and performing a second centrifugation at a temperature of 3.9 to 4.1° C.; removing cell debris, and collecting the supernatant to obtain a tylosin-degrading intracellular enzyme solution;

[0011] Step S2: Preparation of biochar-immobilized degradation enzyme material:

[0012] The biochar is added to the tylosin degradation intracellular enzyme solution obtained in step S1, and the mixture is fully mixed and placed in a constant temperature shaking incubator for adsorption at a temperature of 29 to 31° C.; after the adsorption is completed, the precipitate is collected by centrifugation, washed with phosphate buffer and centrifuged again, and after two more washings and centrifugations, an artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria is obtained.

[0013] The invention relates to an application of an artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria prepared by the above method, and an application of the artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria in the simultaneous removal of antibiotics and resistance genes in soil.

[0014] Principle of the present invention:

[0015] Immobilized enzyme technology not only inherits the efficient catalytic degradation characteristics of free enzymes, but also has the advantages of improved stability, improved bio-enhancement and reusability. Biochar is rich in porous structures and oxygen-containing groups, is stable in nature and inexpensive, and can not only effectively adsorb pollutants such as antibiotics, block the transmission path and frequency of resistance genes, and effectively fix microorganisms or enzymes, but can also be used as a soil conditioner to preserve soil moisture and improve soil properties. It is a highly promising agricultural soil remediation agent and environmentally friendly immobilized carrier. Therefore, the present invention uses biochar to immobilize antibiotic-degrading enzymes, which can not only create a good and stable microenvironment for the enzyme, achieve the effect of integrated adsorption and degradation repair, but also effectively block the transmission path and frequency of resistance genes in the soil system, reduce the risk of drug resistance transmission in the soil environment, and thus provide a new strategy for strengthening the remediation of compound pollution of antibiotics and resistance genes in the soil.

[0016] Biochar-immobilized degradative enzymes have good structure and properties and are a new type of bioreinforced remediation material for soil antibiotic contamination with broad application prospects. Compared with free degradative enzymes, biochar-immobilized degradative enzymes have higher catalytic activity, pH and thermal stability. That is, biochar is selected as a carrier material, and its porous structure and large specific surface area provide a suitable and stable microenvironment for the catalytic degradation of the enzyme, protecting the enzyme from the influence of adverse environmental factors in complex soils. The present invention uses biochar-immobilized degradative enzyme materials to repair soil pollutants, which not only increases the contact efficiency between the enzyme and the pollutant and improves the degradation activity of the enzyme, but also can improve soil nutrient circulation and increase the diversity of indigenous microbial communities, thereby increasing the synergistic degradation of organic pollutants.

[0017] Beneficial effects of the present invention:

[0018] (1) The biochar-immobilized degradative enzyme of the present invention can completely degrade tylosin with an initial concentration of 10 mg / kg in soil and 99.85% of tylosin with an initial concentration of 20 mg / kg in soil after 7 days, and the degradation products are non-toxic;

[0019] (2) The position of the tylosin degrading enzyme in the present invention is optimized, making the preparation of the artificially synthesized biochar degrading enzyme more targeted and efficient;

[0020] (3) The present invention uses the immobilization method to significantly improve the degradation performance and stability of the biochar-immobilized degrading enzyme;

[0021] (4) The present invention is directed to the synthesis and preparation of biochar immobilized degrading enzymes, and verifies the efficient degradability and application stability of biochar immobilized degrading enzymes, as well as the efficacy and application safety of biochar immobilized degrading enzymes in repairing soil tylosin and blocking resistance gene residues;

[0022] (5) The biochar-immobilized degradation enzyme material synthesized in the present invention has a good effect on the simultaneous reduction of tylosin and resistance genes in the soil.

[0023] The present invention can obtain a preparation method and application of an artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A scanning electron microscope (SEM) image showing the artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria in Example 1;

[0025] Figure 2 The figure shows the comparison of Fourier transform infrared spectra (FTIR) of different treatment groups in Example 1;

[0026] Figure 3 A graph showing the thermal stability experiment of the artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria in Example 1;

[0027] Figure 4 A graph showing the acid-base stability experiment of the artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria in Example 1;

[0028] Figure 5 This figure shows the comparison of the degradation efficiency of tylosin in different treatment groups in Example 1;

[0029] Figure 6 This figure shows the comparison of the effectiveness of tylosin in soil remediation in different treatment groups in Example 1;

[0030] Figure 7 It shows the resistance gene control diagram in the repaired soil of different treatment groups compared in Example 1;

[0031] Figure 8 A graph showing the effect of tylosin-contaminated soil remediation on soil bacterial community diversity in different treatment groups in Example 1;

[0032] Figure 9 The phylogenetic tree of Klebsiella sp. TN-T1 in Example 1 is shown. DETAILED DESCRIPTION

[0033] Specific embodiment 1: This embodiment provides a tylosin-degrading bacterium, which is Klebsiella sp. TN-T1, deposited in the China Center for Type Culture Collection on October 28, 2024, with a deposit number of CCTCC NO: M 20242349.

[0034] Specific embodiment 2: This embodiment is based on the artificially synthesized biochar-immobilized degradation enzyme material of the tylosin-degrading bacteria.

[0035] Specific embodiment 3: This embodiment is a method for preparing an artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria, which is carried out according to the following steps:

[0036] Step S1, extraction and preparation of tylosin degrading enzyme:

[0037] The tylosin degrading bacteria were inoculated into the inorganic salt liquid culture medium at a volume fraction of 1%, and then placed in a constant temperature shaking shaker, and cultured at a temperature of 29-31 ° C and a speed of 125-135 r / min until the bacterial liquid OD 600 The method comprises the steps of: the step of: the tylosin-degrading bacterial solution having a pH value of 0.4 to 0.6, obtaining a tylosin-degrading bacterial solution in a logarithmic phase; centrifuging the tylosin-degrading bacterial solution once at a temperature of 3.9 to 4.1° C.; collecting the supernatant, continuously adding ammonium sulfate powder while stirring until the saturation reaches 9.9 to 10.1%, and standing at a temperature of 3.9 to 4.1° C. for salt precipitation; collecting the precipitate by centrifugation to obtain an extracellular enzyme solution; washing the extracellular enzyme solution with a phosphate buffer solution, resuspending the solution in a phosphate buffer solution, performing ultrasonic disruption in an ice bath, and performing a second centrifugation at a temperature of 3.9 to 4.1° C.; removing cell debris, and collecting the supernatant to obtain a tylosin-degrading intracellular enzyme solution;

[0038] Step S2: Preparation of biochar-immobilized degradation enzyme material:

[0039] The biochar is added to the tylosin degradation intracellular enzyme solution obtained in step S1, and the mixture is fully mixed and placed in a constant temperature shaking incubator for adsorption at a temperature of 29 to 31° C.; after the adsorption, the precipitate is collected by centrifugation, washed with phosphate buffer and centrifuged again, and after two more washings and centrifugations, a method for preparing an artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria is obtained.

[0040] Specific embodiment 4: This embodiment differs from specific embodiment 3 in that: the rotation speed of the centrifugation in step S1 is 7990-8010 r / min, and the centrifugation time is 9.5-10.5 min.

[0041] The other steps are the same as those in the third embodiment.

[0042] Specific embodiment 5: This embodiment differs from specific embodiment 3 or 4 in that: in step S1 , the salting out is allowed to stand at a temperature of 3.9 to 4.1° C. for 23 to 25 hours.

[0043] The other steps are the same as those in the third or fourth embodiment.

[0044] Specific embodiment 6: This embodiment differs from specific embodiments 3 to 5 in that: the inorganic salt liquid culture medium is composed of 1.5g K2HPO4, 0.5g KH2PO4, 0.03g MgSO4, 1g NaCl and 1g (NH4)2SO4;

[0045] The phosphate buffer solution is prepared as follows: 8.0 g of NaCl, 0.2 g of KH2PO4, 2.9 g of Na2HPO4·12H2O, and 0.2 g of KCl are sequentially added to a container, distilled water is added to dissolve the solution, the volume is adjusted to 1000 mL, the pH is adjusted to 7.3-7.5, and the solution is sterilized at a temperature of 120-122° C. and a pressure of 105-119 kPa for 20-30 minutes. The solution is cooled and stored in a refrigerator at 3.9-4.1° C.

[0046] The other steps are the same as those in Specific Embodiments 3 to 5.

[0047] Specific embodiment seven: This embodiment differs from any one of specific embodiments three to six in that: the rotation speed of the secondary centrifugation in step S1 is 9990-10010 r / min, and the centrifugation time is 14.5-15.5 min.

[0048] The other steps are the same as those in Specific Embodiments 3 to 6.

[0049] Specific embodiment eight: This embodiment differs from specific embodiments three to seven in that: the rotation speed of the constant temperature oscillating shaker in step S2 is 125-135 r / min.

[0050] The other steps are the same as those in Specific Embodiments 3 to 7.

[0051] Specific embodiment 9: This embodiment differs from specific embodiments 3 to 8 in that the centrifugal speed in step S2 is 2490-2510 r / min and the centrifugal time is 4.5-5.5 min.

[0052] The other steps are the same as those in Specific Embodiments 3 to 8.

[0053] Specific embodiment ten: This embodiment is an application of an artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria, and the application of the artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria in the simultaneous removal of antibiotics and resistance genes in soil.

[0054] The following examples are used to verify the beneficial effects of the present invention:

[0055] Example 1:

[0056] 1. Required drugs: tylosin, tylosin residue, K2HPO4, KH2PO4, MgSO4, NaCl, (NH4)2SO4, Na2HPO4·12H2O, KCl and agar.

[0057] 2. Solutions and culture media used:

[0058] (1) Mushroom residue culture medium: Mix tylosin residue with distilled water in a ratio of 1:10 (w / w), stir evenly, and filter to obtain the filtrate. Adjust the tylosin content as needed.

[0059] (2) Inorganic salt culture medium: K2HPO4 1.5 g, KH2PO4 0.5 g, MgSO4 0.03 g, NaCl 1 g, (NH4)2SO4 1 g, initial tylosin concentration 200 mg / L, distilled water 1000 mL, adjust the pH value of the inorganic salt culture medium to 7.0.

[0060] (3) Inorganic salt solid culture medium: Add 1.5% to 2.0% agar to the above inorganic salt liquid culture medium, heat to 100°C to dissolve, and cool and solidify to obtain a solid culture medium.

[0061] (4) Phosphate buffer saline (PBS): NaCl 8.0 g, KH2PO4 0.2 g, Na2HPO4·12H2O 2.9 g, KCl 0.2 g. Add the above reagents into a quantitative container in order, add appropriate amount of distilled water to dissolve, and then adjust the volume to 1000 mL. Adjust the pH to 7.4, sterilize at high temperature at 112 kPa for 20 min, cool, and store in a refrigerator at 4 °C until used.

[0062] Note: The above culture medium and equipment required for strain culture must be sterilized by high-pressure steam at 121°C for 20 minutes and sterilized under ultraviolet light for 30 minutes.

[0063] 3. Screening of tylosin-degrading bacteria:

[0064] The tylosin residue was dissolved, broken up, and filtered and leached with distilled water. The above residue extract was added to the residue culture medium and cultured in a constant temperature shaking incubator at 30°C and 130r / min for 7 days in each cycle. Screening was performed by gradually increasing the amount of tylosin added, with a total of 4 cycles, each with three replicates. After 4 cycles of screening and acclimation culture, a dominant strain of Klebsiella TN-T1 with strong degradation ability was isolated. After purification, a single colony was picked and inoculated into a slant culture medium and stored in a refrigerator at 4°C. All the above operations were performed under a sterile environment.

[0065] The strain identification was carried out using the Sanger sequencing method, that is, after obtaining the genomic DNA of the target strain, it was amplified using 27F / 1492R, and the amplified product was sequenced using the ABI3730 sequencing platform. The spliced sequence was blast-matched with the NCBI nt library to obtain the species annotation classification results. The tylosin-degrading bacteria is Klebsiella sp. TN-T1, which was deposited in the China Center for Type Culture Collection on October 28, 2024, with a deposit number of CCTCC NO: M 20242349. The NCBI accession number is SUB15312546 SeqID1PV628020.

[0066] Strain TN-1 was physiologically and biochemically characterized according to the "Handbook of Common Bacterial Systematic Identification." The results, shown in Table 1, indicate that strain TN-1 is Gram-negative, non-motile, positive in the VP reaction, incapable of indole production, negative in the starch hydrolysis reaction, negative in the oxidase test, positive in the catalase test, and negative in the methyl red test. It can degrade lincomycin and tolerate 1% NaCl. Experiments examining the effect of temperature on the growth of strain TN-1 revealed that its optimal growth temperature is 37±1°C. The strain exhibits strong adaptability to temperatures, capable of growing in environments ranging from 20°C to 60°C.

[0067] Table 1: Physiological and biochemical characteristics of Klebsiella sp. TN-T1;

[0068]

[0069]

[0070] Note: + represents positive, - represents negative.

[0071] 4. Extraction and preparation of tylosin degrading enzyme:

[0072] The bacterial suspension growing in the logarithmic phase was centrifuged at 4°C and 8000 rpm for 10 minutes in a high-speed refrigerated centrifuge. The supernatant was collected and solid (NH4)2SO4 powder was added to the suspension while stirring until the saturation reached 10%. The suspension was allowed to stand at 4°C for 24 hours for salt precipitation. The precipitate was collected by centrifugation as the extracellular enzyme solution. The precipitated bacterial cells obtained by the above centrifugation were washed three times with a pH 7.4 phosphate buffer (PBS) solution. The precipitate was resuspended in PBS buffer solution and ultrasonically disrupted in an ice bath. The supernatant was then centrifuged at 4°C and 10000 rpm for 15 minutes to remove cell debris. The supernatant was the intracellular enzyme solution. After localization, it was determined that the key enzyme that degrades tylosin is an intracellular enzyme.

[0073] 5. Preparation of biochar-immobilized degradation enzyme materials:

[0074] The biochar-immobilized enzyme was prepared using an adsorption method. Biochar was used as a carrier and added to the tylosin-degrading intracellular enzyme solution obtained in the previous step at a specific carrier dosage. The mixture was thoroughly mixed and incubated in a constant-temperature shaker at 30°C and 130 rpm for a specific period of time. After adsorption, the biochar-immobilized enzyme solution was transferred to a centrifuge tube and centrifuged at 2500 rpm for 5 minutes to separate the supernatant and precipitate. The precipitate was washed with a small amount of phosphate buffer, and the remaining solid was centrifuged again for 5 minutes. This washing was repeated twice, resulting in the immobilized biochar-immobilized enzyme, which was then stored in a refrigerator at 4°C.

[0075] Figure 1 The scanning electron microscope (SEM) image of the artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria in Example 1 is shown. Figure 2 The FTIR spectra of the different treatment groups in Example 1 are compared. Figure 1-2 As shown in the figure, it was observed that a large number of degradation enzymes were attached to the surface of biochar, that is, the large number of pore structures inside the biochar could provide the required space and physical barrier for the enzyme, thereby protecting the enzyme from desorption caused by external environmental pressure, thereby improving the performance stability of the biochar-immobilized degradation enzyme; in addition, according to the FTIR graph, it can be seen from the changes in functional groups that biochar can be better adsorbed and bound to the degradation enzyme.

[0076] 6. Thermal and acid-base stability experiments of biochar-immobilized degradation enzymes:

[0077] Thermal stability: The temperatures of the constant temperature water bath were set to 20°C, 30°C, 40°C, 50°C, and 60°C, and the prepared free degradation enzyme and biochar immobilized degradation enzyme were placed therein, respectively. After the temperature of the degradation enzyme solution reached the set value, they were maintained under the above conditions for 0.5 h, and the activity of the untreated enzyme solution was set to 100%. The relative activity of the enzyme at each temperature was measured, and a curve was drawn with temperature as the horizontal axis and the relative activity of tylosin degradation enzyme as the vertical axis to determine the thermal stability of tylosin degradation enzyme and biochar immobilized degradation enzyme.

[0078] Acid-base stability: Aqueous solutions with pH values of 4.0, 6.0, 8.0, 10.0, and 12.0 were prepared, and free degrading enzyme and immobilized degrading enzyme were added respectively. After stirring evenly, the relative enzyme activity at each pH value was measured after a certain period of time. The activity of the untreated enzyme solution was set as 100%, and the pH value was used as the horizontal axis and the relative enzyme activity of tylosin degrading enzyme was used as the vertical axis to draw the pH acid-base stability curve.

[0079] Figure 3 This is a graph showing the thermal stability experiment of the artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria in Example 1. Figure 4 The figure shows the acid-base stability experiment of the artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria in Example 1; Figure 3-4 As shown, biochar-immobilized degradation enzymes have a wider pH and temperature adaptation spectrum, that is, compared with single degradation bacteria and degradation enzymes, the material has better environmental resistance.

[0080] 7. Tylosin degradation performance experiment:

[0081] To each 50 mL conical flask, 20 mL of an inorganic salt medium (pH = 7) containing 200 mg / L tylosin was added. The following treatment groups were set up in the experiment: ① Degradation bacteria group: 10% of the degradation strain was added to the conical flask; ② Free degradative enzyme group: Degradative enzyme was added to the conical flask according to the same principle as the inoculum size of the degradation strain; ③ Biochar immobilized degradative enzyme group: The same amount of biochar immobilized degradative enzyme as in ② was added to the conical flask. The above treatment groups were placed in a constant temperature shaking incubator at 30°C and 130 r / min. Samples were taken at regular intervals and the tylosin concentration was measured.

[0082] The concentration of tylosin was determined by spectrophotometry at an absorption wavelength of λ = 290 nm.

[0083] Figure 5 The figure shows the degradation efficiency of tylosin in different treatment groups compared in Example 1; Figure 5 As shown in the figure, under the optimal conditions, the biochar-immobilized degradation enzyme can degrade 86.47% of tylosin within 8 h. In comparison, the degradation rates of tylosin by free tylosin degradation enzyme and tylosin-degrading bacteria are only 64.16% and 28.10% respectively.

[0084] 8. Tylosin contaminated soil remediation experiment:

[0085] Soil that had not been treated with tylosin was pre-cultured in a 30°C greenhouse for one week to restore microbial activity in the soil. The soil simulation experiment began with 350g of soil in each pot. Tylosin solutions of a certain concentration, based on the residual status of tylosin in the soil, were sprayed onto the soil surface and mixed thoroughly to achieve 10mg / kg and 20mg / kg tylosin concentrations, respectively. Water was then added until the soil moisture content reached 60% of its maximum water holding capacity. Under laboratory conditions, the prepared degradation bacteria, free degradation enzymes, and biochar-immobilized degradation enzymes were sprayed at appropriate volumes (1%, v / w) onto the surface of soil contaminated with tylosin and mixed thoroughly to investigate the remediation effects of these three simulated remediation materials on tylosin-contaminated soil.

[0086] Figure 6 This is a graph showing the effectiveness of tylosin in soil remediation compared with different treatment groups in Example 1. Figure 7The figure shows the resistance gene control diagram in the repaired soil of different treatment groups in Example 1; Figure 6-7 As shown in the actual tylosin contaminated soil remediation, biochar degrading enzyme has a high tylosin degradation efficiency without generating toxic degradation products, and can achieve the effect of synchronous reduction of resistance genes.

[0087] 9. Determination of soil bacterial community diversity and resistance genes:

[0088] Take samples from different treatment groups at different times in the above soil experiment and follow the kit Fast Following the SpinKit for Soil instructions, 0.4 g of sample was weighed and total microbial DNA was extracted. DNA concentration was determined using a NanoDrop ND-2000c (NanoDrop Technologies, Wilmington, DE). The extracted DNA was then analyzed by high-throughput PCR and quantitative fluorescence PCR.

[0089] The V3-V4 region of the 16S rRNA was amplified by PCR. Sequencing was performed on the raw data from high-throughput sequencing using Illumina NovaSeq / MiSeq and PacBioSequel II. Sequence quality was initially screened, and raw sequences that met quality requirements were divided into libraries and samples based on index and barcode information. The barcode sequence was removed, and quality control screening was performed on each sample data to obtain valid data for each sample. Using the Vsearch software analysis process, methods such as sequence denoising and OTU clustering were employed to visualize the specific composition of each sample (group) at different taxonomic levels, thereby providing an overall overview. The alpha diversity level of each sample was evaluated based on the distribution of ASV / OTUs across samples.

[0090] Fluorescence quantitative PCR reactions were performed on an Applied Biosystems ViiA™ 7 Real-Time PCR System. The PCR reaction volume was 10 μL, consisting of 5 μL of Master (2×), 0.75 μL of each primer (10 μM), 0.5 μL of DNA (1–10 ng), and 3 μL of ddH₂O. PCR reaction conditions were: initial denaturation at 95°C for 10 min, followed by 32 cycles of annealing at 95°C for 60 s, annealing for 60 s, and 72°C for 1 min, followed by a final extension at 72°C for 6 min. Three technical replicates and three biological replicates were performed for each sample.

[0091] Figure 8 The figure shows the effect of different treatment groups on the diversity of soil bacterial communities in the restoration of tylosin-contaminated soil in Example 1; Figure 8 As shown in the actual tylosin-contaminated soil remediation, biochar-immobilized degradation enzymes increased the diversity of soil bacterial communities and contributed to the rapid recovery and improvement of the soil microenvironment.

Claims

1. A tylosin-degrading bacterium, characterized in that The tylosin-degrading bacteria is Klebsiella sp. TN-T1, which is deposited in the China Center for Type Culture Collection on October 28, 2024, with a deposit number of CCTCC NO: M 20242349.

2. An artificially synthesized biochar-immobilized degradation enzyme material based on the tylosin-degrading bacteria as claimed in claim 1.

3. The method for preparing an artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria according to claim 2, characterized in that The preparation method is carried out according to the following steps: Step S1, extraction and preparation of tylosin degrading enzyme: The tylosin degrading bacteria were inoculated into the inorganic salt liquid culture medium at a volume fraction of 1%, and then placed in a constant temperature shaking shaker, and cultured at a temperature of 29-31 ° C and a speed of 125-135 r / min until the bacterial liquid OD 600 The method comprises the steps of: the step of: the tylosin-degrading bacterial solution having a pH value of 0.4 to 0.6, obtaining a tylosin-degrading bacterial solution in a logarithmic phase; centrifuging the tylosin-degrading bacterial solution once at a temperature of 3.9 to 4.1° C.; collecting the supernatant, continuously adding ammonium sulfate powder while stirring until the saturation reaches 9.9 to 10.1%, and standing at a temperature of 3.9 to 4.1° C. for salt precipitation; collecting the precipitate by centrifugation to obtain an extracellular enzyme solution; washing the extracellular enzyme solution with a phosphate buffer solution, resuspending the solution in a phosphate buffer solution, performing ultrasonic disruption in an ice bath, and performing a second centrifugation at a temperature of 3.9 to 4.1° C.; removing cell debris, and collecting the supernatant to obtain a tylosin-degrading intracellular enzyme solution; Step S2: Preparation of biochar-immobilized degradation enzyme material: The biochar is added to the tylosin degradation intracellular enzyme solution obtained in step S1, and the mixture is fully mixed and placed in a constant temperature shaking incubator for adsorption at a temperature of 29 to 31° C.; after the adsorption is completed, the precipitate is collected by centrifugation, washed with phosphate buffer and centrifuged again, and after two more washings and centrifugations, an artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria is obtained.

4. The method for preparing an artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria according to claim 3, characterized in that The rotation speed of one centrifugation in step S1 is 7990-8010 r / min, and the centrifugation time is 9.5-10.5 min.

5. The method for preparing an artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria according to claim 3, characterized in that In step S1, the salting-out is allowed to stand at a temperature of 3.9 to 4.1° C. for 23 to 25 hours.

6. The method for preparing an artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria according to claim 3, characterized in that The inorganic salt liquid culture medium consists of 1.5g K2HPO4, 0.5g KH2PO4, 0.03g MgSO4, 1g NaCl and 1g (NH4)2SO4; The phosphate buffer solution is prepared as follows: 8.0 g of NaCl, 0.2 g of KH2PO4, 2.9 g of Na2HPO4·12H2O, and 0.2 g of KCl are sequentially added to a container, distilled water is added to dissolve the solution, the volume is adjusted to 1000 mL, the pH is adjusted to 7.3-7.5, and the solution is sterilized at a temperature of 120-122° C. and a pressure of 105-119 kPa for 20-30 minutes. The solution is cooled and stored in a refrigerator at 3.9-4.1° C.

7. The method for preparing an artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria according to claim 3, characterized in that The rotation speed of the secondary centrifugation in step S1 is 9990-10010 r / min, and the centrifugation time is 14.5-15.5 min.

8. The method for preparing an artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria according to claim 3, characterized in that The rotation speed of the constant temperature oscillating shaker in step S2 is 125-135 r / min.

9. The method for preparing an artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria according to claim 3, characterized in that The centrifugal speed in step S2 is 2490-2510 r / min, and the centrifugal time is 4.5-5.5 min.

10. Use of an artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria prepared by the method according to any one of claims 3 to 9, characterized in that The application of the artificially synthesized biochar-immobilized degradation enzyme material based on tylosin-degrading bacteria in the simultaneous removal of antibiotics and resistance genes in soil.

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