Strain degrading antibiotic and method for preparing immobilized particles thereof

Abstract

The application provides a strain degrading antibiotics and a preparation method of immobilized particles of the strain, in particular, the application fixes high-efficiency antibiotic-degrading bacteria through an embedding process, and can improve the activity maintenance ability and the anti-environmental interference ability of the degrading bacteria. The application obtains a strain with excellent colonization ability and extremely high degrading activity through mutagenesis and screening, and the strain is particularly suitable for preparing a bacterial agent immobilized particle.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. Specifically, this invention relates to strains that degrade antibiotics and methods for preparing their immobilized particles. Background Technology

[0002] Antibiotics, as broad-spectrum antibacterial drugs, are widely used to treat infectious diseases in humans and animals. After being ingested by organisms, antibiotics are difficult to completely metabolize in the body; most are excreted into the environment through urine and feces as parent bacteria or active metabolites, frequently detected in drinking water, surface water, and soil. While most antibiotics have short half-lives, due to overuse and inadequate treatment, large quantities enter the environment each year, causing a phenomenon known as "pseudo-persistence." In this context, the persistent presence of antibiotics in the environment not only threatens the survival of plants and animals but also promotes the emergence of antibiotic-resistant bacteria and antibiotic-resistant genes, potentially leading to the emergence of superbugs. Furthermore, these superbugs can accumulate in the food chain and enter the human body, threatening human health.

[0003] Antibiotics are ubiquitous in the environment and have become a significant pollutant. Tylosin, sulfonamides, macrolides, and fluoroquinolones are commonly found in surface water. Tetracyclines and aminoglycosides are mainly detected in soil, and their residues lead to a high average abundance of antibiotic resistance genes in topsoil. Furthermore, in areas surrounding pharmaceutical plants, the concentration of sulfonamide antibiotics in groundwater is very high, indicating serious localized pollution. As the problem of antibiotic residues in the environment gains attention, research on various degrading bacteria is gradually underway. These bacteria hold the promise of effectively decomposing residual antibiotics in the environment through biodegradation, mitigating their potential harm to the ecological environment and human health.

[0004] However, free-floating degrading bacteria exhibit significant instability during antibiotic degradation. In terms of their physical form, these bacteria are in a free state, lacking a fixed attachment carrier. When in a flowing water environment, the scouring force of the water directly acts on the bacteria, easily carrying them away and making it difficult for them to persist in the target degradation area. Regarding the sustainability of their action, due to being constantly carried away by the water flow, the bacteria cannot exert their degradation effect in antibiotic-containing water bodies or other environments for extended periods. The degradation process is frequently interrupted due to the loss of bacteria, resulting in a significant reduction in antibiotic degradation efficiency and making it difficult to stably and continuously complete the degradation task.

[0005] To address this deficiency, an encapsulation process can be used to immobilize highly efficient antibiotic-degrading bacteria. Encapsulation provides a "protective barrier" for the bacteria, resisting water erosion and ensuring stable residence, while also blocking harmful interference and maintaining a suitable environment for bacterial survival. This enhances the bacteria's activity retention and resistance to environmental disturbances. Furthermore, the encapsulation carrier can orderly immobilize the bacteria, allowing them to form a collaborative degradation system and continuously supply nutrients, further enhancing their antibiotic degradation activity. It also facilitates subsequent recycling and reuse, reducing treatment costs. Summary of the Invention

[0006] The purpose of this invention is to provide a strain that degrades antibiotics and a method for preparing its immobilized particles.

[0007] In a first aspect of the invention, a strain of Kutella (…) is provided. Kurthia. sp.), the Kurtella bacteria is: Strain 1: Kurtella ( Kurthia. sp.) YJT4, deposited at Guangdong Microbial Culture Collection Center (GDMCC), accession number GDMCC No:67149; or Strain 2: Strain 2 is a derivative strain of strain 1; for example, strain 1 obtained through gene mutation or genetic engineering.

[0008] In a second aspect, the present invention provides a microbial inoculant comprising the *Kutracheid* strain described in the first aspect of the present invention.

[0009] In another preferred embodiment, the microbial agent is a liquid or solid agent.

[0010] In another preferred embodiment, the microbial agent further includes one or more microorganisms selected from the group consisting of: lactic acid bacteria, agrobacterium, rhizobium, yeast, aspergillus, streptomyces, Bacillus subtilis, pseudomonas, Bacillus amyloliquefaciens, nitrogen-fixing bacteria, Bacillus licheniformis, Bacillus laterosporus, Penicillium, Bacillus polymyxa, Trichoderma, Bacillus coagulans, Bacillus marineus, Rhizopus, endophytic Bacillus, Bacillus megaterium, and Bacillus thuringiensis.

[0011] In another preferred embodiment, the microbial agent is an immobilized microbial agent particle.

[0012] A third aspect of the present invention provides the application of the *Kutracheidella* strain described in the first aspect of the present invention in the degradation of antibiotics.

[0013] In another preferred embodiment, the antibiotic is tylosin.

[0014] A fourth aspect of the present invention provides a method for preparing immobilized microbial agents, the method comprising the steps of: (a) Providing the *Kurstella* strain as described in the first aspect of the present invention; (b) The *Kutella* bacteria and the immobilization substrate are mixed and cross-linked to obtain the immobilized bacterial agent particles.

[0015] In another preferred embodiment, in step (b), the immobilization substrate comprises polyvinyl alcohol and sodium alginate.

[0016] In another preferred embodiment, the immobilized substrate also includes mushroom residue biochar.

[0017] In another preferred embodiment, in step (a), the *Kutracheid* strain described in the first aspect of the present invention is fermented using a liquid fermentation method to obtain a fermentation broth of the *Kutracheid* strain.

[0018] In another preferred embodiment, the cell density of the *Kootierella* fermentation broth is approximately 10-1. 5 -10 10 per mL.

[0019] In another preferred embodiment, the method includes the following steps: (1) Liquid fermentation of the *Kouterella* to obtain bacterial solution I; (2) Dissolve the sodium alginate and polyvinyl alcohol and mix them with mushroom residue biochar to obtain mixture II; (3) Mix bacterial solution I with mixed solution II to prepare mixed solution III; (4) Add the mixture III dropwise into the calcium chloride solution for cross-linking and fixation to obtain the immobilized microbial agent particles.

[0020] In another preferred embodiment, in step (1), the *Kutracheid* is cultured in LB liquid medium and the culture solution is collected to obtain bacterial solution I.

[0021] In another preferred embodiment, in step (1): the strain is inoculated into LB liquid medium at an inoculum of 2%-6%, and cultured at 150 r / min, 30℃ in the dark with shaking until the logarithmic growth phase is reached. The bacterial solution is centrifuged at 7000-8000 r / min for 3-5 min, the medium is poured out, sterile water is added, and the mixture is vortexed for 2-5 min. The OD of the bacterial solution is adjusted with sterile water. 600 The concentration is 1-1.3, and it is used to prepare a microbial agent.

[0022] In another preferred embodiment, the polyvinyl alcohol in the mixture II of step (2) has a molecular weight of 72,000-78,000 Da, a viscosity of 12.0-16.0 mPa·s, and a content of 5.0-10.0 wt.%, the sodium alginate content is 1.0-5.0 wt.%, and the mushroom residue biochar has a mesh size of 20-80 mesh.

[0023] In another preferred embodiment, the method for preparing mushroom residue biochar involves rinsing the mushroom residue 2-3 times with ultrapure water, drying it in a 65℃ oven for 24-36 hours, pulverizing it with a pulverizer, and passing it through an 80-mesh sieve. The sieved mushroom residue powder is weighed, placed in a quartz boat, and then placed in a tube furnace for pyrolysis at 500℃ for 2 hours under a nitrogen atmosphere to obtain mushroom residue biochar.

[0024] In another preferred embodiment, the polyvinyl alcohol and sodium alginate are dissolved in water bath shakers at 90°C and 60°C, respectively. After dissolution, the two are mixed together, and mushroom residue biochar is added at the same time. After the mixture is evenly mixed, the shaking is stopped and the mixture is cooled to room temperature.

[0025] In another preferred embodiment, step (3) involves mixing bacterial solution I and mixed solution II in a volume ratio of 1:1-3 to prepare mixed solution III.

[0026] In another preferred embodiment, in step (4), the mixture III from step (3) is drawn dropwise into a 3.0-5.0 wt.% calcium chloride solution using a syringe, crosslinked and fixed for 24-72 h, and finally washed 1-3 times with sterile water to obtain the immobilized microbial agent.

[0027] In a fifth aspect, the present invention provides a method for treating antibiotic-containing wastewater, the method comprising the steps of: treating antibiotic-containing wastewater with bacterial agent immobilized particles prepared by the method described in the fourth aspect of the present invention, thereby achieving the degradation of antibiotics in the wastewater; preferably, the antibiotic is tylosin.

[0028] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0029] Figure 1 This is a colony morphology diagram of the wild-type strain YJT4. Figure 2 The colony morphology of the YJT4 mutant strain (45) is shown. The growth rate of the mutant strain is much better than that of the wild-type strain. Figure 3 A visual representation of the immobilized bacterial strain particles; Figure 4The effect of immobilized strain particles on the degradation of tylosin. Detailed Implementation

[0030] The inventors investigated and analyzed TYL-resistant bacteria in aquatic environments of aquaculture, conducted statistical analysis of TYL-resistant bacteria in aquatic soil and water environments of aquaculture, screened TYL-degrading bacteria, and performed 16S rDNA sequencing analysis on the isolated strains. The screened strain YJT4 showed good degradation activity (see literature: Research on Biodegradation and Application of Tylosin in Aquatic Environments, Master's Thesis, Shandong Jianzhu University). However, in antibiotic wastewater treatment, free-floating degrading bacteria exhibit significant instability. Immobilizing highly efficient antibiotic-degrading bacteria through an encapsulation process can improve the activity retention capacity and resistance to environmental interference of the degrading bacteria. This invention obtained strains with excellent colonization ability and extremely high degradation activity through mutagenesis and screening, which are particularly suitable for preparing immobilized bacterial agents. Based on this, this invention was completed.

[0031] Before describing this invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. As used herein, when referring to a specifically enumerated numerical value, the term “about” means that the value can vary from the enumerated value by no more than 1%. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0033] While any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention, preferred methods and materials are exemplified herein.

[0034] The strain of this invention was obtained as follows: The original starting strain YJT4 was obtained from wastewater surrounding a chicken farm in Laiwu, Shandong Province, through enrichment, separation, and purification. Related work can be found in the literature: "Research on Biodegradation and Application of Tylosin in Aquatic Environments," Master's Thesis, Shandong Jianzhu University. Based on this original starting strain, mutagenesis screening was performed to obtain the *Kutracheidella* strain of this invention. Kurthia. (sp.) strain YJT4 mutant strain.

[0035] strain preservation The above-mentioned strain of the present invention was deposited on October 21, 2025, at the Guangdong Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 67149 and name: Kurthia sp.YJT4.

[0036] The original YJT4 strain exhibits a typical round structure, with its colonies showing a distinct raised shape and a pale yellow color. The YJT4 mutant strain differs from the wild type in its cell characteristics, exhibiting faster colony growth and a slightly darker color.

[0037] In a preferred embodiment of the present invention, the present invention provides a method for preparing immobilized particles of microbial strains that degrade antibiotics.

[0038] The method for preparing the above-mentioned strain immobilized particles includes the following steps: (1) The bacterial culture solution I was obtained by culturing the antibiotic-degrading strain; (2) Dissolve the sodium alginate and polyvinyl alcohol and mix them with mushroom residue biochar to obtain mixture II; (3) Mix bacterial solution I with mixed solution II to prepare mixed solution III; (4) Add the mixture III dropwise to the pre-prepared calcium chloride solution for cross-linking and fixation to obtain the immobilized particles.

[0039] The preparation method of the antibiotic-degrading bacterial agent includes conventionally activating and culturing YJT4 in LB liquid medium and then collecting the culture medium. The LB liquid culture medium is prepared by adding 10.0 g tryptone, 5.0 g yeast extract, 5.0 g sodium chloride, 4.0 g glucose, 1.0 mL HEPES buffer, and 1.0 g magnesium chloride to distilled water, making up to 1 L, adjusting the pH to 7.0, and sterilizing at 121℃ for 20 min. The LB liquid culture medium of the present invention adds glucose, HEPES buffer, magnesium chloride and other substances to the original LB culture medium. Compared with the original LB culture medium, it can promote the growth of strains, increase the growth rate of bacterial solution, and obtain high-density bacterial solution. The bacterial suspension is prepared as follows: The bacterial strain is inoculated into LB liquid medium at an inoculum volume of 2%-6%, and cultured at 150 rpm and 15°C in the dark with shaking until the logarithmic growth phase. The suspension is then centrifuged at 7000-8000 rpm for 3-5 minutes, the supernatant is discarded, sterile water is added, and the suspension is vortexed for 2-5 minutes. The OD value of the suspension is adjusted with sterile water. 600 The concentration is 1-1.3, and it is used to prepare a microbial agent.

[0040] In step (2), the polyvinyl alcohol in mixture II has a molecular weight of 72,000-78,000 Da, a viscosity of 12.0-16.0 mPa·s, and a content of 5.0-10.0 wt.%, while the sodium alginate content is 1.0-5.0 wt.%. The mushroom residue biochar can have a mesh size of 20-80 mesh. The method for preparing mushroom residue biochar is characterized by rinsing the mushroom residue 2-3 times with ultrapure water, drying it in a 65℃ oven for 24-36 h, pulverizing it, and passing it through an 80-mesh sieve. The sieved mushroom residue powder is weighed, placed in a quartz boat, and then placed in a tube furnace for pyrolysis at 500℃ for 2 h under a nitrogen atmosphere to obtain mushroom residue biochar. The polyvinyl alcohol and sodium alginate were dissolved in water bath shakers at 90°C and 60°C, respectively. After dissolution, the two were mixed together, and mushroom residue biochar was added at the same time. After the mixture was evenly mixed, the shaking was stopped and the mixture was cooled to room temperature.

[0041] In step (3), bacterial solution I and mixed solution II are mixed in a volume ratio of 1:(1-3) to prepare mixed solution III.

[0042] The specific steps in step (4) are as follows: In a clean bench, use a syringe to draw up the mixture III from step (3) drop by drop into a 3.0-5.0 wt.% calcium chloride solution, crosslink and fix for 24-72 h, and finally wash with sterile water 1-3 times to obtain the immobilized particles.

[0043] In a preferred embodiment of the present invention, the present invention also provides the application of an antibiotic-degrading strain immobilized particle as described above in the degradation of antibiotics.

[0044] This immobilization and encapsulation technology offers significant advantages. The YJT4 mutant strain exhibits strong degradation capabilities, particularly for antibiotics (especially tylosin). The encapsulation material creates a stable microenvironment for the strain, protecting it from adverse external conditions and maintaining its high activity level. This allows for continuous and efficient decomposition of antibiotics and other pollutants. Furthermore, the encapsulation process stabilizes the immobilized bacterial particles, ensuring a sustained and stable treatment process. Notably, it achieves higher antibiotic removal efficiency than traditional methods, providing superior purification of antibiotics in water.

[0045] The main advantages of this invention are: (1) The YJT4 mutant strain showed significantly improved colonization ability compared with the wild type; (2) Immobilized particles prepared from the YJT4 mutant strain showed significantly improved efficiency in degrading antibiotics.

[0046] (3) The YJT4 mutant strain has strong resistance. When using it for sewage treatment, there is no need to pre-treat the sewage. The effect is stable when it is reused multiple times.

[0047] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in *Molecular Cloning: A Laboratory Manual* by Sambrook J. et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, all experimental materials and reagents used in the following embodiments are commercially available.

[0048] Example 1. Mutagenesis and screening of strains The inventors discovered that the original YJT4 strain had poor colonization ability on biochar, making it difficult to prepare effective immobilized particles. Therefore, the inventors conducted mutagenesis and selection on the original YJT4 strain.

[0049] The culture media mainly include liquid LB (Luria-Bertani) medium and solid LB medium, namely LA (Luria-Bertani Agar) agar medium.

[0050] The preparation method is as follows: LB liquid culture medium is prepared by adding 10.0 g tryptone, 5.0 g yeast extract, 5.0 g sodium chloride, 4.0 g glucose, 1.0 mL HEPES buffer, and 1.0 g magnesium chloride to distilled water, bringing the volume to 1 L, adjusting the pH to 7.0, and sterilizing at 121℃ for 20 min.

[0051] Preparation of solid LB medium: Add 15.0 g / 1000 ml of agar powder to LB liquid medium and sterilize at 121℃ for 20 min.

[0052] Preparation of bacterial suspension Pick a single colony of the wild-type YJT4 strain from a fresh plate and inoculate it into 5 mL of liquid culture medium.

[0053] Incubate at 28°C with shaking until OD 600The concentration is 0.6. Take an appropriate amount of bacterial suspension, centrifuge at 5000-8000 rpm for 5-10 minutes at 4℃, and collect the bacterial cells. Wash the bacterial cells twice with sterile physiological saline, resuspend the bacterial cells with an appropriate amount of physiological saline, and dilute appropriately to make the bacterial suspension concentration approximately 10. 8 CFU / mL.

[0054] Add 5 mL of bacterial suspension to a sterile culture dish. Place the magnetic stir bar in the dish and place it on a magnetic stirrer (turn on low speed to ensure uniform irradiation). Turn on the UV lamp (wavelength 254 nm) and preheat for 20 minutes. Remove the dish lid and immediately start timing the irradiation. Set multiple irradiation time gradients: 0 s (control), 15 s, 30 s, 45 s, 60 s, 90 s, and 120 s.

[0055] Dilution and coating: After sampling at each time point, immediately perform serial dilutions with physiological saline (10-10). -1 Up to 10 -3 Take 100 μL and spread it on a solid plate.

[0056] Cultivation and Counting: Wrap the coated plates with black paper or aluminum foil and incubate in the dark for 24-48 hours. Count the colonies and calculate the survival rate at each time point. Based on preliminary experimental results, the lethality rate of 60 seconds of UV irradiation is approximately 80%. Subsequent formal mutagenesis will be performed using 60 seconds of UV irradiation.

[0057] After formal mutagenesis, the plates (containing 150 mg / L tylosin) were wrapped in aluminum foil and incubated at 28°C in the dark until colonies grew. Based on the colony growth, single colonies with excellent growth were selected for secondary screening.

[0058] Preliminary degradation activity tests were conducted on the 62 single colonies obtained from the secondary screening. In a culture medium (pH 6.0) containing 150 mg / L tylosin, at an inoculum concentration of 2% and a temperature of 28°C, the degradation rate of tylosin by each mutant strain was tested after 12 h of culture.

[0059] Table 1. Screening for Tylosin Degradation Rate

[0060] Seventeen strains with a degradation rate of over 50% were selected and passaged six times consecutively on non-selective plates. The degradation activity was assessed by inoculating each generation into a medium containing 150 mg / L tylosin.

[0061] After six generations, eight YJT4 mutant strains were obtained that could still maintain a degradation rate of over 50%, indicating that they have good genetic stability.

[0062] Using the wild-type YJT4 strain as a control, the colonization ability of each YJT4 mutant strain on biochar was tested.

[0063] Each strain was inoculated into LB liquid medium and cultured at 28℃ and 220 rpm for 36 h to obtain the fermentation broth. The fermentation broth was then prepared to a concentration of 1.0 × 10⁻⁶. 9 CFU / mL bacterial suspension.

[0064] The mushroom residue biochar was sterilized at high temperature to produce a sterile carrier.

[0065] Add 5% by weight of sterile mushroom residue biochar carrier to a culture medium (pH 6.0) containing 150 mg / L tylosin, and incubate at 28°C for 48 h with 5% inoculum.

[0066] After culturing, the biochar carriers were filtered out and rinsed at least five times with sterile saline until the saline was clear and free of obvious turbidity. Each biochar carrier was then centrifuged at 4000 rpm for 15 minutes to remove moisture. After grinding each biochar carrier, the number of viable bacteria per gram of biochar carrier was determined by colony counting. Each strain was tested in triplicate.

[0067] The test results are as follows: Table 2. Colonization capacity determination

[0068] The test results showed that the YJT4 wild-type strain had poor colonization ability on biochar, while the YJT4 mutant strains No. 45, No. 9, and No. 20 had significantly improved colonization ability compared with the wild-type strain.

[0069] Example 2. Preparation of strain immobilization particles This embodiment provides an immobilized particle of antibiotic-degrading strain, which is prepared from antibiotic-degrading strain, polyvinyl alcohol, sodium alginate, and mushroom residue biochar.

[0070] The method for preparing antibiotic-degrading strain immobilized particles is as follows: (1) The strain that degrades antibiotics is cultured and prepared into a bacterial agent to obtain bacterial solution I; Each strain was activated and cultured separately in LB liquid medium, and the culture solution was collected. The LB liquid culture medium is prepared as follows: 10.0 g tryptone, 5.0 g yeast extract, 5.0 g sodium chloride, 4.0 g glucose, 1.0 mL HEPES buffer, and 1.0 g magnesium chloride are added to distilled water, and the volume is adjusted to 1 L. The pH is adjusted to 7.0, and the medium is sterilized at 121℃ for 20 min. The LB liquid medium in this embodiment adds glucose, HEPES buffer, magnesium chloride and other substances to the original LB medium. Compared with the original LB medium, it can promote the growth of strains, increase the growth rate of bacterial solution, and obtain high-density bacterial solution. The bacterial suspension was prepared as follows: The bacterial strain was inoculated at a 5% inoculum into 100 mL of liquid culture medium and cultured at 150 rpm and 30°C in the dark with shaking until the logarithmic growth phase. The suspension was then centrifuged at 8000 rpm for 5 min, the supernatant was discarded, sterile water was added, and the suspension was vortexed for 3 min. The OD value of the suspension was adjusted with sterile water. 600 The concentration is 1.5, which is used to prepare a microbial agent.

[0071] (2) Dissolve the sodium alginate and polyvinyl alcohol and mix them with mushroom residue biochar to obtain mixture II; Specifically, the sodium alginate content is 1.0-5.0 wt.%, the polyvinyl alcohol has a molecular weight of 72,000-78,000 Da, a viscosity of 12.0-16.0 mPa·s, and a content of 5.0-10.0 wt.%, and the mushroom residue biochar can have a mesh size of 20-80 mesh. The method for preparing mushroom residue biochar is as follows: The mushroom residue is washed 2-3 times with ultrapure water, dried in a 65℃ oven for 24-36 hours, pulverized, and passed through an 80-mesh sieve. The sieved mushroom residue powder is weighed, placed in a quartz boat, and then placed in a tube furnace for pyrolysis at 500℃ for 2 hours under a nitrogen atmosphere to obtain mushroom residue biochar. The preparation method of Mixture II is as follows: A certain amount of sodium alginate and polyvinyl alcohol are dissolved separately in water bath shakers at 60℃ and 90℃, respectively. After dissolution, the two are mixed in a water bath shaker at 60℃ to continue dissolving. Simultaneously, biochar from mushroom residue is added. Dissolution is stopped once the solution is uniformly mixed, and the mixture is cooled to room temperature to obtain Mixture II. In this embodiment, the amounts added are: 3% (w / v) sodium alginate, 6% (w / v) polyvinyl alcohol, and 1% (w / v) mushroom residue biochar.

[0072] (3) Mix bacterial solution I with mixed solution II to prepare mixed solution III; Mixture I and Mixture II in a 1:1 volume ratio to prepare Mixture III; (4) Add the mixture III dropwise to the pre-prepared calcium chloride solution for cross-linking and fixation to obtain immobilized particles.

[0073] The calcium chloride solution is prepared by mixing 4 g of calcium chloride with 100 mL of ultrapure water and stirring until completely dissolved to obtain a calcium chloride solution with a concentration of 4% (w / v). The specific laboratory pilot steps in step (4) are as follows: In a clean bench, use a syringe to draw up the mixture III from step (3) drop by drop into an excess of calcium chloride solution, crosslink and fix for 48 h, and finally wash with sterile water 3 times to obtain the immobilized particles. An excess of calcium chloride solution is used to ensure that all added mixture III can be cross-linked and fixed into particles in the calcium chloride solution. For example, the calcium chloride solution can be more than 5 times the amount of the mixture in step (3).

[0074] The prepared immobilized particles, such as Figure 3 As shown.

[0075] Performance testing of immobilized particles: The performance of the prepared immobilized particles was tested. The test methods are shown below, and the results of each parameter measurement are shown in Table 1.

[0076] Diameter measurement: The diameter of the prepared immobilized particles was measured using vernier calipers, and the average value was calculated for 30 particles.

[0077] Mechanical strength test: Prepare inorganic salt basal culture medium, and take 5 mL of phosphate buffer (KH2PO4) to test the mechanical strength. 8.5g·L -1 K2HPO4·H2O 21.75 g·L -1 Na₂HPO₄·12H₂O 33.4 g·L -1 NH4Cl 5.0 g·L -1 ); 3 mL MgSO4 aqueous solution (22.5 g·L⁻¹) -1 ); 1 mL CaCl2 aqueous solution (36.4 g·L⁻¹) -1 ); 1 mL FeCl3 aqueous solution (0.25 g·L⁻¹) -1 ); 1 mL of trace elements (MnSO4·H2O 39.9 mg·L) -1 ZnSO4·H2O 42.8 mg·L -1 (NH4)6Mo7O 24 ·4H2O 34.7 mg·L -1 The volume was adjusted to 1 L, and the pH of the culture medium was adjusted to 6.8-7.0. All glassware and solutions used in this experiment were autoclaved at 121℃ for 30 min before use. Under aseptic conditions, the immobilized particles were immersed in the sterilized and cooled culture medium, sealed, and placed on a shaker for shaking. The degree of particle breakage was observed after 10 days.

[0078] Expansion coefficient detection: The diameter of the immobilized particles was measured after shaking in an inorganic salt culture medium for 10 days. The ratio of the measured average diameter to the original particle diameter is the expansion coefficient.

[0079] Mass transfer performance testing: Prepare a 2% methylene blue ethanol solution (freshly prepared and used immediately). Add 30 drops to 500 mL of distilled water, mix thoroughly, and then use this solution to determine the mass transfer performance of the immobilized particles. Add 50 immobilized particles to this solution and shake on a shaker. After 24 h, measure the absorbance of the methylene blue solution with the particles and the original methylene blue solution at a wavelength of 665 nm. Compare the measured absorbance values; the change in absorbance value indirectly reflects the mass transfer performance of the immobilized particles.

[0080] Cell embedding rate detection: The embedding rate was estimated using colony counting. Before embedding, the bacterial suspension was diluted and spread on LB agar medium for colony counting. After embedding, 30 immobilized particles were taken, washed with physiological saline to release the unembedded cells, diluted, spread, and counted again. The colony counts were compared before and after embedding to calculate the cell embedding rate.

[0081] Table 3. Performance parameters of immobilized particles

[0082] In this embodiment, the composition of the immobilized particles was also studied. The study found that the prepared immobilized particles, using polyvinyl alcohol as the main raw material, have high viscosity and are easy to mold. Combining them with sodium alginate can significantly improve the molding effect. The mushroom residue biochar has large pores, providing more attachment area for microorganisms, increasing the specific surface area, and improving the biocompatibility and hydrophilicity of the biological filler surface. This leads to increased microbial proliferation rate and adhesion, and also promotes microbial activity.

[0083] Example 3. Application of immobilized particles in the treatment of antibiotics The immobilized particles of this invention were added to the tylosin-treated wastewater at a dosage of 3-10%. This embodiment uses a dosage of 5% for the experiment. (1) Set up a sample of wastewater to be treated without the addition of tylosin as a blank control group 1; (2) 10 g of sterile immobilized material particles prepared by replacing the bacterial agent with sterile water in the same proportion were added to 100 mL of tylosin-treated wastewater (200 mg / L) as control group two; (3) Directly inoculate strain YJT4 wild type, with an inoculation amount of 5%, that is, inoculate 5 mL of the above bacterial agent into 100 mL of tylosin-treated wastewater (200 mg / L) as control group three; (4) 10 g of immobilized particles were added to 100 mL of tylosin-treated wastewater (200 mg / L) as the experimental group.

[0084] It should be noted that the control group 3 was inoculated with a bacterial suspension of the wild type YJT4 strain, that is, it was added in the form of a bacterial suspension, while the experimental group was inoculated with immobilized particles, which were added in the form of solid particles.

[0085] In this embodiment, the immobilized particles were prepared from 50% bacterial suspension + 50% mixture II. To ensure that the added free bacteria and the strains in the immobilized particles were added at the same mass as possible, 5 g of free bacteria were added in control group three, and 10 g of immobilized particles were added, with 5 g of the 10 g particles being the weight of the microbial agent.

[0086] The control groups 1, 2, 3 and each experimental group were placed in a constant temperature incubator at 30℃ and cultured for 0, 3, 5 and 7 days. The concentration of residual tylosin in control groups 1, 2, 3 and each experimental group was measured by high performance liquid chromatography (Agilent) to obtain the degradation effect.

[0087] Specific measurement method: At 0h, 12h, 24h, 36h, and 48h, 10 mL of water samples were collected from each of the control groups (1, 2, and 3) and the experimental groups using 15 mL centrifuge tubes. The samples were centrifuged at 8000 r / min for 5 min, repeated twice. 2 mL of the supernatant was extracted, filtered through a 0.22 μm pinhole filter, and transferred to a 2 mL HPLC vial for HPLC analysis.

[0088] Tylosin HPLC conditions: An Agilent C18 column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase was acetonitrile:methanol:0.025 mol / L ammonium dihydrogen phosphate = 30:25:45. The column temperature was set at 30℃, the flow rate at 0.6 mL / min, the injection volume at 15 μL, and the detection wavelength at 290 nm.

[0089] Results analysis: Results of immobilized particles on the treatment of tylosin-containing wastewater: Figure 4 As shown.

[0090] The results showed that in the blank control group without any inoculation, tylosin underwent natural degradation.

[0091] In control group two, which incorporated sterile immobilization material, the degradation rate was higher than that in control group one because biochar itself has certain adsorption properties for antibiotics.

[0092] In the control group 3, which was inoculated with free YJT4 wild-type bacterial suspension, the degradation rate of tylosin was accelerated.

[0093] Among the experimental groups that incorporated the immobilized particles of this invention, the tylosin degradation rate of strain 45 particles was the fastest, with an antibiotic degradation rate of over 90% within 24 hours, which was more than double that of wild-type particles.

[0094] Each immobilized bacterial agent particle was used three more times, with a degradation time of 24 hours each time. The degradation effect of tylosin after 24 hours of degradation was tested as follows: Table 4. Repeated Degradation Test

[0095] The immobilized particles prepared from YJT4 mutant strain 45 exhibited the highest degradation efficiency, and the degradation efficiency increased slightly after three reuses, indicating that the strong colonization ability of YJT4 mutant strain 45 is beneficial for maintaining a good degradation level of the immobilized particles under repeated use.

[0096] The YJT4 mutant strain 45 was deposited at the Guangdong Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 67149 and name: Kurthia sp.YJT4.

[0097] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A type of Kurtella ( Kurthia. sp.), characterized in that, The Kurtella bacteria mentioned are: Strain 1: Kurtella ( Kurthia. sp.) YJT4, deposited at Guangdong Microbial Culture Collection Center (GDMCC), accession number GDMCC No:67149; or Strain 2: Strain 2 is a derivative strain of strain 1; for example, strain 1 obtained through gene mutation or genetic engineering.

2. A microbial inoculant, characterized in that, The microbial agent includes the Kuterella as described in claim 1.

3. The microbial agent as described in claim 2, characterized in that, The microbial agent can be in liquid or solid form.

4. The microbial agent as described in claim 3, characterized in that, The microbial agent is an immobilized microbial agent particle.

5. The use of Kuterella as described in claim 1 in the degradation of antibiotics; preferably, the antibiotic is tylosin.

6. A method for preparing immobilized microbial agent particles, characterized in that, The method includes the following steps: (a) Providing the Kuterella as described in claim 1; (b) The *Kutella* bacteria and the immobilization substrate are mixed and cross-linked to obtain the immobilized bacterial agent particles.

7. The method as described in claim 6, characterized in that, In step (b), the immobilization substrate includes polyvinyl alcohol and sodium alginate.

8. The method as described in claim 7, characterized in that, The immobilized substrate also includes mushroom residue biochar.

9. The method as described in claim 6, characterized in that, The method includes the following steps: (1) Liquid fermentation of the *Kouterella* to obtain bacterial solution I; (2) Dissolve the sodium alginate and polyvinyl alcohol and mix them with mushroom residue biochar to obtain mixture II; (3) Mix bacterial solution I with mixed solution II to prepare mixed solution III; (4) Add the mixture III dropwise into the calcium chloride solution for cross-linking and fixation to obtain the immobilized microbial agent particles.

10. A method for treating antibiotic-containing wastewater, characterized in that, The method includes the steps of: treating antibiotic-containing wastewater with bacterial agent immobilized particles prepared by the method of claim 6, thereby achieving the degradation of antibiotics in the wastewater; preferably, the antibiotic is tylosin.

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