Strain for efficiently degrading doxycycline in water body and application thereof

Abstract

The invention discloses a strain for efficiently degrading doxycycline in a water body and application of the strain. The Sphingobacterium hotanense DX14 screened by the invention can be used for efficiently degrading the doxycycline in the water body, so that the strain has the potential of being developed into a microbial agent for degrading the doxycycline in the water body, and has a good application prospect for solving the problems that the doxycycline in the antibiotic-containing water body exceeds the standard and the like. Meanwhile, the invention further provides an environment-friendly application method of the sphingobacterium mesophilum DX14, the sphingobacterium mesophilum DX14 is recovered, and genome DNA of the sphingobacterium mesophilum DX14 is completely degraded, so that the doxycycline resistance gene carried by the sphingobacterium mesophilum DX14 is effectively prevented from being diffused in a water body, and the yield of the doxycycline resistance gene is improved. And the effect of comprehensively protecting the environment is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of wastewater treatment of new pollutants, and particularly relates to a bacterial strain capable of efficiently degrading doxycycline in water and application thereof. Background Art

[0002] Doxycycline (DC, doxycycline), also known as doxycycline and deoxycycline, belongs to the second generation of tetracycline antibiotics. Its molecular formula is C 22 H 24 N2O8, with a relative molecular mass of 444.44. Tetracycline antibiotics are the second most produced and used antibiotics worldwide. Compared to first-generation tetracyclines, doxycycline has higher lipid solubility, improved bioavailability, a shorter half-life, stronger antibacterial efficacy, and lower toxicity. As a result, doxycycline has become a commonly used tetracycline antibiotic in Europe and China in recent years and has been widely used in livestock and human medicine. After entering the body, doxycycline, while a portion remains in tissues, 30-90% is excreted unchanged or as metabolites in feces and urine.

[0003] While antibiotics have made significant contributions to the prevention and treatment of infectious diseases in humans and animals, their misuse and overuse can also lead to challenging environmental problems. For example, residual antibiotics and some of their metabolites can migrate and spread within aquatic ecosystems, driving the spread of antibiotic-resistant genes and posing a significant and potential threat to ecological stability and human health. Doxycycline residues have been detected in a variety of environmental media, including wastewater treatment plants, livestock and poultry manure, soil, recycled water and groundwater, rivers, and streams and sediments.

[0004] Antibiotics and antibiotic resistance genes have been classified as emerging pollutants, and their management has become a key task in current ecological and environmental protection. Using microorganisms to degrade antibiotic residues in the environment is low-cost and environmentally friendly compared to other physical or chemical methods, making it a promising remediation method. However, it is worth noting that when microorganisms degrade their target antibiotics, they also carry the antibiotic-degrading genes (antibiotic resistance genes) that they carry, which in turn poses the ecological risk of contamination by drug-resistant bacteria and antibiotic resistance genes. Therefore, to address this issue, restrictions on the use of antibiotic-degrading microbial agents can be implemented to reduce their ecological risks in the environment. Summary of the Invention

[0005] The purpose of the present invention is to provide a strain that can efficiently degrade doxycycline in water and an environmentally friendly method of using the strain, which not only completes the degradation of new antibiotic pollutants in water, but also limits the spread of drug-resistant bacteria and the antibiotic resistance genes they carry, thereby comprehensively protecting the ecological environment of the water body.

[0006] The inventors of the present invention obtained a Gram-negative bacterium capable of degrading doxycycline in water by repeated screening from sludge of a sewage treatment plant in Suzhou City, Jiangsu Province.

[0007] This strain was selected for morphological observation, biological characteristics study, taxonomic status determination, and growth medium optimization. 16S rDNA identification confirmed that this strain was Sphingobacterium hotanense The similarity reached 100%. Therefore, combined with the morphological characteristics, Gram staining, physiological and biochemical reactions and 16S rDNA sequence analysis of strain DX14, strain DX14 can be identified as mesophilic sphingobacterium ( Sphingobacterium hotanense ) a new species, named Mesophilic Sphingobacterium ( Sphingobacterium hotanense )DX14 (CGMCC NO.33265).

[0008] The present invention provides a mesophilic Sphingobacterium DX14, which has been deposited in the China General Microorganism Culture Collection (CGMCC) on January 2, 2025, with a deposit number of CGMCC NO.33265, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The sequence of the 16S rDNA gene fragment of the mesophilic Sphingobacterium DX14 is shown in SEQ ID NO: 1.

[0010] The present invention provides a microbial agent containing the mesophilic Sphingobacterium DX14.

[0011] Furthermore, the microbial agent contains living cells of mesophilic Sphingobacterium DX14 bacteria, dry cells of mesophilic Sphingobacterium DX14 obtained by freeze-drying, immobilized mesophilic Sphingobacterium DX14 cells, liquid agent of mesophilic Sphingobacterium DX14, solid agent of mesophilic Sphingobacterium DX14, or mesophilic Sphingobacterium DX14 strains in any other form.

[0012] The present invention also provides the use of the mesophilic Sphingobacillus DX14 or the microbial agent in degrading novel pollutant antibiotics in water bodies.

[0013] Furthermore, the application is for degrading doxycycline in water.

[0014] The present invention further provides an environmentally friendly application method of the mesophilic Sphingobacillus DX14 or the microbial agent, comprising the following steps: Step 1) preparing a DX14 active seed bacterial solution of the mesophilic Sphingobacterium DX14, and detecting the bacterial content and activity in the seed bacterial solution; Step 2) adding the prepared DX14 active seed bacterial solution to the wastewater containing doxycycline at a certain inoculum size, and culturing the solution in the dark for several days under certain temperature and rotation speed conditions; Step 3) centrifuging the treated wastewater at a certain speed for several minutes, and collecting the reacted mesophilic Sphingobacterium DX14 bacteria; Step 4) The collected mesophilic Sphingobacterium DX14 bacteria are sterilized by high pressure for a period of time, and then DNase I is added for a certain period of time to completely degrade the DX14 genome, thereby preventing the spread of the doxycycline resistance gene carried by the mesophilic Sphingobacterium DX14 in the water body.

[0015] Furthermore, in step 1, the DX14 active seed bacterial liquid is quantified.

[0016] Furthermore, in step 2, the prepared DX14 active seed bacterial liquid was added into the water containing doxycycline at an inoculum size of 1%, and cultured at 30°C, 150 rpm, under constant temperature, shaking, and in the dark for 6 days.

[0017] Furthermore, in step 3, the treated wastewater was centrifuged at 8000 rpm for 10 minutes, and the mesophilic Sphingobacterium DX14 bacteria were collected.

[0018] Furthermore, in step 4, the collected reacted mesophilic Sphingobacterium DX14 cells are sterilized by high pressure at 121° C. for 20 minutes, and then a sufficient amount of DNase I is added and allowed to react for a period of time to ensure that the genomic DNA of the mesophilic Sphingobacterium DX14 cells is fully degraded.

[0019] The beneficial effects of the present invention are: The mesophilic sphingobacterium ( Sphingobacterium hotanense )DX14 can efficiently degrade doxycycline in water bodies. Therefore, this strain has the potential to be developed as a microbial agent for degrading doxycycline in water bodies, and has good application prospects in solving problems such as excessive doxycycline in water bodies containing antibiotics.

[0020] At the same time, the present invention also provides an environmentally friendly application method of the mesophilic sphingobacterium DX14. The method not only recovers the mesophilic sphingobacterium DX14, but also completely degrades the genomic DNA of the mesophilic sphingobacterium DX14, thereby effectively preventing the spread of the doxycycline resistance gene carried by the mesophilic sphingobacterium DX14 in the water body, thereby achieving the effect of all-round environmental protection.

[0021] Biomaterial Deposit The mesophilic sphingobacterium of the present invention ( Sphingobacterium hotanense )DX14, deposited in China General Microbiological Culture Collection Center on January 2, 2025, and named taxonomically Sphingobacterium hotanense DX14, the deposit number is CGMCC NO.33265, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0022] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the invention and to implement it according to the contents of the description, the following preferred embodiments of the present invention are described in detail with reference to the accompanying drawings. The specific implementation methods of the present invention are given in detail by the following embodiments and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 This is a colony morphology diagram of the thermophilic Sphingobacterium DX14 screened by the present invention after culture; Figure 2 This is a Gram-stained colony image of the mesophilic Sphingobacterium DX14 cultured in the present invention; Figure 3 The diagram shows the results of 16S rDNA gene phylogeny analysis of the mesophilic Sphingobacterium DX14 screened in the present invention; Figure 4 This is a growth curve of the mesophilic Sphingobacterium DX14 screened by the present invention; Figure 5 This is a graph showing the doxycycline degradation performance test results of the mesophilic Sphingobacterium DX14 screened by the present invention; Figure 6 A graph showing the test results of the present invention on the degradation effect of dissolved oxygen on doxycycline by the screened mesophilic Sphingobacterium DX14; Figure 7 This is a graph showing the test results of the effect of temperature on the degradation of doxycycline by the screened mesophilic Sphingobacterium DX14 conducted in the present invention; Figure 8This is a graph showing the experimental results of the degradation of doxycycline in simulated aquaculture tail water by the mesophilic Sphingobacterium DX14 screened in the present invention; Figure 9 This is a diagram showing the DNA agarose gel electrophoresis verification results of the mesophilic Sphingobacterium DX14 screened in the present invention after treatment with doxycycline in simulated aquaculture tail water and complete genome degradation. DETAILED DESCRIPTION

[0024] The following will be described in detail with reference to the accompanying drawings to better understand the purpose, features and advantages of the invention. It should be understood that the embodiments shown in the accompanying drawings are not intended to limit the scope of the invention, but are only intended to illustrate the essential spirit of the technical solution of the invention.

[0025] In the following description, for the purpose of illustrating the various disclosed embodiments, certain specific details are set forth in order to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the relevant art will recognize that the embodiments may be practiced without one or more of these specific details. In other cases, well-known devices, structures, and techniques associated with this application may not be shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0026] Unless the context requires otherwise, throughout the specification and claims, the word "comprise" and variations such as "include" and "have" should be construed in an open, inclusive sense, that is, should be interpreted to mean "including, but not limited to."

[0027] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any manner in one or more embodiments.

[0028] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be noted that the term "or" is generally employed in its sense including "and / or" unless the context clearly dictates otherwise.

[0029] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0030] The inventors of the present invention obtained a Gram-negative bacterium capable of degrading doxycycline in water by repeated screening from sludge of a sewage treatment plant in Suzhou City, Jiangsu Province.

[0031] 1. Isolation and purification of strains: 0.1 L of sludge water collected from a sewage treatment plant in Suzhou, Jiangsu Province was added to a 0.5 L conical flask wrapped in tin foil (containing 0.2 L of 5 mg·L -1 The culture medium was prepared by adding doxycycline hydrochloride enrichment medium (the main components of the enrichment medium are ( / L): sodium acetate 10 g, NH4Cl 1.5 g, K2HPO4 1.5 g, KH2PO4 0.5 g, MgSO4·7H2O 0.2 g, NaCl 0.5 g, trace element solution 1 mL, pH = 7.0-7.5). After sealing the conical flask with sealing film, the conical flask was placed in a shaking incubator at 30°C and 150 rpm in the dark and cultured for 4 days. Then, 0.1 L of the culture medium was transferred to a new 0.5 L conical flask wrapped in aluminum foil. The conical flask contained 0.2 L of freshly prepared enrichment medium and doxycycline hydrochloride at a concentration of 10 mg·L -1 The cells were inoculated with doxycycline hydrochloride and cultured under the same conditions. New inoculation and acclimation were performed in sequence, and the concentration of doxycycline hydrochloride was increased by 5 mg·L -1 After several inoculations, the concentration of doxycycline hydrochloride in the enrichment medium reached 60 mg·L -1 After about a month of culturing, repeated acclimatization and culturing, the sludge suspension is removed and graded diluted, streaked onto LB agar solid medium plates, and cultured in a constant temperature incubator (30°C). After colonies grow, use an inoculation loop on a sterile workbench to pick up a single colony and place it on a sterile LB agar solid plate for repeated streaking. After repeated streaking and purification, the single colony is saved for later use.

[0032] Degradation experiments were carried out in a sterile screening liquid culture medium, and the strain with the highest degradation efficiency for doxycycline hydrochloride was named DX14 and stored in a -80°C ultra-low temperature freezer.

[0033] 2. Identification of strains: (1) Morphological characteristics of strain DX14: The strain DX14 was spread on LB solid medium and cultured in a constant temperature incubator at 30°C for 48 h. Figure 1 Colony morphology shown.

[0034] Fix the fresh strain DX14 on a glass slide, add a drop of ammonium oxalate crystal violet stain, stain for 1 min and then wash with water, add a drop of iodine solution to stain for 1 min and then wash with water, decolorize with 95% ethanol for 30 s, wash with water and then dry with filter paper, add a drop of safranin stain to counterstain for 10-30 s, wash with water and dry and then examine under a microscope to obtain the following: Figure 2 Colony diagram shown.

[0035] See also Figure 1As shown, the strain DX14 colonies on LB solid medium are round, opaque, yellow, smooth, with a raised center and neat edges. Figure 2 As shown in the figure, Gram staining and microscopic observation showed that DX14 was a Gram-negative bacterium with a rod-shaped body.

[0036] (2) Physiological and biochemical characteristics of strain DX14: The biological characteristics of strain DX14 were then studied, and its physiological and biochemical characteristics are shown in Table 1: Table 1 Physiological and biochemical identification results of strain DX14

[0037] The results of physiological and biochemical identification showed that the strain DX14 was positive in arginine test, esculin test, sucrose test, maltose test, glucose test and oxidase test, and negative in Gram staining test, ornithine test, lysine test, citrate test, nitrate reduction test, urea test, indigo test, ONPG test, xylose test and hydrogen sulfide test.

[0038] (3) Identification of the 16S rDNA gene of strain DX14: The total genomic DNA of strain DX14 was extracted, and the 16S rDNA gene fragment of the strain was amplified using bacterial 16S rDNA universal primers and sequenced. The sequence obtained was compared with the homologous sequence in the NCBI system by BLAST, and the phylogenetic tree was constructed by the neighbor joining (NJ) method using MEGA7.0 software. The results of the phylogenetic analysis are shown in Figure 2. Figure 3 shown.

[0039] The universal primers for bacterial 16S rDNA are as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-ACGGCTACCTTGTTACGACTT-3'.

[0040] The sequence of the 16S rDNA gene fragment of strain DX14 is shown in SEQ ID NO: 1.

[0041] SEQ ID NO: 1:

[0042] See also Figure 3 As shown in the figure, the results of 16S rDNA gene phylogenetic analysis showed that strain DX14 was closely related to the thermophilic Sphingobacterium ( Sphingobacterium hotanense ) similarity reached 100%. Therefore, combined with the morphological characteristics, Gram staining, physiological and biochemical reactions and 16S rDNA sequence analysis of strain DX14, strain DX14 can be identified as mesophilic sphingobacterium ( Sphingobacterium hotanense ) and named it Mesophilic Sphingobacterium ( Sphingobacterium hotanense )DX14.

[0043] The mesophilic sphingobacterium of the present invention ( Sphingobacterium hotanense )DX14, deposited in China General Microbiological Culture Collection Center on January 2, 2025, and named taxonomically Sphingobacterium hotanense DX14, the deposit number is CGMCC NO.33265, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0044] 3. Growth curve drawing of strain DX14: Adjust the bacterial solution to OD 600 =0.8. Strain DX14 was inoculated into LB liquid medium at a 2% inoculum size and incubated at 30°C with a shaker at 150 rpm for 36 h. The bacterial concentration was measured every 4 h, and a growth curve of strain DX14 was plotted. Three replicates were set up, and the results were averaged.

[0045] See also Figure 4 As shown in the figure, the growth of strain DX14 can be seen. 0~4 h is the lag phase, when the bacteria adapt to the new environment and the strain grows slowly; 4~24 h is the logarithmic growth phase, when the bacteria proliferate rapidly; 24~36 h is the stationary phase, when the number of bacterial reproduction and death tends to be balanced, and the number of bacteria is in a relatively stable state.

[0046] 4. Degradation performance test of strain DX14 on doxycycline hydrochloride: (1) Preparation of seed solution: The strain DX14 was inoculated into LB liquid medium and cultured at 30°C and 150 rpm for 16-20 h. The cells were centrifuged at 8000 rpm for 10 min to obtain the cells. The cells were then washed twice with sterile water and resuspended in sterile water to form a bacterial solution. The OD value of the bacterial solution was adjusted with sterile water. 600 The value was 0.8, and the seed solution of strain DX14 was obtained.

[0047] (2) Degradation performance of strain DX14 on doxycycline hydrochloride: Transfer the seed solution at a 2% inoculum volume to 30 mL of degradation medium containing 60 mg / L doxycycline hydrochloride (the main components of the degradation medium are ( / L): tryptone 10 g, NH4Cl 1.5 g, K2HPO4 1.5 g, KH2PO4 0.5 g, MgSO4·7H2O 0.2 g, NaCl 0.5 g, trace element solution 1 mL, pH = 7.0-7.5). Set up an uninoculated blank control. Incubate at 30°C, 150 rpm in the dark for 24 h, and then take 1 mL of the culture medium as the test sample. Set up three replicates and average the results. Detect the residual concentration of doxycycline hydrochloride in the test sample and calculate its degradation rate. See [ 1 mL of the culture medium ] for details. Figure 5 As shown, the results showed that the degradation rate of doxycycline hydrochloride in the control group was less than 20%, while the degradation rate of doxycycline hydrochloride in the strain DX14 was more than 90%.

[0048] (3) Effect of dissolved oxygen on the degradation of doxycycline hydrochloride by degrading bacteria: The seed solution was inoculated at a 2% inoculum into 30 mL of degradation culture medium containing 60 mg / L doxycycline hydrochloride. A blank control (containing only doxycycline hydrochloride without degrading bacteria) was set up for each treatment group. The culture medium was then placed in a shaker at 50 rpm, 100 rpm, 150 rpm, and 200 rpm at 30°C in the dark for 24 h. 1 mL of the culture medium was taken as the test sample. Three replicates were set up, and the results were averaged. To determine the degradation rate of doxycycline hydrochloride in the test sample, refer to [ 15 ]. Figure 6 As shown in the figure, the results show that the degradation rate of doxycycline hydrochloride by strain DX14 gradually increases with the increase of rotation speed, which proves that within a certain range, the higher the dissolved oxygen, the better the degradation effect of doxycycline hydrochloride by strain DX14.

[0049] (4) Effect of temperature on degradation of doxycycline hydrochloride by degrading bacteria: The seed liquid was inoculated at a rate of 2% into 30 mL of degradation culture medium containing 60 mg / L doxycycline hydrochloride. A blank control (containing only doxycycline hydrochloride without degrading bacteria) was set up for each treatment group. The cells were then shaken at 150 rpm in a shaker at 15°C, 20°C, 25°C, 30°C, and 35°C in the dark for 24 h. 1 mL of the culture medium was taken as the test sample. Three replicates were set up, and the results were averaged to detect the degradation rate of doxycycline hydrochloride in the test samples. See [1]. Figure 7 As shown, the results showed that the degradation rate of doxycycline hydrochloride by strain DX14 first increased and then decreased with the increase of temperature. Therefore, it was proved that before the temperature did not exceed 30°C, the degradation effect of doxycycline hydrochloride by strain DX14 gradually increased with the increase of temperature. When the temperature exceeded 30°C, the degradation effect of doxycycline hydrochloride by strain DX14 gradually deteriorated with the increase of temperature.

[0050] The above experiments can fully prove that the mesophilic Sphingobacterium DX14 selected in the present invention has a high efficiency in degrading doxycycline in water.

[0051] Based on the above properties, the mesophilic Sphingobacillus DX14 selected in the present invention has the potential to be developed into a microbial agent, specifically, it is expected to be prepared into a doxycycline-degrading microbial agent suitable for efficiently degrading doxycycline in water bodies.

[0052] Preferably, the doxycycline-degrading microbial agent may contain living cells of mesophilic Sphingobacterium DX14 bacteria, freeze-dried dry cells of mesophilic Sphingobacterium DX14, immobilized cells of mesophilic Sphingobacterium DX14, liquid agent of mesophilic Sphingobacterium DX14, solid agent of mesophilic Sphingobacterium DX14, or mesophilic Sphingobacterium DX14 strains in any other form.

[0053] In addition, the mesophilic Sphingobacillus DX14 selected by the present invention and the doxycycline-degrading microbial agent prepared using the strain DX14 can be used to degrade new pollutant antibiotics in water bodies, especially for degrading doxycycline in water bodies containing antibiotics, and have good application prospects for solving the problem of doxycycline residues in water bodies containing antibiotics.

[0054] 5. Application, recovery and resistance genome digestion process of strain DX14 in simulated aquaculture tail water: Because DX14 carries the doxycycline resistance gene, in order to prevent it from spreading in the environment during use, we recovered the strain itself and completely degraded its genomic DNA before discarding it in the environment, achieving the effect of all-round environmental protection.

[0055] (1) Prepare simulated aquaculture tail water: (NH4)2SO4 135 mg, K2HPO4 78 mg, K2HPO4 31 mg, MgSO4·7H2O 98 mg, KCl 37 mg, trace element solution 2 mL, glucose 550 mg, and dilute to 1 L. Sterilize the simulated aquaculture tail water in 1 L conical flasks, 800 mL per flask. After sterilization, add doxycycline hydrochloride to a final concentration of 60 mg / L.

[0056] (2) Adjust the DX14 bacterial solution to OD 600 =0.8, the bacterial solution was transferred to the simulated aquaculture tail water at a 1% inoculum volume and cultured in a constant temperature shaking incubator at 30°C and 150 rpm in the dark. Three experimental groups and three control groups were set up in the experiment. After 6 days, samples were taken to test the content of doxycycline hydrochloride and calculate the degradation rate. Figure 8As shown, the results showed that the degradation rate of doxycycline hydrochloride in the control group was just over 40%, while the degradation rate of doxycycline hydrochloride in the strain DX14 was over 80%.

[0057] (3) Collection of working strain DX14: Centrifuge the bacterial solution after the above reaction at 8000 rpm for 10 minutes, and collect the bacterial cells, weighing approximately 4.316 g.

[0058] (4) Degradation of the resistance genome of strain DX14: 4.316 g of the above bacteria were sterilized by high pressure (121°C, 20 min), 2400 U of DNase I was added, and the reaction was continued for 60 min. The DX14 genome was detected by UV spectrophotometer every 10 min to see if it was completely degraded. When the OD 260 When the value no longer increases, take a sample and verify it by DNA agarose gel electrophoresis. Figure 9 As shown in the figure, M represents a marker. Lane 1 represents the sterilized sample, Lane 2 represents the sample 30 minutes after enzyme addition, Lane 3 represents the sample 40 minutes after enzyme addition, Lane 4 represents the sample 50 minutes after enzyme addition, and Lane 5 represents the sample 60 minutes after enzyme addition. Combined UV spectrophotometric analysis and gel electrophoresis results showed that the DX14 genome was completely degraded after 40 minutes of exposure and no longer contained doxycycline resistance genes, posing no risk of increasing the environmental resistance burden.

[0059] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A thermophilic Sphingobacterium Sphingobacterium hotanense )DX14, which is characterized by, The thermophilic sphingobacterium DX14 was deposited in the China General Microorganism Culture Collection Center on January 2, 2025, with the deposit number CGMCC NO.33265.

2. A microbial agent containing the mesophilic Sphingobacterium DX14 strain according to claim 1.

3. The microbial agent according to claim 2, characterized in that The microbial agent contains living cells of mesophilic Sphingobacterium DX14 bacteria, dry cells of mesophilic Sphingobacterium DX14 obtained by freeze-drying, immobilized cells of mesophilic Sphingobacterium DX14, liquid agent of mesophilic Sphingobacterium DX14, solid agent of mesophilic Sphingobacterium DX14, or mesophilic Sphingobacterium DX14 strains in any other form.

4. Use of the mesophilic Sphingobacillus DX14 according to claim 1 or the microbial agent according to claim 2 in degrading antibiotics, a new pollutant in water.

5. The use according to claim 4, characterized in that Used to degrade doxycycline in water bodies.

6. An environmentally friendly method for using the mesophilic Sphingobacterium DX14 according to claim 1 or the microbial agent according to claim 2, characterized in that: The steps include: Step 1) preparing the preserved mesophilic Sphingobacterium DX14 into a DX14 active seed bacterial solution, and detecting the bacterial content and activity in the seed solution; Step 2) adding the prepared DX14 active seed bacterial solution to the wastewater containing doxycycline at a certain inoculum size, and culturing the solution in the dark for several days under certain temperature and rotation speed conditions; Step 3) centrifuging the treated wastewater at a certain speed for several minutes, and collecting the reacted mesophilic Sphingobacterium DX14 bacteria; Step 4) The collected mesophilic Sphingobacterium DX14 bacteria are sterilized by high pressure for a period of time, and then DNase I is added for a certain period of time to completely degrade the DX14 genome, thereby preventing the spread of the doxycycline resistance gene carried by the mesophilic Sphingobacterium DX14 in the water body.

7. The environmentally friendly application method according to claim 6, characterized in that: In step 1, the DX14 active seed bacterial liquid is quantified.

8. The environmentally friendly application method according to claim 6, characterized in that: In step 2, the prepared DX14 active seed bacterial liquid was added to the water containing doxycycline at a 1% inoculation rate, and cultured at 30°C, 150 rpm, under constant temperature, shaking, and in the dark for 6 days.

9. The environmentally friendly application method according to claim 6, characterized in that: In step 3, the treated wastewater is centrifuged at 8000 rpm for 10 minutes, and the mesophilic Sphingobacterium DX14 bacteria are collected.

10. The environmentally friendly application method according to claim 6, characterized in that: In step 4, the collected reacted mesophilic Sphingobacterium DX14 cells are sterilized by autoclaving at 121° C. for 20 minutes, and then a sufficient amount of DNase I is added and allowed to react for a period of time to ensure that the genomic DNA of the mesophilic Sphingobacterium DX14 cells is fully degraded.

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