Microbial composition for degrading doxycycline and application
Through the composition of Acinetobacter Guilin, Adx-3, and Plant Proliferation Bacteria JX15, the degradation problem of doxycycline in the environment is solved, efficient degradation effect is achieved, and the health of the ecological environment is guaranteed.
Patent Information
- Application Number
- CN202510609607.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-13
- Publication Date
- 2025-08-15
Smart Images
Figure BDA0005399280790000071
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of antibiotic pollution prevention and control, and particularly relates to a microbial composition for degrading doxycycline and an application thereof. Background Art
[0002] Doxycycline is a semisynthetic tetracycline antibiotic derived from oxytetracycline via deoxygenation at the 6α-position. While relatively stable in aquatic environments, doxycycline can be toxic to aquatic organisms. Studies have shown that it can affect the growth, development, and reproduction of fish, interfere with their endocrine systems, and negatively impact the behavior and survival of aquatic invertebrates, thereby disrupting the balance of aquatic ecosystems. Once introduced into the soil, doxycycline alters the community structure and function of soil microorganisms. It inhibits the growth of certain beneficial microorganisms, such as those involved in nitrogen cycling and soil nutrient transformation, thereby impacting soil fertility and ecological function. It can also promote the growth of some drug-resistant microorganisms, increasing the risk of the spread of drug-resistant bacteria in the soil.
[0003] Reducing doxycycline residues in the environment can reduce the risk of bacterial resistance and its spread, ensuring the effectiveness of antibiotics in clinical treatment, thereby better protecting human health and preventing the threat to human life posed by difficult-to-treat infectious diseases caused by drug-resistant bacteria. Therefore, developing relevant degradation technologies can help address the environmental problems caused by doxycycline and is an important measure for achieving environmental protection and sustainable development. It is in line with today's society's pursuit of green and environmentally friendly development and is conducive to building a healthier and more harmonious ecological environment.
[0004] Currently, the main focus of developing strains for doxycycline degradation is screening for novel strains, such as the Acinetobacter LDH10-1 and LDH6-2 strains identified by Yangzhou University, and Acinetobacter AC-tetX identified by the Jiangsu Academy of Agricultural Sciences. However, there are relatively few reports on the development of microbial consortia using known strains to degrade doxycycline. Summary of the Invention
[0005] In view of the shortcomings of the existing technology, the purpose of the present invention is to screen a microbial composition suitable for degrading doxycycline among known microorganisms, so as to improve the industrial use value of known microorganisms.
[0006] The present invention provides the following technical solutions to achieve the aforementioned objectives.
[0007] A microbial composition for degrading doxycycline, comprising the following strains:
[0008] Acinetobacter guiliniensis HZJK-2, Achromobacter adx-3, and plant growth-promoting bacteria JX15;
[0009] Among them, the preservation number of Acinetobacter HZJK-2 is CGMCC No. 22143; the preservation number of Achromobacter adx-3 is CCTCC NO: M 208176; and the preservation number of plant growth-promoting bacteria JX15 is CGMCC No. 5622.
[0010] Preferably, the addition amounts of Guilin Acinetobacter HZJK-2, Colorless Bacterium adx-3 and Plant Growth-Promoting Bacteria JX15 are 1:1 to 3:1 based on the bacterial cell number ratio.
[0011] More preferably, the addition amounts of Guilin Acinetobacter HZJK-2, Colorless Bacterium adx-3 and plant growth-promoting bacteria JX15 are 1:2:1 based on the ratio of bacterial cell numbers.
[0012] Application of the aforementioned microbial composition in degrading doxycycline.
[0013] At the beginning of the research for this invention, the inventors discovered that Chen Chong and others from Yangzhou University had screened out the Acinetobacter strains LDH10-1 and LDH6-2, which can efficiently degrade a variety of antibiotics, including doxycycline. He Tao and others from the Jiangsu Academy of Agricultural Sciences had screened out Acinetobacter AC-tetX, which also had a good degradation effect on a variety of antibiotics, including doxycycline. Based on this, the inventors searched for Acinetobacter strains that could degrade doxycycline and found that few strains had this function. In addition, because screening strains often involves extremely high uncertainty, the inventors considered combining existing Acinetobacter strains with other strains to investigate their doxycycline degradation.
[0014] During the screening process, the inventors gave priority to strains that can grow in the same culture medium (such as LB culture medium). Through screening, the inventors found that when Guilin Acinetobacter HZJK-2 and Colorless Bacterium adx-3 were mixed and cultured, not only no growth antagonism was found, but also a certain degradation effect on doxycycline was found, which is a function that a single strain does not have. Inspired by this experimental result, the inventors tried to add other strains to conduct exploratory experiments on the basis of the combination of these two strains, and found that after adding plant growth-promoting bacteria JX15, no growth antagonism was produced, and a very excellent doxycycline degradation effect could be obtained. So far, the inventors have completed the research and development of the aforementioned microbial composition.
[0015] Beneficial effects of the present invention:
[0016] The microbial composition provided by the present invention has an excellent degradation effect on doxycycline, with a degradation rate exceeding 93%. DETAILED DESCRIPTION
[0017] The following descriptions of specific embodiments are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.
[0018] Example 1
[0019] 1.1 Acquisition and culture of Guilin Acinetobacter HZJK-2:
[0020] It was purchased from the General Microbiology Center of China Culture Collection Administration of Microorganisms with the accession number CGMCC No.22143.
[0021] Use LB solid medium to streak culture of Guilin Acinetobacter HZJK-2, take a single colony, and then transfer it to LB liquid medium for culture. Cultivate at 25°C until the stationary phase, collect the bacteria by centrifugation, and resuspend the bacteria in PBS for later use.
[0022] 1.2 Acquisition and cultivation of Achromobacter adx-3:
[0023] It was purchased from China Center for Type Culture Collection with the accession number CCTCC NO: M 208176.
[0024] Achromobacter adx-3 was streaked using LB solid medium, and a single colony was taken and then transferred to LB liquid medium for culture. The culture was cultured at 30°C until the stationary phase, and the bacteria were collected by centrifugation and resuspended in PBS for later use.
[0025] 1.3 Acquisition and cultivation of plant growth-promoting bacteria JX15:
[0026] It was purchased from the General Microbiology Center of China Culture Collection Administration of Microorganisms with the accession number of CGMCC No.5622.
[0027] Use LB solid medium to streak culture of plant growth-promoting bacteria JX15, take a single colony, and then transfer it to LB liquid medium for culture. Cultivate at 30°C until the stationary phase, collect the bacteria by centrifugation, and resuspend the bacteria in PBS for later use.
[0028] 1.4 Mix equal volumes of PBS bacterial suspensions of Guilin Acinetobacter HZJK-2, Colorless Bacterium adx-3 and Plant Growth-Promoting Bacteria JX15 at a bacterial cell number ratio of 1:2:1 and use them as treatment agents.
[0029] Example 2
[0030] 1.1 Acquisition and culture of Guilin Acinetobacter HZJK-2:
[0031] It was purchased from the General Microbiology Center of China Culture Collection Administration of Microorganisms with the accession number of CGMCC No.22143.
[0032] Use LB solid medium to streak culture of Guilin Acinetobacter HZJK-2, take a single colony, and then transfer it to LB liquid medium for culture. Cultivate at 25°C until the stationary phase, collect the bacteria by centrifugation, and resuspend the bacteria in PBS for later use.
[0033] 1.2 Acquisition and cultivation of Achromobacter adx-3:
[0034] It was purchased from China Center for Type Culture Collection with the accession number CCTCC NO: M 208176.
[0035] Achromobacter adx-3 was streaked using LB solid medium, and a single colony was taken and then transferred to LB liquid medium for culture. The culture was cultured at 30°C until the stationary phase, and the bacteria were collected by centrifugation and resuspended in PBS for later use.
[0036] 1.3 Acquisition and cultivation of plant growth-promoting bacteria JX15:
[0037] It was purchased from the General Microbiology Center of China Culture Collection Administration of Microorganisms with the accession number of CGMCC No.5622.
[0038] Use LB solid medium to streak culture of plant growth-promoting bacteria JX15, take a single colony, and then transfer it to LB liquid medium for culture. Cultivate at 30°C until the stationary phase, collect the bacteria by centrifugation, and resuspend the bacteria in PBS for later use.
[0039] 1.4 Mix equal volumes of PBS bacterial suspensions of Guilin Acinetobacter HZJK-2, Colorless Bacterium adx-3 and Plant Growth-Promoting Bacteria JX15 at a bacterial cell number ratio of 1:1:1 and use as a treatment agent.
[0040] Example 3
[0041] 1.1 Acquisition and culture of Guilin Acinetobacter HZJK-2:
[0042] It was purchased from the General Microbiology Center of China Culture Collection Administration of Microorganisms with the accession number of CGMCC No.22143.
[0043] Use LB solid medium to streak culture of Guilin Acinetobacter HZJK-2, take a single colony, and then transfer it to LB liquid medium for culture. Cultivate at 25°C until the stationary phase, collect the bacteria by centrifugation, and resuspend the bacteria in PBS for later use.
[0044] 1.2 Acquisition and cultivation of Achromobacter adx-3:
[0045] It was purchased from China Center for Type Culture Collection with the accession number CCTCC NO: M 208176.
[0046] Achromobacter adx-3 was streaked using LB solid medium, and a single colony was taken and then transferred to LB liquid medium for culture. The culture was cultured at 30°C until the stationary phase, and the bacteria were collected by centrifugation and resuspended in PBS for later use.
[0047] 1.3 Acquisition and cultivation of plant growth-promoting bacteria JX15:
[0048] It was purchased from the General Microbiology Center of China Culture Collection Administration of Microorganisms with the accession number of CGMCC No.5622.
[0049] Use LB solid medium to streak culture of plant growth-promoting bacteria JX15, take a single colony, and then transfer it to LB liquid medium for culture. Cultivate at 30°C until the stationary phase, collect the bacteria by centrifugation, and resuspend the bacteria in PBS for later use.
[0050] 1.4 Mix equal volumes of PBS bacterial suspensions of Guilin Acinetobacter HZJK-2, Achromobacter adx-3, and plant growth-promoting bacteria JX15 at a bacterial cell number ratio of 1:3:1 and use them as treatment agents.
[0051] Experimental Example 1
[0052] Soil not containing doxycycline was taken and placed in an experimental pot (a square pot with a length, width and height of 50 cm), and doxycycline was mixed with the soil so that the concentration of doxycycline was 10 mg / kg.
[0053] The treatment agents obtained in Examples 1 to 3 were prepared to have a total bacterial cell concentration of 10 8 A solution containing 100 CFU / mL of PBS was added to the doxycycline-mixed soil at a ratio of 100 mL / kg. The soil was incubated at room temperature, and samples were taken on the fourth day to measure the doxycycline content (using HPLC-MS / MS). A control group containing the same volume of PBS was used. The experimental results are shown in Table 1.
[0054] Table 1
[0055]
[0056] Experimental Example 2
[0057] This experimental example records the inventor's exploratory experiments in finding microbial combinations before carrying out Experimental Example 1.
[0058] The inventors used Achromobacter adx-3, already purchased in the laboratory, as one of the test strains. They also selected various Acinetobacter species for mixed culture to investigate whether there was growth antagonism between the strains. The experimental results, shown in Table 2, indicate that Achromobacter adx-3 exhibited growth antagonism with both Acinetobacter LDH6-2 and Acinetobacter T1, making the combination unsuitable.
[0059] Table 2
[0060] Whether growth antagonism occurs adx-3 and HZJK-2 - adx-3 and LDH6-2 +++ adx-3 and T1 ++
[0061] Note: “-” indicates that after equal amounts of the two strains were inoculated and grown at 30°C to the plateau phase, there was no clear difference in the number of each cell compared with that in single-strain culture; “++” indicates that after equal amounts of the two strains were inoculated and grown at 30°C to the plateau phase, the number of each cell decreased by 2 orders of magnitude compared with that in single-strain culture, indicating an antagonistic effect; “+++” indicates that after equal amounts of the two strains were inoculated and grown at 30°C to the plateau phase, the number of each cell decreased by 3 orders of magnitude compared with that in single-strain culture, indicating an antagonistic effect; “adx-3” indicates Achromobacter adx-3, “HZJK-2” indicates Acinetobacter guilinensis HZJK-2, “LDH6-2” indicates Acinetobacter LDH6-2 (accession number GDMCC NO: 64034, purchased from Guangdong Provincial Center for Microbial Culture Collection), and “T1” indicates Acinetobacter T1 (accession number CCTCC NO.M 2014558, purchased from China Center for Type Culture Collection).
[0062] Based on the results in Table 2, the inventors investigated the degradation of doxycycline in soil using a mixture of Achromobacter adx-3 and Acinetobacter guiliniensis HZJK-2, as well as individual strains of the two bacteria. The experimental process followed the method described in Experimental Example 1. After four days of incubation, it was found that the two strains had no effect on doxycycline degradation, while the mixture of the two strains produced a 12.7% degradation of doxycycline.
[0063] Before carrying out the experiment of degrading doxycycline by mixing three strains in Experimental Example 1, the inventors mixed and cultured Guilin Acinetobacter HZJK-2, Colorless Bacterium adx-3 and plant growth-promoting bacteria JX15 (mixed cultured at 30°C and 25°C, respectively), and no obvious antagonistic effect was found (the difference in the number of bacteria did not exceed 1 order of magnitude).
Claims
1. A microbial composition for degrading doxycycline, characterized in that: The microbial composition consists of the following strains: Acinetobacter guiliniensis HZJK-2, Achromobacter adx-3, and plant growth-promoting bacteria JX15; Among them, the preservation number of Guilin Acinetobacter HZJK-2 is CGMCC No.22143; the preservation number of Colorless Bacterium adx-3 is CCTCC NO: M 208176; and the preservation number of Plant Growth-Promoting Bacteria JX15 is CGMCC No.5622.
2. A microbial composition for degrading doxycycline according to claim 1, characterized in that: According to the ratio of bacterial numbers, the addition amounts of Guilin Acinetobacter HZJK-2, Colorless Bacterium adx-3 and plant growth-promoting bacteria JX15 are 1:1 to 3:
1.
3. The microbial composition for degrading doxycycline according to claim 1, characterized in that: According to the ratio of bacterial numbers, the addition amounts of Guilin Acinetobacter HZJK-2, Colorless Bacterium adx-3 and plant growth-promoting bacteria JX15 were 1:2:
1.
4. Use of the microbial composition according to any one of claims 1 to 3 in degrading doxycycline.