Microbacterium sp. and application thereof in degrading chloramphenicol and microplastics

Abstract

This invention provides a strain of *Exiguobacterium* and its application in the degradation of chloramphenicol and microplastics. The bacterium was collected from a duck farm in Longping Town, Guangdong Province, and obtained through artificial enrichment culture, isolation, and purification. It is named *Exiguobacterium sp. CAP4*. Quantitative analysis using high-performance liquid chromatography (HPLC) and gas chromatography (GC) revealed that the strain *Exiguobacterium sp. CAP4* possesses the ability to efficiently degrade chloramphenicol, mineralize PE microplastics, and various types of plastics. It can mineralize different types of plastics into CO2, indicating that this bacterium has the potential to efficiently degrade chloramphenicol, mineralize PE microplastics, and various types of plastics in environments such as livestock and poultry farms and municipal wastewater, achieving the biodegradation of chloramphenicol, microplastics, and various types of plastics.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to a microbacterium and its application in the degradation of chloramphenicol and microplastics. Background Technology

[0002] Chloramphenicol (CAP) is a white to grayish-white or yellowish-white fine crystalline powder or crystal with a bitter taste. It readily diffuses through bacterial cell walls and binds to the 50S ribosomal subunit, thereby blocking the activity of peptidyl transferases, halting peptide chain transfer, and inhibiting protein synthesis. Therefore, chloramphenicol is a broad-spectrum antibiotic with inhibitory activity against Gram-positive bacteria, Gram-negative bacteria, and certain anaerobic microorganisms.

[0003] With the increasing demand for plastic products in daily life and the development of the plastics industry, large quantities of plastic are produced and discarded. Once in the environment, plastic waste gradually decomposes and breaks down into fine particles, forming microplastics (MPs). Microplastics are typically plastic fragments smaller than 5 millimeters in size, often resulting from the crushing of large pieces of plastic or from small plastic particles directly produced in plastic factories. Due to the recalcitrant nature of plastics, large quantities of discarded microplastics can persist in the environment for extended periods. Therefore, microplastics have been detected in various environmental media and foods, such as seawater, river water, seabed / riverbed sediments, municipal sewage, arable soil, lettuce, barley, rice, human blood, breast milk, and neonatal feces. Microplastics can adsorb organic matter, heavy metals, and organic pollutants from the environment, and recruit environmental microorganisms to form microbial films, establishing an independent ecological niche (microplastic space); altering pollutants and their own migration and transformation. Furthermore, microplastics can provide ecological niches for antibiotic resistance genes and resistant pathogens, enriching the abundance of antibiotic resistance genes and pathogens, altering their migration patterns, and posing a potential threat to the environment and human health. Therefore, reducing or eliminating microplastic pollution in the environment is urgent, and there is a pressing need to find an efficient and green method for microplastic removal. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a strain of microbacteria. Small bacterium sp. CAP4, collected from a duck farm in Longping Town, Guangdong Province, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 19, 2024, with accession number GDMCC No: 64770.

[0005] Another object of the present invention is to provide the application of the above-mentioned Aquabacterium in the degradation of chloramphenicol and microplastics.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A strain of microbacteria ( Small bacteriumThe sample was collected from a duck farm in Longping Town, Guangdong Province, and obtained through artificial enrichment culture, separation and purification. Its name is... Small bacterium sp. CAP4.

[0008] The aforementioned microbacterium, with accession number GDMCC No: 64770, was deposited on June 19, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0009] The aforementioned microbacteria are rod-shaped, Gram-negative, and have a cell size of 0.6–1.5 μm.

[0010] The nucleotide sequence of the 16S rRNA of the aforementioned microbacterium is shown in SEQ ID NO: 1.

[0011] The application of the aforementioned microbacteria in the degradation of chloramphenicol.

[0012] The application of the aforementioned microbacteria in the degradation of chloramphenicol includes the following steps:

[0013] Microbes were inoculated into a culture medium to obtain a seed culture. The seed culture was then added to a system containing chloramphenicol for further culturing to degrade the chloramphenicol.

[0014] The culture conditions are 28–32℃ and shaking culture at 100–200 rpm.

[0015] The application of the aforementioned microbacteria in degrading plastics.

[0016] The plastic includes at least one of polyethylene, polyvinyl chloride, polypropylene, and polyethylene terephthalate.

[0017] The present invention has the following advantages and effects compared with the prior art:

[0018] This invention provides a strain of microbacterium ( Small bacterium ) strain Small bacterium sp. CAP4 was quantitatively analyzed using high-performance liquid chromatography (HPLC), and the degradation ability of CAP was found to be... Small bacterium sp. CAP4 can efficiently remove CAP; quantitative analysis of the degradation capacity of microplastics and different types of plastics using gas chromatography (GC) revealed... Small bacterium sp. CAP4 possesses the ability to mineralize microplastics and various types of plastics, converting them into CO2. Therefore, Small bacterium sp. CAP4 has the potential to be applied in environments such as livestock and poultry farms and municipal wastewater to achieve the biodegradation of chloramphenicol, microplastics and various types of plastics. Attached Figure Description

[0019] Figure 1 yes Small bacterium Scanning electron microscope image of sp. CAP4 strain.

[0020] Figure 2 yes Small bacterium Phylogenetic analysis diagram of sp. CAP4 strain.

[0021] Figure 3 In Example 3 Small bacterium Concentration trend of CAP degradation by sp. CAP4 strain.

[0022] Figure 4 In Example 3 Small bacterium Figure showing the degradation efficiency of sp. CAP4 strain at different concentrations of CAP.

[0023] Figure 5 It is in Example 4 Small bacterium A trend graph showing the mineralization of microplastics into CO2 by sp. CAP4 strain.

[0024] Figure 6 It is in Example 5 Small bacterium A trend graph showing the mineralization of different types of plastics into CO2 by the sp. CAP4 strain. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0026] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0027] Example 1: Isolation and purification of strains

[0028] (1) Duck farm wastewater was collected from a duck farm in Longping Town, Guangdong Province. 100 mL of duck farm wastewater sample was placed in a 500 mL conical flask, 10 mg / L of chloramphenicol (CAP) and 5 g of PE microplastics (particle size 3-1000 μm) were added, and the sample was placed in a constant temperature shaker at 30 ℃ and shaken at 150 rpm in the dark.

[0029] (2) During the culture process, samples were taken and tested regularly. After the CAP concentration dropped to 0 mg / L, 10 mg / L of CAP was added to the conical flask, and the culture was repeated for 4 cycles. After about 30 days, the culture medium was centrifuged at 10,000 rpm to remove the supernatant and obtain the sediment. The sediment was washed 3 times with 0.9% sterile physiological saline, then suspended in saturated sterile NaCl, and centrifuged again at 10,000 rpm. After the sediment was resuspended, the microplastics suspended on the surface were collected and placed in 0.9% sterile physiological saline for 15 min of sonication to dissociate the microorganisms on the plastic and obtain a microbial mixture on the surface of the microplastics. The microbial mixture was diluted 10 -1 10 -2 10 -3 10 -4 and 10 -5 Then, spread the culture evenly onto solid LB agar plates and incubate them at 30 ℃. After colonies have grown, pick colonies of different sizes and shapes and streak them onto new LB plates. Repeat this step 4 to 5 times until single purified colonies are obtained and stored.

[0030] Example 2 Identification of the strain

[0031] (1) Colony morphology

[0032] The strain isolated and purified in Example 1 was a short rod-shaped, Gram-negative strain, as shown in the SEM image. Figure 1 As shown, the bacterial cell size is 0.6–1.5 μm.

[0033] (2) Molecular biological identification

[0034] Genomic DNA was extracted from the purified strain obtained in Example 1 and used as a DNA template. The bacterial 16S rRNA gene was amplified using universal primers 27F (5'-agagtttgatcmtggctcag-3') and 1492R (5'-ggytaccttgttacgactt-3'). The amplified 16S rRNA gene sequence was sequenced and uploaded to the NCBI database for comparison and analysis.

[0035] The obtained 16S rRNA gene sequence was submitted to the National Center for Biotechnology Information (NCBI) website for BLAST comparison. Sequences of highly homologous type strains were also downloaded for BLAST alignment and homology analysis. A phylogenetic tree was constructed using Mega 6.0 software with the Neighbour-Joining method. Comparison of the 16S rRNA gene sequences revealed that the 16S rRNA of the strain isolated and purified in Example 1 was similar to... Exiguobacterium profundum The sequence is closest.

[0036] Based on the results of the above two aspects, it was determined that the strain isolated in Example 1 belongs to the genus Microbacterium ( ). Small bacterium ), named Small bacterium sp. CAP4 was deposited on June 19, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No: 64770), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

[0037] Example 3 Small bacterium sp. CAP4 degradation performance of CAP

[0038] 3.1 Chloramphenicol Degradation Kinetics

[0039] To clarify Small bacterium The degradation performance of sp. CAP4 for chloramphenicol was investigated. The degradation kinetics of chloramphenicol were determined in this experiment, and the specific steps are as follows:

[0040] (1) Small bacterium sp. CAP4 was streaked onto LB plates and incubated at 30 ℃ for 20 h. Single colonies were picked and inoculated into LB liquid medium and incubated at 30 ℃ and 150 rpm for 20 h to obtain CAP4 seed culture.

[0041] (2) CAP4 seed culture was inoculated into LB liquid medium containing 10 mg / L chloramphenicol at a ratio of 2% (v / v) and cultured at 30 ℃ with shaking at 150 rpm. Samples were collected at 0, 0.25, 0.5, 1, 2, 4 and 6 days (d). CAP4 strain without the addition of CAP4 was used as a blank control (CK). Three replicates were set up. The samples were analyzed by HPLC. Small bacterium The amount of chloramphenicol removed by sp. CAP4 and the removal efficiency were calculated.

[0042] Experimental results are as follows Figure 3 As shown, the removal efficiencies in the chloramphenicol degradation kinetics were 0, 7.3, 16.3, 28, 48.5, 80.3, and 100%, respectively. Small bacterium The degradation kinetics of sp. CAP4 on chloramphenicol follows a first-order kinetic model, and the degradation curve is c=14.4(1-exp(-0.26t)).

[0043] 3.2 Gradient concentration verification of chloramphenicol degradation performance

[0044] This experiment verified that Small bacterium The degradation performance of sp. CAP4 on different concentrations of chloramphenicol was investigated through the following steps:

[0045] CAP4 seed culture was inoculated at a ratio of 2% (v / v) into LB liquid medium containing 5, 10, 20, 50, 100, and 200 mg / L chloramphenicol, and cultured at 30 ℃ with shaking at 150 rpm. Three replicates were set up. Samples were taken after 2 days of culture, and the samples were analyzed by HPLC. Small bacterium The amount of chloramphenicol removed by sp. CAP4 and the removal efficiency were calculated.

[0046] Experimental results are as follows Figure 4 As shown, the removal efficiencies of chloramphenicol at initial concentrations of 5, 10, 20, 50, 100, and 200 mg / L were 53.7%, 51.8%, 38.3%, 37.7%, 13.8%, and 4.8%, respectively. This demonstrates... Small bacterium sp. CAP4 still exhibits certain activity and degradation effects under high concentrations of chloramphenicol, proving that this bacterium can tolerate high concentrations of chloramphenicol and simultaneously achieve degradation, and can be applied to purify chloramphenicol in soil and water.

[0047] Example 4 Small bacterium The mineralization properties of sp. CAP4 on PE microplastics

[0048] The study on the mineralization properties of microplastics included three groups: a group with added bacteria, a group without added bacteria, and a group with added bacteria but no microplastics. The specific procedures were as follows:

[0049] Microplastic pretreatment: The PE microplastics used in this embodiment were placed in an oven at 55 ℃ overnight, then surface-sterilized three times with 75% alcohol in a laminar flow hood, and then sterilized overnight under ultraviolet light in a laminar flow hood.

[0050] In the bacterial addition group: 30 mg of pretreated PE microplastics were added to a 50 mL vial, and the vial was filled with MSM medium. 20 mL of syngas (nitrogen:hydrogen:helium = 1:2:7) was added using the water displacement method, followed by 2% of the culture medium obtained in Example 3. Small bacterium sp. CAP4 seed solution.

[0051] No bacterial culture group: Prepared according to the method of the bacterial culture group, but without adding CAP4 seed liquid.

[0052] Microplastic-free group with added bacteria: Prepared according to the method of the group with added bacteria, but without adding microplastics.

[0053] After preparing the experimental culture flasks, they were placed in a shaker at 30 ℃ and cultured at 150 rpm. The CO2 concentration in the headspace of the system was detected by gas chromatography at 0, 2, 5, 10, 17, 27, and 37 days.

[0054] The headspace CO2 concentrations of the bacteria-added group, the bacteria-added but microplastic-free group, and the group without bacteria were plotted as concentration change curves. Figure 5As shown in the figure, the CO2 concentration in the untreated group ranged from 204 to 298 ppm, with no significant difference. In the sterilized group without microplastics, the CO2 concentration increased from 280 ppm to around 700 ppm and then remained constant. However, in the sterilized and microplastic-treated groups, the CO2 concentration increased from 280 ppm to 2512 ppm and then decreased to 1600 ppm, which was significantly higher than that in the untreated group and the sterilized and microplastic-treated group, indicating that the strain... Small bacterium sp. CAP4 has the ability to mineralize microplastics.

[0055] Example 5 Small bacterium The mineralization properties of sp. CAP4 on different types of plastics

[0056] The study on the mineralization properties of different types of plastics included both groups with and without bacteria. The specific procedures were as follows:

[0057] Pretreatment of different types of plastics: All different types of plastics used in this embodiment (polyethylene, polyvinyl chloride, polypropylene, and polyethylene terephthalate; all of which are 1 mm thick sheet materials) were placed in an oven at 55 ℃ overnight, then surface-sterilized three times with 75% alcohol in a laminar flow hood, and then sterilized overnight under ultraviolet light in a laminar flow hood.

[0058] Add five 2×2 cm pieces of pretreated plastic of different types to a 50 mL vial, then fill with MSM medium. Add 20 mL of syngas (nitrogen:hydrogen:helium = 1:2:7) using the water displacement method; then add... Small bacterium sp. CAP4 seed culture. In addition, a corresponding control group without bacterial culture was set up for each type of plastic experimental group, prepared according to the same method as the group with bacterial culture, except that CAP seed culture was not added. The mixture was placed in a shaker at 30 ℃ and incubated with shaking at 150 rpm. The CO2 concentration in the headspace of the system was detected by gas chromatography at 0, 7, 15, 30, 45, and 60 days.

[0059] Plot the headspace CO2 concentration changes in the group with and without bacteria as follows: Figure 6 As shown in the figure, the CO2 concentration in the untreated group ranged from 0 to 450 ppm with no significant difference. In the treated group, the CO2 concentration increased from 150 ppm to approximately 980 ppm, then decreased slightly, but remained significantly higher than that in the untreated group, indicating that the strain... Small bacterium sp. CAP4 has the ability to mineralize different types of plastics (polyethylene, polyvinyl chloride, polypropylene, polyethylene terephthalate).

[0060] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of microbacterium, characterized by: Name is Exiguobacterium sp. CAP4, with accession number GDMCC No: 64770, was deposited on June 19, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

2. The application of the microbacteria described in claim 1 in the degradation of chloramphenicol.

3. The application of the microbacteria according to claim 2 in the degradation of chloramphenicol, characterized in that... Includes the following steps: Microbes were inoculated into a culture medium to obtain a seed culture. The seed culture was then added to the system containing chloramphenicol to be treated, and culture was continued to degrade chloramphenicol. The culture conditions are 28–32 °C and shaking culture at 100–200 rpm.

4. The application of the microbacteria of claim 1 in the degradation of at least one of polyethylene, polyvinyl chloride, polypropylene, and polyethylene terephthalate.

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