Exiguobacterium sp. And application thereof in degradation of chloramphenicol and microplastics

Abstract

The invention provides an Exiguobacterium sp. And an application thereof in degradation of chloramphenicol and microplastics. The strain is collected from a duck farm in Longping town, Guangdong province, is obtained through artificial enrichment culture, separation and purification, and is named as Exiguobacterium sp.CAP4. A high performance liquid chromatograph and a gas chromatograph are used for quantitative analysis, and the strain Exiguobacterium sp.CAP4 is found to have the capabilities of efficiently degrading chloramphenicol and mineralizing PE micro-plastics and different types of plastics, and can mineralize different types of plastics into CO2, so that the strain has the capabilities of efficiently degrading chloramphenicol and mineralizing PE micro-plastics and different types of plastics, and can be used for preparing CO2. The strain has the potential of being applied to environments such as livestock and poultry, municipal sewage and the like to realize biodegradation of chloramphenicol, micro-plastics and different types of plastics.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a strain of Exiguobacterium and application thereof in degrading chloramphenicol and microplastics. Background Art

[0002] Chloramphenicol (CAP) is a white to off-white or yellow-white fine crystalline powder or fine crystals with a bitter taste. It can easily diffuse through the bacterial cell wall and bind to the bacterial 50S ribosomal subunit, thereby blocking the activity of peptidyl transferase, causing the transfer of peptide chains to stop and inhibiting protein synthesis. Therefore, chloramphenicol, as a broad-spectrum antibiotic, has inhibitory activity against Gram-positive bacteria, Gram-negative bacteria and certain anaerobic microorganisms. Chloramphenicol is widely used as a veterinary drug and feed additive in animal husbandry and aquaculture due to its low price and wide antibacterial activity. However, due to the incomplete absorption and metabolic characteristics of chloramphenicol by animals, 30-90% of chloramphenicol is excreted from the body through feces and urine, and eventually enters the environment, endangering the environment and human health. For example, it promotes the drift and abundance of antibiotic resistance genes, endangers the activity and diversity of soil microorganisms, and reduces soil fertility. Due to the abuse and improper handling of chloramphenicol, chloramphenicol residues have been detected in a variety of environmental media and foods, such as cultivated soil, river sediments, municipal sewage, livestock and poultry feces, radish, rapeseed, eggs, rabbits, etc. Therefore, it is imperative to reduce or eliminate chloramphenicol pollution in the environment, which has important practical significance for food safety and green ecological environment construction.

[0003] With the demand for plastic products in people's daily lives and the development of the plastic industry, a large amount of plastic is produced and discarded. After plastic waste enters the environment, it gradually decomposes and breaks into fine particles to form microplastics (MP). Microplastics are usually plastic fragments less than 5 mm in size, often small plastic particles produced directly by large pieces of plastic or directly produced by plastic factories. Due to the difficult degradation of plastics, a large amount of discarded microplastics can exist in the environment for a long time. Therefore, the presence of microplastics has been detected in a variety of environmental media and foods, such as seawater, river water, seabed / river bottom sediments, municipal sewage, cultivated soil, lettuce, barley, rice, human blood, breast milk and newborn feces. Microplastics can absorb organic matter, heavy metals and organic pollutants in the environment, recruit environmental microorganisms, form microbial films, establish an independent ecological niche (microplastic world), and change the migration and transformation of pollutants and themselves. In addition, microplastics can provide ecological niches for antibiotic resistance genes and resistant pathogens, enrich the abundance of antibiotic resistance genes and pathogens, change their migration and fate, and bring potential threats to the environment and human health. Therefore, it is urgent to reduce or eliminate the pollution of microplastics in the environment, and there is an urgent need to find an efficient and green method for removing microplastics. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a strain of Exiguobacterium sp.CAP4, which was collected from a duck farm in Longping Town, Guangdong Province, and was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 19, 2024, with a deposit number of GDMCC No: 64770.

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

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A strain of Exiguobacterium was collected from a duck farm in Longping Town, Guangdong Province. It was obtained through artificial enrichment, culture, separation and purification and was named Exiguobacterium sp.CAP4.

[0008] The deposit number of the microbacterium is GDMCC No: 64770, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 19, 2024. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0009] The microbacterium is rod-shaped, Gram-negative, and has a size of 0.6 to 1.5 μm.

[0010] The nucleotide sequence of the 16s rRNA of the Exiguobacterium is shown in SEQ ID NO:1.

[0011] The application of the microbacterium in degrading chloramphenicol.

[0012] The application of the Exiguobacterium tumefaciens in degrading chloramphenicol comprises the following steps:

[0013] The microbacterium is inoculated into a culture medium and cultured to obtain a seed solution, and then the seed solution is added into a system containing chloramphenicol to be treated and cultured continuously to degrade the chloramphenicol.

[0014] The culture conditions are 28-32° C. and 100-200 rpm shaking culture.

[0015] The application of the microbacterium in degrading plastics.

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

[0017] Compared with the prior art, the present invention has the following advantages and effects:

[0018] The present invention provides an Exiguobacterium sp.CAP4 strain. The CAP degradation ability is quantitatively analyzed by high performance liquid chromatography (HPLC), and it is found that Exiguobacterium sp.CAP4 can efficiently remove CAP. The degradation ability of microplastics and different types of plastics is quantitatively analyzed by gas chromatograph (GC), and it is found that Exiguobacterium sp.CAP4 has the ability to mineralize microplastics and different types of plastics, and can mineralize microplastics and different types of plastics into CO2. Therefore, Exiguobacterium sp.CAP4 has the potential to be applied to livestock and poultry and municipal sewage environments to achieve biodegradation of chloramphenicol, microplastics and different types of plastics. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a scanning electron micrograph of Exiguobacterium sp.CAP4 strain.

[0020] Figure 2 This is the phylogenetic analysis diagram of Exiguobacterium sp.CAP4 strain.

[0021] Figure 3 This is a concentration trend diagram of CAP degradation by Exiguobacterium sp.CAP4 strain in Example 3.

[0022] Figure 4 This is a graph showing the degradation efficiency of Exiguobacterium sp.CAP4 strain in Example 3 for degrading CAP of different concentrations.

[0023] Figure 5 This is a trend chart of the mineralization of microplastics into CO2 by the Exiguobacterium sp.CAP4 strain in Example 4.

[0024] Figure 6 This is a trend chart of the mineralization of different types of plastics into CO2 by the Exiguobacterium sp.CAP4 strain in Example 5. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0026] If no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[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 30 °C constant temperature shaker with an oscillation rate of 150 rpm to avoid light;

[0029] (2) During the culture process, samples were taken regularly for testing. When 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 solution was centrifuged at 10,000 rpm, and the supernatant was removed to obtain the sediment; it was washed three times with 0.9% sterile saline, and then the sediment was suspended with 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 saline for 15 minutes of ultrasound to dissociate the microorganisms on the plastics to obtain a microplastic surface microbial mixture. Take the microbial mixture and dilute it by 10 -1 ,10 -2 ,10 -3 ,10 -4 and 10 -5 Afterwards, they were evenly spread on solid LB medium plates and placed in a constant temperature incubator at 30°C. After the colonies grew, colonies of different sizes and shapes were picked and inoculated on new LB plates by streaking. This step was repeated 4 to 5 times until a single purified colony was obtained and preserved.

[0030] Example 2 Identification of strains

[0031] (1) Colony morphology

[0032] The strain isolated and purified in Example 1 is short rod-shaped, Gram-negative, and the SEM photo is as follows: Figure 1 As shown, the bacterial body size is 0.6 to 1.5 μm.

[0033] (2) Molecular Biological Identification

[0034] The genomic DNA of the purified strain obtained by separation and purification in Example 1 was extracted and used as a DNA template for amplification using the universal primers 27F (5'-agagtttgatcmtggctcag-3') and 1492R (5'-ggytaccttgttacgactt-3') of the bacterial 16S rRNA gene. 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 Biological Information (NCBI) website for BLAST comparison, and the model strain sequence with high homology was downloaded for BLAST comparison and homology analysis, and the phylogenetic tree was constructed using the Neighbour-Joining method using Mega 6.0 software. The 16S rRNA gene sequence was compared and it was found that the 16S rRNA of the isolated and purified strain in Example 1 was closest to the sequence of Exiguobacterium profundum.

[0036] Based on the results of the above two aspects, it was determined that the strain isolated in Example 1 was Exiguobacterium, named Exiguobacterium sp.CAP4, which was deposited in Guangdong Microbiological Culture Collection Center on June 19, 2024, with a deposit number of GDMCC No: 64770, and the deposit address was the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0037] Example 3: Degradation performance of Exiguobacterium sp.CAP4 on CAP

[0038] 3.1 Chloramphenicol degradation kinetics

[0039] In order to clarify the chloramphenicol degradation performance of Exiguobacterium sp.CAP4, the chloramphenicol degradation kinetics was determined in this experiment. The specific steps are as follows:

[0040] (1) Streak Exiguobacterium sp.CAP4 on an LB plate, culture at 30°C for 20 h, pick a single colony and inoculate it into LB liquid culture medium, culture it in a shaker at 30°C and 150 rpm for 20 h to obtain CAP4 seed solution;

[0041] (2) CAP4 seed liquid was inoculated into LB liquid culture medium containing 10 mg / L chloramphenicol at a ratio of 2% (v / v), and cultured at 30°C and 150 rpm with shaking; samples were collected at 0, 0.25, 0.5, 1, 2, 4 and 6 days (d), and the blank control (CK) without adding strain CAP4 was used, and three replicates were set up; the samples were determined by HPLC to determine the removal amount of chloramphenicol by Exiguobacterium sp.CAP4 and the removal efficiency was calculated.

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

[0043] 3.2 Gradient concentration verification of chloramphenicol degradation performance

[0044] This experiment verifies the degradation performance of Exiguobacterium sp.CAP4 on chloramphenicol at different concentrations. The specific steps are as follows:

[0045] The CAP4 seed liquid was inoculated into LB liquid culture medium containing 5, 10, 20, 50, 100, and 200 mg / L chloramphenicol at a ratio of 2% (v / v), and cultured at 30°C and 150 rpm with shaking, and three repeated experiments were set up. Samples were taken after 2 days of culture, and the samples were used to determine the removal amount of chloramphenicol by Exiguobacterium sp.CAP4 using HPLC and the removal efficiency was calculated.

[0046] The experimental results are as follows Figure 4 As shown, the removal efficiency of chloramphenicol at initial concentrations of 5, 10, 20, 50, 100, and 200 mg / L was 53.7, 51.8, 38.3, 37.7, 13.8, and 4.8%, respectively. This proves that Exiguobacterium sp.CAP4 still has certain activity and degradation effect under high concentrations of chloramphenicol, and proves that the bacteria can tolerate high concentrations of chloramphenicol and achieve degradation at the same time, and can be used to purify chloramphenicol in soil and water.

[0047] Example 4 Mineralization performance of Exiguobacterium sp.CAP4 on PE microplastics

[0048] The study on the mineralization performance of microplastics set up a group with bacteria, a group without bacteria, and a group with bacteria but without microplastics. The specific operations are as follows:

[0049] Microplastic pretreatment: The PE microplastics used in this example were placed in a 55°C oven overnight, then surface-sterilized three times with 75% alcohol in a clean bench, and sterilized overnight under ultraviolet light in the clean bench.

[0050] Bacteria-added group: 30 mg of the pretreated PE microplastics were added to a 50 mL vial, and the MSM culture medium was filled up. 20 mL of synthesis gas (nitrogen: hydrogen: helium = 1:2:7) was added by drainage method, and then 2% of the Exiguobacterium sp.CAP4 seed solution obtained in Example 3 was added.

[0051] Group without bacteria: Prepared according to the method of group with bacteria, but without adding CAP4 seed liquid.

[0052] Bacteria-added and microplastic-free group: The preparation method is similar to that of the bacteria-added group, but no microplastics are added.

[0053] After the experimental culture bottles were prepared, they were placed in a shaker at 30°C 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 concentration change curve of the headspace CO2 concentration of the bacteria-added group, the bacteria-added group without microplastics, and the group without bacteria was drawn, as shown in Figure 5 In the group without bacteria, the CO2 concentration ranged from 204 to 298 ppm, with no significant difference; in the group with bacteria and no microplastics, the CO2 concentration increased from 280 ppm to about 700, and then remained constant; in the group with bacteria and microplastics, the CO2 concentration increased from 280 ppm to 2512 ppm, and then decreased to 1600 ppm, which was significantly higher than that of the group without bacteria and the group with bacteria and no microplastics, indicating that the strain Exiguobacterium sp.CAP4 has the ability to mineralize microplastics.

[0055] Example 5 Mineralization performance of Exiguobacterium sp.CAP4 on different types of plastics

[0056] The mineralization performance study of different types of plastics set up a group with bacteria and a group without bacteria. The specific operation is as follows:

[0057] Pretreatment of different types of plastics: All the different types of plastics used in this embodiment (polyethylene, polyvinyl chloride, polypropylene, polyethylene terephthalate; all 1 mm thick sheet materials) were placed in a 55°C oven overnight, then surface-sterilized three times with 75% alcohol in a clean bench, and sterilized under ultraviolet light in the clean bench overnight.

[0058] Add 5 pieces of 2×2cm pretreated plastics of different types to a 50mL vial, fill it with MSM medium, add 20mL of synthetic gas (nitrogen: hydrogen: helium = 1:2:7) by drainage method; then add Exiguobacterium sp. CAP4 seed solution. In addition, a corresponding control group without bacteria was set for each plastic experimental group. The preparation method was similar to the bacteria group, except that CAP seed solution was not added. Place in a 30℃ shaker, shake and culture at 150rpm, and use a gas chromatograph to detect the CO2 concentration in the headspace of the system at 0, 7, 15, 30, 45, and 60 days.

[0059] The CO2 concentration in the headspace of the bacteria-added group and the group without bacteria was plotted as a concentration change curve. Figure 6In the group without bacteria, the CO2 concentration ranged from 0 to 450 ppm, with no significant difference; in the group with bacteria, the CO2 concentration increased from 150 ppm to about 980 ppm, and then decreased slightly, which was significantly higher than that of the group without bacteria, indicating that the strain Exiguobacterium sp.CAP4 has the ability to mineralize different types of plastics (polyethylene, polyvinyl chloride, polypropylene, polyethylene terephthalate).

[0060] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Exiguobacterium, characterized in that: The name is Exiguobacterium sp.CAP4, the deposit number is GDMCC No: 64770, and it was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 19, 2024. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

2. The Exiguobacterium according to claim 1, characterized in that: The microbacterium is rod-shaped, Gram-negative, and has a size of 0.6 to 1.5 μm.

3. The Exiguobacterium according to claim 1, characterized in that: The nucleotide sequence of the 16s rRNA of the Exiguobacterium is shown in SEQ ID NO:

1.

4. Use of the Exiguobacterium according to any one of claims 1 to 3 in degrading chloramphenicol.

5. The use of Exiguobacterium according to claim 4 in degrading chloramphenicol, characterized in that The steps include: Inoculating microbacterium into a culture medium, culturing to obtain a seed solution, then adding the seed solution into a system containing chloramphenicol to be treated, and continuing to culture to degrade the chloramphenicol; The culture conditions are 28-32°C and 100-200 rpm shaking culture.

6. Use of the Exiguobacterium according to any one of claims 1 to 3 in degrading plastics.

7. The use of Exiguobacterium spp. in degrading plastics according to claim 6, characterized in that: The plastic includes at least one of polyethylene, polyvinyl chloride, polypropylene and polyethylene terephthalate.

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