A marine-derived Microbacterium foliatum strain CJB-Jou02 and its application

Abstract

The present invention relates to a marine-derived Microbacterium foliatum CJB-Jou02 strain that can efficiently biodegrade PE microplastics, and evaluates its degradation ability. The present invention screened out a Microbacterium foliatum strain (CJB-Jou02) that can degrade PE microplastics from marine mud samples. Microbacterium of leaves ) CJB-Jou02 (strain collection number: CGMCC 29643), the strain treated high-density PE microplastics for 30 days, and the weight loss rate was as high as 16.6±0.46%. The test results based on scanning electron microscopy, Fourier transform infrared spectroscopy, water contact angle meter and X-ray diffractometer confirmed that the strain has the ability to efficiently biodegrade PE microplastics. The dense crystal structure of PE microplastics was destroyed, the particle diameter was significantly reduced, and the hydrophilicity was increased. At the same time, the present invention also found that the leaf microbacterium CJB-Jou02 strain has a certain degradation ability for PE membranes and microplastics such as PA, PP, PS, and PET. The present invention provides a new solution to the environmental pollution problem of PE microplastics.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial engineering, and specifically relates to a marine-derived Microbacterium phylloensis CJB-Jou02 strain and its application in degrading PE microplastics. Background Art

[0002] Polythene (PE) is the most common material in life. PE materials are inexpensive, stable, and have excellent thermodynamic properties. They are used in all walks of life. PE materials are often used in daily necessities such as food packaging bags, garbage bags, disposable gloves, and agricultural mulch. However, since PE materials are not easy to degrade, discarded polyethylene plastics have not been effectively disposed of. After a long period of accumulation, their various hazards are manifested in two aspects: "visual pollution" and "potential pollution". PE plastic products will not be degraded in the natural environment for dozens or even hundreds of years, seriously polluting the ecological environment and causing serious interference to the ecosystem. With the increase in PE waste and the decrease in landfill capacity, the environmental degradation rate of PE has slowed down, resulting in current research being more inclined to reduce waste. Microplastics (MPs) are chemically stable plastic particles with a diameter usually less than 5 mm. MPs are not only stored in large quantities in marine ecosystems, but also in freshwater ecosystems such as lakes, rivers, and reservoirs. It has been reported that the concentration of MPs in freshwater environments is comparable to that in seawater. Lakes are temporary or long-term gathering places for MPs, while rivers are considered the main channels for MPs to flow into the ocean. Microplastics can exist in the environment for a long time, and at least 80% of microplastics in the terrestrial environment reach the marine environment through rivers. Humans are exposed to microplastics in various ways in their daily lives, which can have a significant impact on human health, such as growth and development toxicity, neurotoxicity, digestive tract damage, inflammation, and damage to the immune system.

[0003] The degradation of plastic refers to the change of structure, loss of performance, reduction of relative molecular mass, reduction of structure or mechanical strength, etc. caused by physical, chemical or biological factors. Degradation mainly includes photodegradation, thermo-oxidative degradation and biodegradation. Photodegradation refers to the degradation of plastic under ultraviolet irradiation, and thermo-oxidative degradation refers to the degradation of polymer caused by the main chain of plastic breaking due to oxidation reaction at high temperature. Biodegradation refers to the degradation of plastic under the action of microorganisms (bacteria, fungi, algae, etc.). The essence of biodegradation is to convert chain polymers into monomers or oligomers, and finally achieve complete mineralization of plastics. Although plastics can be degraded by various mechanisms such as heat and photo-oxidation, their degradation rate is extremely slow. The conditions that increase their degradation rate are often too harsh, and they are prone to produce highly harmful secondary pollutants. Therefore, biodegradation is an environmentally friendly and green sustainable development technology, and marine microbial degradation of microplastics has broad research prospects, which can repair the marine ecological environment without adverse effects. Using microorganisms to degrade PE microplastics in situ is the preferred means to solve the problem of microplastic pollution. However, due to the dense structure and poor accessibility of PE microplastics, there are few strains that can efficiently degrade PE microplastics. Therefore, screening microbial strains that can efficiently degrade PE microplastics is of great significance and research value for achieving carbon resource recycling and improving the ecological environment. Summary of the invention

[0004] The present invention aims at the pollution problem caused by the above-mentioned microplastics. By sampling and screening from marine samples, a strain of PE microplastics is obtained. The PE degradation microorganisms reported at home and abroad are mainly bacteria and fungi, and actinomycetes are rarely reported to be isolated as PE degradation strains. The new actinomycetes that can degrade PE microplastics screened out from marine soil by the present invention, i.e., Microbacterium foliorum CJB-Jou02, have fewer reports on the degradation of microplastics by this genus, and experiments show that this strain also has different degrees of degradation effects on microplastics such as PA, PP, PS, and PET. Among them, CJB-Jou02 has the best degradation effect on PE, the most vigorous growth trend, and the highest weight loss rate, so this strain is used for the degradation of PE microplastics.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The first object of the present invention is to propose a Microbacterium foliorum CJB-Jou02 strain, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on January 17, 2024, with a deposit number of CGMCC NO: 29643;

[0007] Microbacterium foliorum (Microbacterium foliorum) CJB-Jou02 is a 16S rDNA sequence with SEQ IDNo:1.

[0008] TGCAAGTCGAACGGTGAACACGGAGCTTGCTCTGTGGGATCAGTGGCGAACGGGTGAGTAACACGTGAGC

[0009] AACCTACCCCTGACTCTGGGATAAGCGCTGGAAACGGCGTCTAATACTGGATACGAGTGGCGACCGCATG

[0010] GTCAGCTACTGGAAAGATTTATTGGTTGGGGATGGGCTCGCGGCCTATCAGCTTGTTGGTGAGGTAATGG

[0011] CTCACCAAGGCGTCGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCA

[0012] GACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCAACGCCGCGTG

[0013] AGGGATGACGGCCTTCGGGTTGTAAACCTCTTTTAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAA

[0014] AAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGAATTATTG

[0015] GGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGCTGTGAAATCCGGAGGCTCAACCTCCGGCCTGCAG

[0016] TGGGTACGGGCAGACTAGAGTGCGGTAGGGGAGATTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATA

[0017] TCAGGAGGAACACCGATGGCGAAGGCAGATCTCTGGGCCGTAACTGACGCTGAGGAGCGAAAGGGTGGGG

[0018] AGCAAACAGGCTTAGATACCCTGGTAGTCCACCCCGTAAACGTTGGGAACTAGTTGTGGGGTCCATTCCA

[0019] CGGATTCCGTGACGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAA

[0020] AGGAATTGACGGGGACCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTAC

[0021] CAAGGCTTGACATATACGAGAACGGGCCAGAAATGGTCAACTCTTTGGACACTCGTAAACAGGTGGTGCA

[0022] TGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGT

[0023] TGCCAGCACGTAATGGTGGGAACTCATGGGATACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGT

[0024] CAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGCTGCAATAC

[0025] CGCGAGGTGGAGCGAATCCCAAAAAGCCGGTCCCAGTTCGGATTGAGGTCTGCAACTCGACCTCATGAAG

[0026] TCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCC

[0027] CGTCAAGTCATGAAAGTCGGTAACACCTGAAGCCGGTGGCCTAACCCTTGTGGAGGGAGCCGTCGAAGG.

[0028] The serial number of the present invention is at the website https: / / www.ncbi.nlm.nih.gov / The name in the text is Microbacterium foliorum strain CJB-Su2. The latter part of the name is self-named. The difference in the name does not lead to different strains. They still belong to the same bacteria.

[0029] Acquisition of Microbacterium foliorum CJB-Jou02:

[0030] 1 g of marine soil sample was weighed and added to LCFBM liquid culture medium containing 0.6 g of PE microplastics, and cultured in a constant temperature shaking incubator at 180 rpm and 30 °C. After enrichment several times, the sample was diluted to 10 -4 , 10 -5 , 10 -6 、 10 -7 、 10 -8 The dilution gradients were spread on CFBAM solid medium, cultured at 30℃ for 7 days, and the growth of colonies on CFBAM solid medium was observed;

[0031] The strains with better growth were selected as the objects of subsequent research, and they were separated and purified by streaking multiple times on LB medium plates;

[0032] A single colony was inoculated on a slant medium and cultured in pure form. After culturing in a 30°C incubator for 2 days, it was moved to a 4°C refrigerator for storage.

[0033] The second purpose of the present invention is to use the Microbacterium foliorum CJB-Jou02 strain or bacterial solution in the biodegradation of PE microplastics.

[0034] The third object of the present invention is to propose a method for biodegrading PE microplastics, wherein the method is to first prepare a bacterial solution of the Microbacterium foliorum CJB-Jou02 strain, and then add the bacterial solution to the sterilized microplastic PE.

[0035] The specific steps are as follows:

[0036] (1) Activation of bacterial strains: The bacterial strains stored on the LB medium slant at 4°C were moved to a 30°C biochemical incubator for activation for 12 h. A single bacterial strain was picked and inoculated into NB liquid medium and cultured at 180 rpm and 30°C.

[0037] (2) Pretreatment of PE microplastics: Accurately weigh 2 g of 150 mesh PE microplastics and sterilize them in a UV clean bench for more than 12 h before use.

[0038] (3) Determination of weight loss rate: The bacterial liquid cultured in NB medium for 2 days was centrifuged at 3000 rpm for 5 min, and the bacteria were resuspended in LCFBM medium. This process was repeated three times to completely remove the remaining NB. The recovered bacteria were added to 50 mL of LCFBM and 2 g of sterilized microplastic PE were added. The cells were cultured in a constant temperature shaking incubator at 180 rpm and 30°C for 30 days.

[0039] To determine the weight loss rate of microplastic PE degradation by leaf microbacteria, the biodegradation rate was evaluated by comparing the initial dry weight of polyethylene before and after cultivation. The plastic was washed with 2% SDS to remove the bacteria adsorbed on it, rinsed with anhydrous ethanol three times, and treated in an oven at 60°C for 24h. The weight of the dried microplastic PE was measured independently three times.

[0040] The degradation rate of microplastics is determined using the following formula:

[0041] w% = ab / a × 100%

[0042] a = weight of plastic before degradation

[0043] b = weight of plastic after degradation

[0044] The leaf microbacterium CJB-Jou02 obtained by the present invention is used for the biodegradation characteristics evaluation method of PE microplastics. Usually, a scanning electron microscope (SEM) is used to observe the obvious pits and wrinkles on the plastic after degradation, or an atomic force microscope (AFM) is used to observe the roughness of the surface of the plastic after degradation; Fourier transform infrared spectroscopy (FTIR) is used to analyze the changes in functional groups of the plastic before and after degradation; a water contact angle meter (WCA) is used to measure the changes in hydrophilicity / hydrophobicity before and after degradation; and an X-ray diffractometer (XRD) is used to analyze its crystallinity.

[0045] The fourth object of the present invention is to propose a bacterial agent for biodegrading PE microplastics, wherein the bacterial agent uses the Microbacterium foliorum CJB-Jou02 strain as the main active ingredient, and the bacterial agent contains the culture solution, culture solution concentrate or culture suspension of the strain.

[0046] The following beneficial effects can be obtained through the above technical solution:

[0047] Compared with other strains with the ability to degrade PE microplastics reported in current literature or patents, Microbacterium phylloides CJB-Jou02 is a new actinomycete that can efficiently degrade PE microplastics. After 30d of processing PE microplastics, the weight loss rate is as high as 16.6±0.46%, the compact structure of PE microplastics is destroyed, and the particle diameter is significantly reduced. The PE microplastics degraded by the CJB-Jou02 strain in the present invention are high-density polyethylene (HDPE), which belongs to the more difficult to degrade PE, which is enough to show that the CJB-Jou02 strain in the present invention has the characteristics of efficient degradation of PE microplastics. At the same time, the present invention also finds that Microbacterium phylloides CJB-Jou02 strain also has a certain degradation ability for PE film, can attach and destroy PE film, and Microbacterium phylloides CJB-Jou02 can also use microplastics such as PA, PP, PS, PET as carbon source, indicating that CJB-Jou02 strain has good application prospects in the subsequent degradation of microplastics and the recycling of its hydrolyzate. The patent of this invention is of great significance for solving white pollution, saving oil resources that are facing depletion, reducing carbon dioxide emissions, and protecting the ecological environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 : Purification and isolation of plastic-degrading strains;

[0049] Figure 2 :CJB-Jou02 colony morphology and electron microscopic cell morphology;

[0050] Figure 3 : Molecular biological identification of CJB-Jou02 strain (left: PCR amplification band of 16S rDNA sequence of CJB-Jou02 strain, left band: DL2000, right band: 16S rDNA fragment of CJB-Jou02; right: phylogenetic tree analysis of CJB-Jou02 strain);

[0051] Figure 4 : Growth curve and optimum temperature of CJB-Jou02 (left: optimum temperature; right: growth curve);

[0052] Figure 5 : FTIR analysis (left: surface microscopic characteristics of PE microplastics before degradation; right: surface microscopic characteristics of PE microplastics after degradation by CJB-Jou02 strain);

[0053] Figure 6 : Water contact angle meter (WCA) measurement (left: contact angle of PE microplastics before degradation; right: contact angle of PE microplastics after degradation by CJB-Jou02 strain);

[0054] Figure 7: X-ray diffractometer (XRD) analysis (a: untreated PE microplastics; b: control group PE microplastics; c: PE microplastics treated with strain CJB-Jou02);

[0055] Figure 8 : Scanning electron microscopy (SEM) observation (attachment of strain CJB-Jou02 on PE microplastics: a-c; surface microscopic characteristics analysis of PE microplastics before degradation: d-f; surface microscopic characteristics analysis of PE microplastics after degradation: g-i);

[0056] Fig. 9 : Scanning electron microscope (SEM) observation (attachment of strain CJB-Jou02 on PE film: a-b; surface microscopic characteristics analysis of PE film before degradation: c-d; surface microscopic characteristics analysis of PE film after degradation: e-f);

[0057] Fig.10 : Growth of CJB-Jou02 on PE film (left: control group; right: experimental group);

[0058] Fig.11 : Growth chart of CJB-Jou02 in PA, PP, PE, PS, and PET LCFBM liquid culture medium. DETAILED DESCRIPTION

[0059] The following is combined with Figure 1-9 The present invention is further described with examples:

[0060] A Microbacterium foliorum CJB-Jou02 strain, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 17, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO: 29643.

[0061] The 16S rDNA sequence of a Microbacterium foliorum strain CJB-Jou02 is shown in SEQ ID No: 1:

[0062] TGCAAGTCGAACGGTGAACACGGAGCTTGCTCTGTGGGATCAGTGGCGAACGGGTGAGTAACACGTGAGC

[0063] AACCTACCCCTGACTCTGGGATAAGCGCTGGAAACGGCGTCTAATACTGGATACGAGTGGCGACCGCATG

[0064] GTCAGCTACTGGAAAGATTTATTGGTTGGGGATGGGCTCGCGGCCTATCAGCTTGTTGGTGAGGTAATGG

[0065] CTCACCAAGGCGTCGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCA

[0066] GACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCAACGCCGCGTG

[0067] AGGGATGACGGCCTTCGGGTTGTAAACCTCTTTTAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAA

[0068] AAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGAATTATTG

[0069] GGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGCTGTGAAATCCGGAGGCTCAACCTCCGGCCTGCAG

[0070] TGGGTACGGGCAGACTAGAGTGCGGTAGGGGAGATTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATA

[0071] TCAGGAGGAACACCGATGGCGAAGGCAGATCTCTGGGCCGTAACTGACGCTGAGGAGCGAAAGGGTGGGG

[0072] AGCAAACAGGCTTAGATACCCTGGTAGTCCACCCCGTAAACGTTGGGAACTAGTTGTGGGGTCCATTCCA

[0073] CGGATTCCGTGACGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAA

[0074] AGGAATTGACGGGGACCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTAC

[0075] CAAGGCTTGACATATACGAGAACGGGCCAGAAATGGTCAACTCTTTGGACACTCGTAAACAGGTGGTGCA

[0076] TGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGT

[0077] TGCCAGCACGTAATGGTGGGAACTCATGGGATACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGT

[0078] CAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGCTGCAATAC

[0079] CGCGAGGTGGAGCGAATCCCAAAAAGCCGGTCCCAGTTCGGATTGAGGTCTGCAACTCGACCTCATGAAG

[0080] TCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCC

[0081] CGTCAAGTCATGAAAGTCGGTAACACCTGAAGCCGGTGGCCTAACCCTTGTGGAGGGAGCCGTCGAAGG.

[0082] Example 1 Strain Screening

[0083] 1 g of marine soil sample was weighed and added to 50 mL of LCFBM liquid culture medium containing 0.6 g of PE microplastics. The culture was placed in a constant temperature shaking incubator at 180 rpm and 30 °C. The enrichment screening was performed for 10 days. After several enrichments, the sample was diluted to 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8The dilution gradients were spread on CFBAM solid medium and cultured at 30℃ for 7 days. The growth of the colonies on CFBAM solid medium was observed. After the strains grew out, they were purified and separated on plates. The strains with better growth were selected as the subsequent research objects. They were separated and purified by streaking on LB medium plates for multiple times until there were no foreign bacteria ( Figure 1 shown).

[0084] After culturing in a 30°C incubator for 2 days, pick a single bacterium and inoculate it into 50 mL NB liquid culture medium and place it in a constant temperature shaking incubator at 180 rpm and culture it at 30°C for 2 days. Accurately weigh 2 g of microplastic 150 mesh PE and sterilize it in a UV clean bench for 12 hours before use. Centrifuge the bacterial solution cultured in NB at 3000 rpm for 5 minutes to remove the NB culture medium and resuspend the precipitated bacteria in LCFBM culture medium. Repeat the above steps three times to completely remove the residual NB liquid culture medium. Resuspend the recovered bacteria in LCFBM and adjust the OD 600 The concentration of PE was 0.2-0.6, and 2 g of microplastic PE was added, and the culture was carried out in a constant temperature shaking incubator at 180 rpm and 30 ° C. After 30 days of culture, the biodegradation rate was evaluated by comparing the initial dry weight of PE before and after culture. The bacteria adsorbed on the plastic were removed by washing with 2% SDS, and rinsed three times with anhydrous ethanol, and treated in a 60 ° C oven for more than 24 hours to constant weight, and the weight of the dry microplastic PE was measured independently 3 times. The degradation rate of the microplastic PE by the strain was measured separately, and the highest microplastic degradation rate of the strain was 16.6 ± 0.46%, which was named CJB-Jou02.

[0085] After the strain CJB-Jou02 was cultured in LB solid medium at 30°C for 48 h, its colonies were round, small, light yellow, with a smooth and rounded surface, slightly raised, and neat edges. It was a Gram-positive bacterium ( Figure 2 shown).

[0086] LB medium: 10 g / L peptone, 5 g / L yeast powder, 20 g / L agar, ultrapure water, pH 7.0. 121°C, high pressure sterilization for 20 min.

[0087] NB medium: 3 g / L beef extract, 1 g / L yeast extract, 10 g / L sucrose, 5 g / L peptone, ultrapure water, pH 7.0. 121°C, high pressure sterilization for 20 min.

[0088] LCFBM liquid medium: According to the ASTM plastics bacterial resistance test standard (G22-76, 1996), the liquid carbon-free basic medium (LCFBM) prepared with ultrapure water contains (per 1000 mL) 0.7 g KH2PO4, 0.7 g K2HPO4, 0.7 g MgSO4·7H2O, 1.0 g NH4NO3, 0.005 g NaCl, 0.002 g FeSO4·7H2O, 0.002 g ZnSO4·7H2O, 0.001 g MnSO4·H2O. Sterilize at 121°C for 20 min.

[0089] CFBAM solid medium: Add 20 g agar to 1000 mL LCFBM medium to prepare carbon-free basic agar medium (CFBAM). Autoclave at 121°C for 20 min.

[0090] Example 2 Bacterial species identification

[0091] 16S rDNA identification of strain CJB-Jou02: Pick a single colony and inoculate it in LB liquid medium at 180rpm and 30℃ for 12h. Collect 0.5mL of bacterial culture solution in a 1.5mL centrifuge tube, centrifuge at 12000rpm for 2min at 4℃, and discard the supernatant. Use a rapid extraction kit to extract the DNA of the CJB-Jou02 genome, use it as a template, and use universal primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' to amplify the 16S rDNA gene.

[0092] PCR amplification conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 25 s, for a total of 35 cycles; extension at 72°C for 5 min.

[0093] PCR results: After PCR, the target gene product, i.e., 16S rDNA (marker: DL2000), was analyzed by 1% agarose gel electrophoresis. Figure 3The 16S rDNA of the strain was sent to Qingdao Qingke Biological Co., Ltd. for sequencing. The obtained sequence was uploaded to the database GenBank (GenBank NO.: PP101514.1), and the sequencing results were analyzed by BLAST, and the strain sequence with higher homology was selected to compare with the strain CJB-Jou02 to determine the type of the strain. The comparison and evolutionary relationship analysis of the strain CJB-Jou02 with other strains showed that the 16S rDNA sequence of the strain and the Microbacterium foliorum were clustered into one branch, with the closest relationship. Based on the results of morphological and molecular biological identification, the strain was preliminarily identified as Microbacterium foliorum, which has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration Committee on January 24, 2024, and the strain deposit number is CGMCC 29643.

[0094] Example 3 Determination of the optimum temperature and growth curve of CJB-Jou02

[0095] The activated CJB-Jou02 strain was picked up with a disposable inoculation loop and inoculated into 5 mL of LB liquid culture medium. After 12 hours in a constant temperature shaking incubator at 180 rpm and 30 °C, it was transferred to 100 mL of LB liquid culture medium. A total of 12 bottles were cultured at 180 rpm at four temperatures: 25 °C, 28 °C, 30 °C, and 37 °C.

[0096] Take 200uL of culture solution from each bottle and inject it into a clean 96-well plate in the clean bench every 4 hours. Take three replicates of each sample and measure it with a full-wavelength microplate reader. Adjust the wavelength to OD 600 , measured with OD 600 The value is the vertical axis, and the culture time is the horizontal axis. The growth curve of CJB-Jou02 strain at four temperatures of 25℃, 28℃, 30℃, and 37℃ is plotted to determine the optimal growth temperature of CJB-Jou02 strain. Using this temperature as the culture temperature, repeat the above steps and take the culture solution from each bottle every 2h to measure the OD 600 , with OD 600 The value is the vertical axis, and the culture time is the horizontal axis. The growth curve of CJB-Jou02 strain under 30℃ temperature condition is drawn ( Figure 4 shown).

[0097] Example 4 Infrared spectroscopy analysis (FTIR)

[0098] FTIR spectroscopy was used to determine the concentration of PE microplastics at 4000-400 cm -1The contraction, vibration, disappearance and growth of groups in the original microplastics and the microplastics biodegraded by strain CJB-Jou02 were further verified to be effective in the degradation of PE microplastics by strain CJB-Jou02. Figure 5 After microorganisms attach to the surface of microplastics, they can change the polymer structure through oxidation reactions. That is, oxidation reactions increase the hydrophilicity of PE by producing functional groups such as carbonyl, carbon-carbon double bonds, and carbon-oxygen double bonds, thereby enhancing its biodegradability. 1723cm -1 The increase in the peak value at is attributed to the stretching vibration of -C=O-, indicating that the surface of the PE particles has aged during the degradation process, causing changes in the properties of the microplastic material and affecting its degradability.

[0099] Example 5 Water Contact Angle Apparatus (WCA) Measurement

[0100] The hydrophilicity and hydrophobicity of PE microplastics can be determined by testing the contact angle between its surface and water. Generally, the smaller the contact angle, the lower the hydrophobicity and the higher the hydrophilicity. Generally, the better the hydrophilicity of PE, the more conducive it is for microorganisms to attach and colonize. The water contact angle of PE microplastics before and after 30 days of degradation by strain CJB-Jou02 was tested. The results were ( Figure 6 As shown in the figure, the contact angles of PE microplastics after degradation by CJB-Jou02 were 114.88° on the left and 112.94° on the right. The average contact angle was 113.91°. The contact angles of PE microplastics in the control group were 146.76° on the left, 146.51° on the right, and 146.635° on the average. Compared with the control group, the water contact angle of PE microplastics became smaller before and after 30 days of degradation by strain CJB-Jou02. The decrease in the contact angle of PE microplastics indicates that the hydrophobicity of PE microplastics has become lower and the hydrophilicity has become higher. It can be preliminarily explained that the surface of PE microplastics is oxidized by strain CJB-Jou02 to produce hydrophilic groups. The production of hydrophilic groups in PE microplastics will facilitate the attachment and colonization of a large number of strains, thereby reducing their ability to resist subsequent degradation by strains.

[0101] Example 6 X-ray diffractometer (XRD) analysis

[0102] XRD (X-ray Diffraction) is a fast, accurate and efficient non-destructive material testing technology. In this study, the XRD spectra of PE microplastics treated with and without strain CJB-Jou02 were compared. In the XRD spectrum of PE microplastics, the peaks at 22° and 25° are the reflections of the 110 and 200 faces of the orthorhombic phase, respectively. These peaks are characteristic peaks of PE. When the peaks of the PE microplastics in the experimental group were compared with those in the control group, it was observed that the intensity and width of these peaks changed ( Figure 7The decrease in the intensity of the XRD characteristic peak is due to the oxidative degradation of PE microplastics by strain CJB-Jou02, which then changes the crystal structure of PE microplastics. The amorphous properties of the test material are represented by the peak width, while the crystalline properties are represented by a peak height. As the degree of degradation increases, the decrease in peak height may be due to the transformation of the crystal structure into an amorphous structure, further demonstrating the feasibility of the strain to degrade PE microplastics.

[0103] Example 7 Scanning electron microscope (SEM) observation

[0104] First, the PE microplastics degraded by the degradation strain CJB-Jou02 were sterilized, treated with 2% SDS for more than 4 hours, washed to remove the bacteria adsorbed on the plastic, and rinsed three times with anhydrous ethanol. After drying in a 60°C oven for more than 24 hours until constant weight, electron scanning microscopy (SEM) observation was performed. In addition, this experiment also conducted electron microscopic observations of the strain CJB-Jou02 before and after the degradation of PE membranes. First, the PE membranes were sterilized, treated with 2% SDS for more than 4 hours, washed to remove the bacteria adsorbed on the PE membranes, and rinsed three times with anhydrous ethanol and dried naturally. The PE microplastics and PE membranes in the treatment group and the control group were sputtered with gold for 60 seconds using an ion sputtering instrument (Edwards Vacuum Ltd., England); they were observed using a scanning electron microscope ((PhenomPro, USA, phenom)) at a voltage of 15KV. The results showed that the surface of PE microplastics and PE films before degradation was relatively flat and smooth. After inoculation with the degradation bacteria CJB-Jou02, the surface became rougher, with local unevenness, obvious gullies, grooves, pits and wrinkles. The degree of damage was significantly greater than that of the microplastics before degradation, and the particle size of the treated microplastics was significantly smaller than that of the PE microplastics before degradation ( Figure 8 , 9 shown).

[0105] The erosion and holes formed on the surface of microplastics and PE films can reflect the colonization and degradation degree of the target plastics by microorganisms. Microscopic detection showed that the microplastics degraded by the PE microplastic-degrading strain CJB-Jou02 showed obvious damage and erosion changes on the surface. This verifies that the polyethylene microplastic-degrading strain CJB-Jou02 can use PE microplastics as a carbon source to meet its own growth and development, thereby achieving the purpose of degrading PE microplastics.

[0106] Example 8 Degradation of PE Film by Strain CJB-Jou02

[0107] Cut the PE plastic film into square pieces, weigh them accurately and then sterilize them. Soak them in 2% SDS and anhydrous ethanol for more than 4 hours respectively. Rinse them with sterile water 3 times after treatment. Use sterile filter paper to absorb the moisture on the surface of the plastic film on the clean bench, and sterilize them with ultraviolet light for 12 hours. Centrifuge the CJB-Jou02 bacterial solution cultured in NB at 3000rpm for 5 minutes to remove the NB culture medium and resuspend the precipitated bacteria in LCFBM culture medium. Repeat the above steps three times to completely remove the residual NB liquid culture medium. Resuspend the recovered bacteria in LCFBM and adjust the OD 600 1.0, sample and dilute to a suitable gradient, draw 200uL and apply it on CFBAM solid culture medium, the control group is sterile water, then cover the PE plastic film in the center of the plate, culture at 30℃ for 30 days, and observe the growth of the colony ( Fig.10 shown).

[0108] The results showed that the area coated with CJB-Jou02 bacterial solution and covered with PE plastic film grew vigorously and showed a distinct goose yellow color. Microscopic examination results showed short rods, which were confirmed to be leaf microbacteria. However, no obvious colonies grew in the area not covered by PE plastic film and the control group, further proving that strain CJB-Jou02 can utilize the decomposed PE plastic film to colonize and grow on its surface.

[0109] Example 9 Degradation of different microplastics by strain CJB-Jou02

[0110] In addition to studying the degradation ability of strain CJB-Jou02 on PE microplastics with PE microplastics as the sole carbon source, this project also conducted a preliminary study on the degradation effect of PA, PP, PS, PET and other microplastics. The results showed that CJB-Jou02 can grow on plates with PA, PP, PS, and PET microplastics as the sole carbon source ( Fig.11 As shown), it shows that strain CJB-Jou02 has great degradation potential in degrading microplastics.

[0111] The above are all preferred embodiments of the present invention. For ordinary technicians in this technical field, without departing from the principle of the present invention, various equivalent modifications to the present invention belong to the protection scope of the claims attached to this application.

Claims

1. A strain of Microbacterium spp. Microbacterium foliorum ) CJB-Jou02 strain, characterized in that The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on January 17, 2024, with the deposit number CGMCC NO: 29643. Microbacterium foliorum ) The 16S rDNA sequence of the CJB-Jou02 strain is shown in SEQ ID No:

1.

2. The leaf microbacterium according to claim 1 ( Microbacterium foliorum ) Application of CJB-Jou02 strain or bacterial liquid in biodegradation of PE microplastics.

3. The leaf microbacterium according to claim 1 ( Microbacterium foliorum ) Application of CJB-Jou02 strain or bacterial solution in biodegradation of PA, PP, PS, and PET microplastics.

4. A method for biodegrading microplastics, characterized in that: The method is to firstly treat the leaf microbacterium ( Microbacterium foliorum ) The CJB-Jou02 strain is prepared into a bacterial liquid, which is then added to the sterilized microplastics PE or PA or PP or PS or PET for degradation.

5. A method for biodegrading microplastics according to claim 4, characterized in that: The specific method is as follows: (1) Activation of bacterial strains: Move the bacterial strains stored on the LB medium slant at 4°C to a 30°C biochemical incubator for activation for 12 h, pick a single strain and inoculate it into NB liquid medium at 180 rpm and 30°C; (2) Pretreatment of PE microplastics: Accurately weigh 2 g of 150 mesh PE microplastics and sterilize them in a UV clean bench for more than 12 h before use; (3) Determination of weight loss rate: The bacterial liquid cultured in NB medium for 2 days was centrifuged at 3000 rpm for 5 min, and the bacteria were resuspended in LCFBM medium. This was repeated three times to completely remove the remaining NB. The recovered bacteria were added to 50 mL LCFBM and 2 g of sterilized microplastic PE were added. The cells were cultured in a constant temperature shaking incubator at 180 rpm and 30°C for 30 days.

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