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CN119351247B - Marine source microbacterium strain CJB-Jou02 and application thereof - Google Patents

Marine source microbacterium strain CJB-Jou02 and application thereof Download PDF

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CN119351247B
CN119351247B CN202411321761.7A CN202411321761A CN119351247B CN 119351247 B CN119351247 B CN 119351247B CN 202411321761 A CN202411321761 A CN 202411321761A CN 119351247 B CN119351247 B CN 119351247B
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jou02
cjb
microplastics
microplastic
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CN119351247A (en
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杨光
胡志红
郭可欣
黄雪萍
吴依雯
俞国豪
崔仟
甘萍
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Jiangsu Ocean University
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Abstract

本发明涉及一株可高效生物降解PE微塑料的海洋源叶片微杆菌CJB‑Jou02菌株,并对其降解能力进行了评价。本发明从海洋泥样中筛选出的可降解PE微塑料叶片微杆菌(Microbacterium foliorum) CJB‑Jou02(菌株保藏号:CGMCC 29643),该菌株处理高密度PE微塑料30 d,失重率高达16.6±0.46%,并基于扫描电子显微镜、傅立叶变换红外光谱、水接触角仪和X‑射线衍射仪测试结果证实该菌株具有高效生物降解PE微塑料的能力,PE微塑料致密的晶体结构被破坏,颗粒直径显著变小,亲水性增加。同时,本发明还发现叶片微杆菌CJB‑Jou02菌株对PE膜片和PA、PP、PS、PET等微塑料菌有一定的降解能力,本发明为PE微塑料的环境污染问题提供了一种新的解决方案。

The present invention relates to a marine source Microbacterium foliorum CJB-Jou02 strain that can efficiently biodegrade PE microplastics, and its degradation ability is evaluated. The present invention screened out the degradable PE microplastic Microbacterium foliorum CJB-Jou02 (strain deposit number: CGMCC 29643) from marine mud samples. 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, and the dense crystal structure of PE microplastics is destroyed, the particle diameter is significantly reduced, and the hydrophilicity is increased. At the same time, the present invention also found that the Microbacterium foliorum 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

Marine source microbacterium strain CJB-Jou02 and application thereof
Technical Field
The invention belongs to the technical field of microbial engineering, and particularly relates to a marine source blade microbacterium CJB-Jou02 strain and application thereof in degradation of PE (polyethylene) microplastic.
Background
Polyethylene (polythene, PE for short) is the most common material in life, and PE materials are low in price, good in stability, excellent in thermodynamic performance and the like, and are applied to various industries, and PE materials can be frequently used in usual living goods such as food packaging bags, garbage bags, disposable gloves, agricultural mulching films and the like. However, the PE material is not easy to degrade, the waste polyethylene plastic is not effectively treated, and various hazards are shown in two aspects of visual pollution and potential pollution after long-time accumulation. PE plastic products cannot be degraded in the natural environment for tens or hundreds of years, so that the ecological environment is seriously polluted, and serious interference is generated to an ecological system. As PE waste increases and landfill capacity decreases, environmental degradation rates of PE are slowed down, making current research more prone to waste reduction. Microplastic (Microplastics, MPs) is a plastic particle with stable chemical properties, the diameter is usually smaller than 5mm, and the MPs are not only stored in a large amount in a marine ecosystem, but also exist in a freshwater ecosystem such as lakes, rivers, reservoirs and the like. It has been reported that the concentration of MPs in fresh water environments is comparable to that of seawater. Lakes are temporary or long-term collections of MPs, and rivers are considered the primary channels through which MPs flow into the ocean. Microplastic can exist in the environment for a long time, and at least 80% of microplastic in land environments is transported from river to sea. Humans are exposed to micro-plastics in daily life through various pathways that can have significant impact on human health, such as growth and development toxicity, neurotoxicity, digestive tract damage, causing inflammation, disrupting the immune system, and the like.
Degradation of plastics refers to the change in structure, loss of properties, reduction in relative molecular mass, reduction in structural or mechanical strength, etc. of a polymer due to physical, chemical or biological factors. Degradation mainly comprises photodegradation, thermal oxidative degradation and biodegradation, wherein photodegradation refers to degradation of plastics under ultraviolet irradiation, and thermal oxidative degradation refers to polymer degradation caused by the fact that main chains of plastics are broken due to oxidation reaction at high temperature. Biodegradation refers to the degradation of plastics by microorganisms (bacteria, fungi, algae, etc.). The essence of biodegradation is that chain polymers are converted into monomers or oligomers, the complete mineralization of plastics is finally realized, the plastics can be degraded through various mechanisms such as heat, photooxidation and the like, but the degradation speed is extremely slow, the condition for increasing the degradation speed is often too harsh, and secondary pollutants with stronger hazard are easily generated, so the biodegradation is an environment-friendly and green sustainable development technology, and the marine microorganism degradation micro-plastics has wide research prospect, can repair the marine ecological environment, and cannot generate adverse effects. The PE microplastic is degraded in situ by microorganisms, so that the method is a preferred means for solving the problem of microplastic pollution. However, due to the compact structure and poor accessibility of PE microplastic, few strains capable of efficiently degrading PE microplastic exist. Therefore, screening the microorganism strain capable of efficiently degrading PE micro-plastics has important significance and research value for realizing recycling of carbon resources and improving ecological environment.
Disclosure of Invention
Aiming at the pollution problem brought by the microplastic, the invention obtains a strain of the degradable PE microplastic by sampling and screening marine samples, and PE degrading microorganisms reported at home and abroad mainly take bacteria and fungi, and actinomycetes are freshly reported as PE degrading strains to be separated. The novel actinomycetes of the degradable PE microplastic screened from the marine soil, namely the microbacterium lamina (Microbacterium foliorum) CJB-Jou02, have fewer reports on the degradation microplastic of the genus, and experiments show that the strain has different degradation effects on the microplastic such as PA, PP, PS, PET and the like. Among them, CJB-Jou02 has the best degradation effect on PE, the most vigorous growth trend and the highest weight loss rate, so the strain is used for PE micro-plastic degradation.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
The first aim of the invention is to provide a microbacterium vane (Microbacterium foliorum) CJB-Jou02 strain which is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 29643 in the 1 st month of 2024;
the 16S rDNA sequence of the microbacterium vane (Microbacterium foliorum) CJB-Jou02 strain is shown in SEQ ID No. 1:
TGCAAGTCGAACGGTGAACACGGAGCTTGCTCTGTGGGATCAGTGGCGAACGGGTGAGTAACACGTGAGC
AACCTACCCCTGACTCTGGGATAAGCGCTGGAAACGGCGTCTAATACTGGATACGAGTGGCGACCGCATG
GTCAGCTACTGGAAAGATTTATTGGTTGGGGATGGGCTCGCGGCCTATCAGCTTGTTGGTGAGGTAATGG
CTCACCAAGGCGTCGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCA
GACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCAACGCCGCGTG
AGGGATGACGGCCTTCGGGTTGTAAACCTCTTTTAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAA
AAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGAATTATTG
GGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGCTGTGAAATCCGGAGGCTCAACCTCCGGCCTGCAG
TGGGTACGGGCAGACTAGAGTGCGGTAGGGGAGATTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATA
TCAGGAGGAACACCGATGGCGAAGGCAGATCTCTGGGCCGTAACTGACGCTGAGGAGCGAAAGGGTGGGG
AGCAAACAGGCTTAGATACCCTGGTAGTCCACCCCGTAAACGTTGGGAACTAGTTGTGGGGTCCATTCCA
CGGATTCCGTGACGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAA
AGGAATTGACGGGGACCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTAC
CAAGGCTTGACATATACGAGAACGGGCCAGAAATGGTCAACTCTTTGGACACTCGTAAACAGGTGGTGCA
TGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGT
TGCCAGCACGTAATGGTGGGAACTCATGGGATACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGT
CAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGCTGCAATAC
CGCGAGGTGGAGCGAATCCCAAAAAGCCGGTCCCAGTTCGGATTGAGGTCTGCAACTCGACCTCATGAAG
TCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCC
CGTCAAGTCATGAAAGTCGGTAACACCTGAAGCCGGTGGCCTAACCCTTGTGGAGGGAGCCGTCGAAGG。
the name of the serial number in the website https:// www.ncbi.nlm.nih.gov/is Microbacterium foliorum strain CJB-Su2, the latter part of the name belongs to self-naming, and the different strains are not caused by the difference of the name parts, but still belong to the same strain.
Acquisition of Microbacterium blade (Microbacterium foliorum) CJB-Jou 02:
Weighing 1g of marine soil sample, adding the marine soil sample into LCFBM liquid culture medium containing 0.6g of PE microplastic, placing the liquid culture medium into a constant-temperature shake culture box at 180rpm and at 30 ℃ for culture, sampling and diluting the liquid culture medium into a dilution gradient of 10 -4、10-5、10-6 10-7 10-8 after enrichment for several times, respectively coating the liquid culture medium on CFBAM solid culture medium, culturing the liquid culture medium at 30 ℃ for 7d, and observing the growth condition of colonies on the CFBAM solid culture medium;
selecting a strain with better growth vigor as a follow-up research object, and carrying out repeated streak separation and purification culture on an LB culture medium plate;
The single colony is inoculated on a slant culture medium and is subjected to pure culture, and after being cultured for 2d in a 30 ℃ incubator, the single colony is moved into a 4 ℃ refrigerator to be preserved for standby.
The second purpose of the invention is the application of the microbacterium vane (Microbacterium foliorum) CJB-Jou02 strain or bacterial liquid in the biodegradation of PE microplastic.
The third purpose of the invention is to provide a method for biodegradation of PE micro-plastics, which comprises the steps of preparing bacterial liquid from a strain of Microbacterium glabrous (Microbacterium foliorum) CJB-Jou02, and then adding the bacterial liquid into sterilized PE micro-plastics.
The method comprises the following specific steps:
(1) And (3) strain activation, namely transferring the strain stored on the inclined plane of the LB culture medium under the condition of 4 ℃ to a 30 ℃ biochemical incubator for activation for 12 hours, and picking single strain to be inoculated to the NB liquid culture medium for 180rpm and culturing at 30 ℃.
(2) PE microplastic pretreatment, namely accurately weighing 150-mesh PE 2g of microplastic, and sterilizing for more than 12 hours by an ultraviolet clean bench for later use.
(3) Determination of the loss of weight the bacterial liquid cultivated in NB medium 2d was centrifuged at 3000rpm for 5min, and the bacterial liquid was resuspended in LCFBM medium, the remaining NB was removed completely three times, the recovered bacterial cells were added to 50mL LCFBM and 2g of the sterilized microplastic PE was added and cultivated in a shaking incubator at 180rpm and 30℃for 30d.
To determine the rate of loss of weight of the micro-plastic PE degraded by the micro-bacilli in the leaves, the rate of biodegradation was evaluated by comparing the initial dry weight of the polyethylene before and after the culture. Bacteria adsorbed on the plastic were removed by washing with 2% SDS, and washed three times with absolute ethanol, and oven-treated at 60℃for 24 hours, and the weight of the dried microplastic PE was independently measured 3 times.
The degradation rate of the microplastic is determined by the following formula:
w%=a-b/a×100%
a = weight before plastic degradation
B=weight after plastic degradation
The characteristic evaluation method of the microbacterium vane CJB-Jou02 for the biodegradation of PE microplastic generally adopts a Scanning Electron Microscope (SEM) to observe pits and folds after the degradation of the plastic or an Atomic Force Microscope (AFM) to observe the roughness of the surface of the plastic after the degradation, a Fourier transform infrared spectrum (FTIR) to analyze the change of functional groups before and after the degradation of the plastic, a water contact angle meter (WCA) to measure the change of hydrophilicity/hydrophobicity before and after the degradation, and an X-ray diffractometer (XRD) to analyze the crystallinity.
The fourth object of the invention is to provide a microbial agent for biodegradation of PE micro-plastics, which takes the micro-bacillus calmette guerin (Microbacterium foliorum) CJB-Jou02 strain as a main active ingredient, and the microbial agent contains a culture solution, a culture solution concentrate or a culture bacterial suspension of the strain.
The following beneficial effects can be obtained through the technical scheme:
Compared with other strains with the capability of degrading PE (polyethylene) microplastic reported in the current literature or patent, the Microbacterium vane CJB-Jou02 is a novel actinomycete capable of degrading PE microplastic efficiently, the weight loss rate of the actinomycete is up to 16.6+/-0.46% after the PE microplastic is treated for 30 days, the compact structure of the PE microplastic is damaged, and the particle diameter is obviously reduced. The PE microplastic degraded by the CJB-Jou02 strain is high-density polyethylene (HDPE), belongs to PE which is more difficult to degrade, and is sufficient to show that the CJB-Jou02 strain has the characteristic of efficiently degrading the PE microplastic. Meanwhile, the invention also discovers that the microbacterium vane CJB-Jou02 strain has certain degradation capability on PE membrane, can attach and destroy the PE membrane, and can also utilize PA, PP, PS, PET and other microplastics as carbon sources, thus showing that the CJB-Jou02 strain has good application prospect in subsequent microplastic degradation and hydrolysate recycling. The invention has important significance for solving white pollution, saving depleted petroleum resources, reducing carbon dioxide emission, protecting ecological environment and the like.
Drawings
FIG. 1 shows the purification and separation of plastic degradation strains;
FIG. 2 CJB-Jou02 colony morphology and electron microscope cell morphology;
FIG. 3: molecular biological identification of CJB-Jou02 strain (left panel: PCR amplified band of 16S rDNA sequence of CJB-Jou02 strain, left band: DL2000, right band: 16S rDNA fragment of CJB-Jou 02; right panel: evolutionary tree analysis of CJB-Jou02 strain);
FIG. 4 CJB-Jou02 growth curve and optimum temperature (left: optimum temperature: right: growth curve);
FIG. 5, infrared spectroscopy (FTIR) analysis (left panel: surface microfeature analysis before PE microplastic degradation; right panel: surface microfeature analysis after PE microplastic degradation by CJB-Jou02 strain);
FIG. 6 is a graph showing the Water Contact Angle (WCA) measurement (left graph: contact angle before PE microplastic degradation; right graph: contact angle after PE microplastic degradation by CJB-Jou02 strain);
FIG. 7:X analysis by radiation diffraction (XRD) (a: untreated PE microplastic; b: control PE microplastic; c: PE microplastic treated with strain CJB-Jou 02);
FIG. 8 shows observation by a Scanning Electron Microscope (SEM) (the adhesion condition of the strain CJB-Jou02 on PE microplastic: a-c; the micro-feature analysis of the surface before PE microplastic degradation: d-f; and the micro-feature analysis of the surface after PE microplastic degradation: g-i);
FIG. 9 shows observation by a Scanning Electron Microscope (SEM) (the adhesion condition of the strain CJB-Jou02 on the PE membrane: a-b; the micro-feature analysis of the surface before the PE membrane is degraded: c-d; and the micro-feature analysis of the surface after the PE membrane is degraded: e-f);
FIG. 10 CJB-Jou02 growth on PE membrane (left panel: control group; right panel: experimental group);
FIG. 11 CJB-Jou02 growth chart at PA, PP, PE, PS, PET LCFBM liquid medium.
Detailed Description
The invention is further illustrated by the following examples in conjunction with figures 1-9:
A strain of Microbacterium lobus (Microbacterium foliorum) CJB-Jou02 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 1 month 17 of 2024, and has a preservation address of North Star Xiyu No. 1, 3 of the Korean region of Beijing, and a preservation number of CGMCC No. 29643.
The 16S rDNA sequence of the microbacterium vane (Microbacterium foliorum) CJB-Jou02 strain is shown in SEQ ID No. 1:
TGCAAGTCGAACGGTGAACACGGAGCTTGCTCTGTGGGATCAGTGGCGAACGGGTGAGTAACACGTGAGC
AACCTACCCCTGACTCTGGGATAAGCGCTGGAAACGGCGTCTAATACTGGATACGAGTGGCGACCGCATG
GTCAGCTACTGGAAAGATTTATTGGTTGGGGATGGGCTCGCGGCCTATCAGCTTGTTGGTGAGGTAATGG
CTCACCAAGGCGTCGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCA
GACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCAACGCCGCGTG
AGGGATGACGGCCTTCGGGTTGTAAACCTCTTTTAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAA
AAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGAATTATTG
GGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGCTGTGAAATCCGGAGGCTCAACCTCCGGCCTGCAG
TGGGTACGGGCAGACTAGAGTGCGGTAGGGGAGATTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATA
TCAGGAGGAACACCGATGGCGAAGGCAGATCTCTGGGCCGTAACTGACGCTGAGGAGCGAAAGGGTGGGG
AGCAAACAGGCTTAGATACCCTGGTAGTCCACCCCGTAAACGTTGGGAACTAGTTGTGGGGTCCATTCCA
CGGATTCCGTGACGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAA
AGGAATTGACGGGGACCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTAC
CAAGGCTTGACATATACGAGAACGGGCCAGAAATGGTCAACTCTTTGGACACTCGTAAACAGGTGGTGCA
TGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGT
TGCCAGCACGTAATGGTGGGAACTCATGGGATACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGT
CAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGCTGCAATAC
CGCGAGGTGGAGCGAATCCCAAAAAGCCGGTCCCAGTTCGGATTGAGGTCTGCAACTCGACCTCATGAAG
TCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCC
CGTCAAGTCATGAAAGTCGGTAACACCTGAAGCCGGTGGCCTAACCCTTGTGGAGGGAGCCGTCGAAGG。
EXAMPLE 1 Strain screening
Weighing 1g of marine soil sample, adding the marine soil sample into 50mL LCFBM liquid culture medium containing 0.6g PE microplastic, placing the liquid culture medium into a constant-temperature shake incubator at 180rpm and at 30 ℃, culturing for 10d for enrichment screening for one period, respectively coating dilution gradients which are sampled and diluted to 10 -4、10-5、10-6、10-7、10-8 after enrichment for several times on CFBAM solid culture medium, culturing for 7d at 30 ℃, observing the growth condition of bacterial colonies on CFBAM solid culture medium, carrying out plate purification and separation when bacterial strains grow out, picking the bacterial strains with better growth vigor as subsequent study objects, and carrying out repeated streak separation and purification on LB culture medium plates until the bacterial strains are free of impurities (shown in figure 1).
After culturing for 2d in a 30 ℃ incubator, picking single bacteria, inoculating to 50mL of NB liquid culture medium, placing in a constant temperature shake incubator at 180rpm, culturing for 2d at 30 ℃, precisely weighing 150-mesh PE 2g of microplastic, sterilizing for 12h in an ultraviolet ultra-clean bench, centrifuging bacterial liquid cultured in NB at 3000rpm for 5min to remove NB culture medium, suspending the precipitated bacterial liquid in LCFBM culture medium, repeating the steps for more than three times, and completely removing residual NB liquid culture medium. The recovered bacteria are resuspended in LCFBM, OD 600 is adjusted to 0.2-0.6, 2g of microplastic PE is added, 180rpm is added, and the culture is carried out in a 30 ℃ constant temperature shake incubator. After 30d of incubation, the biodegradation rate was evaluated by comparing the initial dry weight of PE before and after incubation. Bacteria adsorbed on the plastic were removed by washing with 2% SDS, and washed three times with absolute ethanol, and oven-treated at 60℃for more than 24 hours to constant weight, and the weight of the dried microplastic PE was independently measured 3 times. The degradation rate of the strain on the microplastic PE is measured respectively, and the highest degradation rate of the strain on the microplastic PE is 16.6+/-0.46%, and the strain is named as CJB-Jou02.
After the strain CJB-Jou02 is cultured for 48 hours in LB solid medium at 30 ℃, the colony is round, smaller, light yellow, smooth and round in surface, slightly convex and neat in edge, and is a gram positive bacterium (shown in figure 2).
LB culture medium, peptone 10g/L, yeast powder 5g/L, agar 20g/L, and ultra-pure water, pH 7.0. Autoclaving at 121℃for 20min.
NB medium, beef extract 3g/L, yeast extract 1g/L, sucrose 10g/L, peptone 5g/L, and ultrapure water, pH 7.0. Autoclaving at 121℃for 20min.
LCFBM liquid Medium liquid carbon-free basal Medium (LCFBM) formulated with ultrapure water according to ASTM plastics bacteriological resistance assay standard (G22-76,1996) contained (autoclaved 20min every 1000mL)0.7g KH2PO4、0.7g K2HPO4、0.7gMgSO4·7H2O、1.0g NH4NO3、0.005g NaCl、0.002g FeSO4·7H2O、0.002g ZnSO4·7H2O、0.001g MnSO4·H2O.121℃,).
CFBAM solid Medium 20g agar was added to 1000mL LCFBM medium to prepare a carbon-free basal agar medium (CFBAM). Autoclaving at 121℃for 20min.
Example 2 identification of species
The strain CJB-Jou02 was identified by 16S rDNA, single colony was picked and inoculated in LB liquid medium for 12h at 180rpm and 30 ℃. A1.5 mL centrifuge tube was used to collect 0.5mL of the bacterial culture, centrifuged at 12000rpm at 4℃for 2min, and the supernatant was discarded. The DNA of the genome of the bacterium CJB-Jou02 is extracted by using a rapid extraction kit, and the DNA is used as a template, and the amplification of the 16S rDNA gene is carried out by adopting general primers 27F:5 '-AGAGTTTGATCCTGGGCTCAG-3' and 14992 R:5 '-TACGGCTACCTTGTTACGACTT-3'.
PCR amplification conditions were 95℃for 5min, 95℃for 15s,58℃for 15s,72℃for 25s, and 72℃for 5min.
As a result of PCR, the target gene product, namely, 16S rDNA (Marker DL 2000) was amplified by 1% agarose gel electrophoresis analysis after the completion of PCR, and the target band (shown in FIG. 3) obtained by PCR was sent to Qingdao qing department of Kyoto be sequenced. The resulting sequence was uploaded to a database GenBank (GenBank No.: PP 101514.1), the sequencing result was analyzed by BLAST, and the strain sequence having higher homology was selected and compared with the strain CJB-Jou02 to determine the kind of strain. The CJB-Jou02 performs comparison and evolutionary relationship analysis results of the strain and other strains, and shows that the 16S rDNA sequence of the strain and the microbacterium vanicum Microbacterium foliorum are gathered into one and the relationship is nearest. The method combines morphological and molecular biological identification results, and initially identifies the strain as the microbacterium glabrous Microbacterium foliorum which is preserved in the China general microbiological culture Collection center with the preservation date of 2024, 1 month and 24 number and the strain preservation number of CGMCC 29643.
Example 3CJB-Jou02 optimal temperature measurement and growth Curve
The activated CJB-Jou02 strain is inoculated into 5mL of LB liquid medium by a disposable inoculating loop, transferred to 100mL of LB liquid medium after 12h of shaking incubator at 180rpm and 30 ℃ and cultured at 180rpm under four temperature conditions of 25 ℃, 28 ℃,30 ℃ and 37 ℃ for three bottles.
200UL of culture solution is taken in an ultra clean bench at intervals of 4 hours and injected into a clean 96-well plate, each sample is taken three times in parallel, the measurement is carried out by adopting a full-wavelength enzyme-labeled instrument, the wavelength is regulated to be OD 600, the measured OD 600 value is taken as an ordinate, the culture time is taken as an abscissa, the growth curve of the CJB-Jou02 strain under the four temperature conditions of 25 ℃, 28 ℃, 30 ℃ and 37 ℃ is drawn, and the optimal growth temperature of the CKB-Jou 02 strain is determined. The above procedure was repeated with this temperature as the cultivation temperature, and the culture solution was taken from each flask at an interval of 2 hours to determine OD 600, the OD 600 value was taken as the ordinate, and the cultivation time was taken as the abscissa, and the growth curve of CJB-Jou02 strain at 30℃was plotted (shown in FIG. 4).
Example 4 Infrared Spectrometry (FTIR)
And (3) using FTIR spectrum to measure shrinkage vibration, disappearance, growth and the like of groups in the original microplastic and the microplastic biologically degraded by the strain CJB-Jou02 within the frequency range of 4000-400 cm -1 of the PE microplastic, and further verifying the degradation of the PE microplastic by the strain CJB-Jou02 (shown in figure 5). After microorganism adheres to the surface of the microplastic, the polymer structure can be changed through oxidation reaction, namely, the oxidation reaction increases the hydrophilicity of PE through generating functional groups such as carbonyl, carbon-carbon double bond, carbon-oxygen double bond and the like, thereby enhancing the biodegradability of PE. The increase in peak at 1723cm -1 was due to the stretching vibration of-c=o-indicating that the aging of the PE particle surface during degradation resulted in a change in the properties of the microplastic material, affecting its degradability.
EXAMPLE 5 Water contact Angle appearance (WCA) measurement
The hydrophilicity and hydrophobicity of PE microplastic can be obtained by detecting the contact angle of the surface of PE microplastic with water, generally, the smaller the contact angle is, the lower the hydrophobicity is, the higher the hydrophilicity is, and generally, the better the hydrophilicity is, the more favorable the PE is for adhesion and colonization of microorganisms. The water contact angle of PE microplastic before and after 30 days of degradation of the strain CJB-Jou02 is detected, and as a result (shown in FIG. 6), the contact angle of PE microplastic after CJB-Jou02 degradation is 114.88 DEG on the left contact angle and 112.94 DEG on the right contact angle. The average contact angle is 113.91 °. The contact angle of the PE microplastic of the control group is 146.76 degrees on the left contact angle, 146.51 degrees on the right contact angle and 146.635 degrees on the average. The decrease of the contact angle of PE microplastic before and after 30 days of degradation by the strain CJB-Jou02 shows that the PE microplastic has lower hydrophobicity and higher hydrophilicity, and can initially indicate that the surface of the PE microplastic is oxidized by the strain CJB-Jou02 to generate hydrophilic groups, and the generation of the hydrophilic groups of the PE microplastic is favorable for the mass adhesion and colonization of the strain, so that the subsequent capability of resisting the degradation of the strain is reduced.
EXAMPLE 6X-ray diffractometer (XRD) analysis
XRD (X-ray Diffraction) is a rapid, accurate and efficient material nondestructive testing technology, and the XRD spectrum of PE microplastic treated by the strain CJB-Jou02 is compared in the study. In XRD spectra for PE microplastic, peaks at 22℃and 25℃are respectively 110-plane and 200-plane reflections of the orthorhombic phase, and these peaks are characteristic peaks for PE. When the peaks of the PE microplastic of the experimental group were compared with those of the control group, both the intensity and the width of the peaks were observed to be changed (shown in FIG. 7), and the intensity of the XRD characteristic peak was reduced because the strain CJB-Jou02 caused oxidative degradation of the PE microplastic, thereby changing the crystal structure of the PE microplastic. The amorphous nature of the test material is represented by a peak width, while the crystalline nature is represented by a peak height, and as the degree of degradation increases, the decrease in peak height may be due to the transformation of the crystalline structure into an amorphous structure, further indicating the feasibility of the strain to degrade PE microplastic.
Example 7 Scanning Electron Microscope (SEM) observations
The PE micro-plastic degraded by the degradation strain CJB-Jou02 is sterilized, treated by 2% SDS for more than 4 hours, washed to remove bacteria adsorbed on the plastic, washed three times by absolute ethyl alcohol, dried by a 60 ℃ oven for more than 24 hours to constant weight, and observed by an electron scanning microscope (SEM). In addition, the experiment also carries out electron microscope observation of the strain CJB-Jou02 before and after the degradation of the PE membrane, firstly, the PE membrane is subjected to sterilization treatment, 2% SDS treatment for more than 4 hours, bacteria adsorbed on the PE membrane are removed by washing, and the PE membrane is washed three times by absolute ethyl alcohol and naturally dried. The treated and control PE microplastics and PE films were sputter coated with gold for 60s using an ion sputter (Edwards Vacuum ltd., england) and observed at 15KV using a scanning electron microscope ((Phenom Pro, USA, phenom)). The results show that the surfaces of PE microplastic and PE membrane before degradation are smoother, the surface is rougher after inoculation of degradation bacteria CJB-Jou02, the local height is uneven, obvious ravines, grooves, pits and folds appear, the damage degree is obviously larger than that of the microplastic before degradation, and the particle size of the processed microplastic is obviously smaller than that of the PE microplastic before degradation (shown in figures 8 and 9).
The corrosion and the cavity formed on the surfaces of the microplastic and the PE film can reflect the field planting condition and degradation degree of the target plastic by microorganisms, and the surface of the microplastic degraded by the PE microplastic degradation strain CJB-Jou02 is obviously damaged and corroded by microscopic detection, so that the polyethylene microplastic degradation strain CJB-Jou02 can be verified to utilize the PE microplastic as a carbon source, thereby meeting the growth and development of the microplastic and further achieving the purpose of degrading the PE microplastic.
EXAMPLE 8 degradation of PE Membrane by Strain CJB-Jou02
Cutting PE plastic film into square film, accurately weighing, and performing aseptic treatment. Sequentially soaking in 2% SDS and absolute ethyl alcohol for more than 4 hours respectively, washing with sterile water for 3 times after treatment, sucking water adhered to the surface of the plastic film on an ultra-clean workbench with sterile filter paper, and sterilizing with ultraviolet lamp for 12 hours. The CJB-Jou02 bacterial liquid cultured in NB was centrifuged at 3000rpm for 5min to remove NB medium and the precipitated bacteria were resuspended in LCFBM medium, and the steps were repeated three more times to completely remove the residual NB liquid medium. The recovered strain was resuspended in LCFBM, OD 600 was adjusted to 1.0, sampled and diluted to a suitable gradient, 200uL was aspirated and applied to CFBAM solid medium, control was sterile water, PE plastic film was then placed in the center of the dish, and incubated at 30℃for 30d, and colony growth was observed (FIG. 10).
The result shows that the area coated with the CJB-Jou02 bacterial liquid and covered with the PE plastic film is vigorous in growth, obvious goose yellow is presented, the microscopic examination result is a short rod, which is confirmed to be the microbacterium vane, and the area uncovered by the PE plastic film and the control group have no obvious colony growth, so that the bacterial strain CJB-Jou02 can be colonized and grown on the surface of the bacterial strain CJB-Jou02 by decomposing the PE plastic film.
EXAMPLE 9 degradation of different microplastic by Strain CJB-Jou02
The project researches the degradation capability of the strain CJB-Jou02 on PE microplastic by taking PE microplastic as the sole carbon source, and preliminary researches on degradation effects of PA, PP, PS, PET and other microplastics, and the result shows that CJB-Jou02 can grow on a flat plate by taking PA, PP, PS, PET microplastic as the sole carbon source (shown in figure 11), and the strain CJB-Jou02 has huge degradation potential on degradation of microplastic.
The foregoing is a preferred embodiment of the present application, and modifications, obvious to those skilled in the art, of the various equivalent forms of the present application can be made without departing from the principles of the present application, are intended to be within the scope of the appended claims.

Claims (5)

1. 一株叶片微杆菌(Microbacterium foliorum)CJB-Jou02菌株,其特征在于,所述菌株已于2024年1月17日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC NO:29643,一株叶片微杆菌(Microbacterium foliorum)CJB-Jou02菌株的16S rDNA序列如SEQ ID No:1所示。1. A Microbacterium foliorum CJB-Jou02 strain, characterized in that the strain has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 17, 2024, with a deposit number of CGMCC NO: 29643, and the 16S rDNA sequence of the Microbacterium foliorum CJB-Jou02 strain is shown in SEQ ID No: 1. 2.根据权利要求1所述的叶片微杆菌(Microbacterium foliorum)CJB-Jou02菌株或菌液在生物降解PE微塑料中的应用。2. Use of the Microbacterium foliorum CJB-Jou02 strain or bacterial solution according to claim 1 in biodegradation of PE microplastics. 3.根据权利要求1所述的叶片微杆菌(Microbacterium foliorum)CJB-Jou02菌株或菌液在生物降解PA、PP、PS、PET微塑料中的应用。3. Use of the Microbacterium foliorum CJB-Jou02 strain or bacterial solution according to claim 1 in biodegradation of PA, PP, PS, and PET microplastics. 4.一种生物降解微塑料的方法,其特征在于:所述方法是首先将权利要求1所述的一株叶片微杆菌(Microbacterium foliorum)CJB-Jou02菌株制备成菌液,然后将其加入到灭菌处理的微塑料PE或PA或PP或PS或PET进行降解。4. A method for biodegrading microplastics, characterized in that: the method is to first prepare a bacterial liquid of the Microbacterium foliorum CJB-Jou02 strain described in claim 1, and then add the bacterial liquid to the sterilized microplastics PE or PA or PP or PS or PET for degradation. 5.根据权利要求4所述的一种生物降解微塑料的方法,其特征在于:具体方法如下:5. A method for biodegrading microplastics according to claim 4, characterized in that: the specific method is as follows: (1)菌种活化:将4 ℃条件下保存在LB培养基斜面上的菌种移至30 ℃生化培养箱活化12 h,挑取单菌接至NB液体培养基180 rpm,30 ℃培养;(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)PE微塑料预处理:精确称取微塑料150目PE 2 g,紫外超净台灭菌12 h以上后备用;(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)失重率测定:将培养在NB培养基2 d中的菌液以3000 rpm离心5 min,并将菌体重悬于LCFBM培养基中,重复三次完全除去剩余NB,将回收的菌体加入50 mL LCFBM中,并加入灭菌处理的2 g微塑料PE,180 rpm 30℃恒温震荡培养箱中培养30 d。(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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