Strain for degrading polyethylene plastic and separation method and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a strain for degrading polyethylene plastic and a separation method and application thereof. The strain is PEI LAB L-A-17, which is classified and named as Brevundimonas huaxiensis, and was preserved in the China General Microbiological Culture Collection Center on September 26, 2024, with a preservation number of CGMCC No. 32083. The strain is obtained by screening from the intestinal fluid of Lasioderma serricorne larvae gnawing polyethylene plastic, and can grow by taking polyethylene plastic as the only carbon source and effectively degrade polyethylene. The application provides a new strain resource for the biodegradation of polyethylene, and has important significance for treating polyethylene plastic waste.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain for degrading polyethylene plastics, its isolation method, and its application. Background Technology

[0002] Polyethylene (PE) is a widely used synthetic material made from ethylene monomers. It is non-toxic, odorless, and resistant to oxidation, acids, and alkalis. Due to its excellent physical and chemical properties, it is widely used in packaging, construction, automobiles, and electronics. However, with the large-scale production and consumption of polyethylene plastics, the resulting "white pollution" problem has become increasingly serious. Because of the inefficient recycling of waste plastics, the vast majority of plastic waste is released into the environment, becoming ubiquitous from land to water. At the same time, waste plastics can also become a carrier of disease, entering the human body through the food chain and causing health problems.

[0003] Currently, polyethylene plastics are mostly disposed of using non-biological methods such as landfill and incineration, which easily leads to resource waste and secondary pollution, and the treatment cost is relatively high. Therefore, in addition to developing new environmentally friendly plastic products, research and development of safe and green disposal technologies for polyethylene and other plastic waste are urgently needed. Biodegradation utilizes microorganisms to digest and metabolize polyethylene plastics, breaking them down into smaller molecules through enzymatic reactions, ultimately converting them into harmless water, carbon dioxide, and other inorganic salts. While degrading polyethylene, it can also promote the recycling of carbon resources, which is of great significance for improving the ecological environment. Due to its advantages such as low cost, environmental sustainability, and versatility, biodegradation has become a research hotspot in the environmental protection field. Known microorganisms that can degrade polyethylene plastics mainly include Pseudomonas, Stenotrophomonas, Klebsiella, Acinetobacter, Rhodococcus, Staphylococcus, Streptococcus, and Bacillus. Most of these strains have the ability to degrade PE surfaces and / or form biofilms on PE surfaces.

[0004] Some existing technologies have also reported on polyethylene plastic-degrading bacteria. For example, patent CN117821314A discloses a method for screening polyethylene plastic-degrading bacteria and its application. This patent obtains strains with polyethylene degradation capabilities by acclimating and enriching the samples to be isolated, then transferring them to a culture medium with polyethylene as the sole carbon source and culturing them for a period of time. The obtained polyethylene plastic-degrading bacteria include *Ochrobactrum intermedium* D2-6-1 and *Pseudoxanthomonas* sp. D4-2-1. Patent CN115960782B discloses a marine polyethylene plastic-degrading bacterium and its application. This strain, *Evansella clarkii* SCSIO 43802, was isolated from a seagrass bed ecosystem and grows with polyethylene plastic as the sole carbon source. This strain can degrade polyethylene plastic, especially in marine habitats.

[0005] Although biodegradation methods offer environmental advantages, the availability of effective microbial resources capable of degrading plastic waste such as polyethylene is currently limited, resulting in low treatment efficiency and slow degradation rates, which significantly restricts the widespread application of microbial plastic degradation. Therefore, seeking green and environmentally friendly degradation methods and screening for new, highly efficient plastic-degrading bacteria remains an urgent priority. Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide a strain that has the function of degrading polyethylene plastic, which can effectively degrade polyethylene plastic.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The strain capable of degrading polyethylene plastic, namely PEI LAB LA-17, classified as Brevundimonas huaxiensis, was deposited on September 26, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32083.

[0009] Furthermore, the nucleotide sequence of the 16S rDNA of the strain is shown in SEQ ID NO: 1.

[0010] Furthermore, the strain is a Gram-negative bacterium, in the form of short rods; its colony characteristics and cell morphology are: light yellow, round, protruding, with a relatively viscous texture and opaque.

[0011] Furthermore, the physiological and biochemical characteristics of the strain are as follows: negative for PEI LAB LA-17V.P, positive for glucose acid production test, negative for CIT citrate test, negative for IND indole test, negative for GEL gelatin liquefaction test, and negative for H2S hydrogen sulfide test.

[0012] In recent years, the ingestion of polyolefin plastics by insects has attracted researchers' attention. The most representative examples include the mealworm (Tenebrio molitor), black mealworm (Tenebrio obscurus), and superbug (Zophobas morio) of the order Tenebrio, and the large wax moth (Galleria mellonella) and Indian meal moth (Plodia interpunctella) of the order Pyralidae. Existing research suggests that the gut microbiota of these insects likely plays a significant role in the biodegradation of polyolefin plastics. However, there are few cases of extracting strains from insects and using them for the degradation of polyethylene plastics. This invention isolates and screens a polyethylene plastic-degrading bacterium from the gut fluid of tobacco beetle larvae that ingest polyethylene plastic. This strain can utilize polyethylene plastic as its sole carbon source for growth. This invention provides a new microbial resource for the biodegradation of polyethylene plastic mulch films and offers new resources and ideas for the treatment of polyethylene plastic waste in the environment, with broad application prospects.

[0013] A second objective of this invention is to provide a formulation product for degrading polyethylene plastics.

[0014] To achieve the above objectives, the present invention adopts the following technical solution:

[0015] A formulation product for degrading polyethylene plastics, wherein the aforementioned strain is used as the active ingredient.

[0016] The third objective of this invention is to provide a method for isolating and screening the aforementioned strains.

[0017] To achieve the above objectives, the present invention adopts the following technical solution:

[0018] The method for isolating and screening the aforementioned strains includes the following steps:

[0019] 1) Using insects that gnaw on polyethylene plastic as the strain source, enrichment culture was carried out using a culture medium with polyethylene plastic as the sole carbon source to obtain a culture medium containing plastic-degradable microorganisms.

[0020] 2) The culture medium obtained in step 1) was purified and cultured using the streak plate method on solid TSA medium; then, a culture medium with polyethylene plastic as the sole carbon source was added and cultured for a longer period of time, and the polyethylene degrading bacterium PEI LAB LA-17 was screened out.

[0021] Furthermore, the insect that gnaws on the polyethylene plastic is a tobacco beetle larva.

[0022] Further, in step 1), the culture conditions are: constant temperature shaking culture at 25-35℃ and 140-160r / min for 24-48 days; more preferably, constant temperature shaking culture at 30℃ and 150r / min for 30 days.

[0023] Further, in step 1), the culture medium is a carbon-free mineral salt liquid culture medium, composed of the following components: K2HPO4·3H2O 0.5-1.0 g / L, KH2PO4 0.5-1.0 g / L, NH4NO3 0.7-1.3 g / L, MgSO4·7H2O 0.3-0.7 g / L, NaCl 0.003-0.007 g / L, FeSO4·7H2O 0.001-0.003 g / L, ZnSO4·7H2O 0.001-0.003 g / L, MnSO4·H2O 0.0005-0.002 g / L.

[0024] Preferably, the carbon-free mineral salt liquid culture medium is composed of the following components: K2HPO4·3H2O 0.7 g / L, KH2PO4 0.7 g / L, NH4NO3 1.0 g / L, MgSO4·7H2O 0.5 g / L, NaCl 0.005 g / L, FeSO4·7H2O 0.002 g / L, ZnSO4·7H2O 0.002 g / L, and MnSO4·H2O 0.001 g / L.

[0025] Further, in step 2), the solid TSA culture medium is composed of the following components: tryptone, soybean peptone, sodium chloride, agar powder and distilled water; the ratio of tryptone, soybean peptone, sodium chloride, agar powder and distilled water is 5-7g: 1-3g: 1-3g: 350-500mL: 5-7g.

[0026] Preferably, the ratio of tryptone, soybean peptone, sodium chloride, agar powder and distilled water in the solid TSA culture medium is 6g:2g:2g:400mL:6g.

[0027] Preferably, the method includes the following steps:

[0028] 1. Preparation of a suspension of intestinal bacteria from tobacco beetle larvae;

[0029] 2. Add carbon-free mineral salt liquid culture medium and polyethylene membrane to the obtained intestinal bacterial suspension for culturing to obtain a culture medium;

[0030] 3. Take a small amount of culture medium and spread it on a TSA solid medium plate. After obvious colonies grow on the plate, observe the size, color and morphology of the colonies and mark the strains with different morphologies. Purify the isolated bacteria by streak plating on TSA solid medium. Add carbon-free mineral salt liquid culture with polyethylene film as the sole carbon source and continue culturing to screen and obtain the polyethylene degrading bacterium PEI LAB LA-17.

[0031] The fourth objective of this invention is to provide a method for degrading polyethylene plastic using the aforementioned strains.

[0032] To achieve the above objectives, the present invention adopts the following technical solution:

[0033] The method for degrading polyethylene plastic using the aforementioned strain involves co-culturing the strain with polyethylene plastic to degrade the polyethylene plastic; the strain grows using polyethylene as the sole carbon source.

[0034] Furthermore, the strain was co-cultured with polyethylene plastic in an inorganic salt culture medium.

[0035] Furthermore, a culture medium containing trace elements required by the microbial strains is added during the cultivation process.

[0036] Furthermore, during the degradation process, polyethylene plastic undergoes chemical structural changes, resulting in the appearance of carbon groups, hydroxyl groups, and carboxylic acid groups.

[0037] The fifth objective of this invention is to provide the application of the aforementioned strain and / or the aforementioned formulation product in the preparation of a degradation agent for degrading polyethylene plastics.

[0038] To achieve the above objectives, the present invention adopts the following technical solution:

[0039] The use of the aforementioned strains and / or the aforementioned formulation products in the preparation of degradation agents for degrading polyethylene plastics.

[0040] The beneficial effects of this invention are as follows:

[0041] 1. This invention isolates and screens a strain capable of effectively degrading polyethylene plastic from the intestinal fluid of the tobacco beetle larvae (Lasioderma serricorne), which gnaws at polyethylene plastic: *Brevundimonas huaxiensis* PEI LAB LA-17. This strain can utilize polyethylene plastic as its sole carbon source for growth and effectively degrades polyethylene. Currently, no method for treating polyethylene using this strain has been observed. This invention provides a new microbial resource for the biodegradation of polyethylene plastic mulch films.

[0042] 2. The PEI LAB LA-17 strain of this invention can effectively degrade polyethylene plastic. Scanning electron microscopy revealed a rough surface and obvious cracks on the polyethylene plastic. Fourier transform infrared spectroscopy showed that the polyethylene plastic underwent chemical structural changes after degradation, indicating that this strain has a good degradation effect on polyethylene plastic. This invention provides new resources and ideas for the treatment of polyethylene plastic waste in the environment, and has broad application prospects. Attached Figure Description

[0043] Figure 1 This is a colony morphology diagram of PEI LAB LA-17;

[0044] Figure 2 Image showing the Gram staining results of PEI LAB LA-17;

[0045] Figure 3 A diagram showing the phylogenetic information of PEI LAB LA-17 based on the 16S rDNA sequence;

[0046] Figure 4 The growth curve of PEI LAB LA-17 using polyethylene plastic film as the carbon source;

[0047] Figure 5 Fluorescence pattern of PEI LAB LA-17 on polyethylene plastic film for bacterial viability detection;

[0048] Figure 6 Scanning electron microscope image of PEI LAB LA-17 on a polyethylene plastic film;

[0049] Figure 7 These are scanning electron microscope images showing the morphological characteristics of polyethylene plastic after degradation in PEI LAB LA-17; among them, Figure 7 -A is the morphological feature diagram of the control group; Figure 7 -B shows the morphological characteristics of the PEI LAB LA-17 strain treatment group;

[0050] Figure 8 The infrared spectrum shows the structure of polyethylene plastic after degradation in PEI LAB LA-17. Detailed Implementation

[0051] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0052] In this embodiment of the invention, a strain PEI LAB LA-17 capable of degrading polyethylene plastic is provided. It is classified as Brevundimonas huaxiensis and was deposited on September 26, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 32083.

[0053] In this embodiment of the invention, the sequence information involved is shown in Table 1.

[0054] Table 1. Summary of Sequence Information

[0055]

[0056] Example 1. Enrichment, separation, screening and identification of PEI LAB LA-17

[0057] (1) Enrichment, separation and screening

[0058] The larvae of tobacco beetles that have been feeding on plastic were disinfected until they were clean. Under aseptic conditions, their intestines were dissected, and the intestinal fluid was expelled to prepare an intestinal bacterial suspension. The prepared intestinal bacterial suspension was added to 50 ml of sterile carbon-free mineral salt liquid culture medium in an Erlenmeyer flask, along with a sterile polyethylene film (2×2 cm). 2 The culture was then incubated at 30℃ and 150 rpm for 30 days. A small amount of the culture was plated on TSA solid medium. After obvious colonies grew on the plates, the size, color, and morphology of the colonies were observed, and strains with different morphologies were labeled. The isolated bacteria were then purified and numbered using the TSA solid medium streak method and stored at 4℃ for later use. Then, a carbon-free mineral salt liquid medium with polyethylene film as the sole carbon source was added. The presence of bacterial growth in the medium was confirmed by measuring OD600. Finally, a bacterium capable of efficiently degrading polyethylene plastic was screened and labeled PEI LAB LA-17.

[0059] The formula for the carbon-free mineral salt liquid culture medium is as follows: K2HPO4·3H2O 0.7g / L, KH2PO4 0.7g / L, NH4NO3 1.0g / L, MgSO4·7H2O 0.5g / L, NaCl 0.005g / L, FeSO4·7H2O 0.002g / L, ZnSO4·7H2O 0.002g / L, MnSO4·H2O 0.001g / L;

[0060] TSA medium: 6g tryptone, 2g soybean peptone, 2g sodium chloride, 400mL distilled water. For solid medium, add 6g agar powder.

[0061] (2) Colony characteristics and colony morphology

[0062] The colony morphology of PEI LAB LA-17 is as follows: Figure 1 As shown, PEI LAB LA-17 strain is pale yellow, round, raised, with a relatively viscous texture, and is opaque. After Gram staining, the bacterium can be observed under an oil immersion microscope; it is a Gram-negative bacterium, appearing as short rods. See details... Figure 2 .

[0063] (3) Physiological and biochemical characteristics

[0064] The strain PEI LAB LA-17V.P was negative in assay, positive in glucose acid production assay, negative in CIT citrate assay, negative in IND indole assay, negative in GEL gelatin liquefaction assay, and negative in H2S hydrogen sulfide assay.

[0065] (4) Molecular biological identification

[0066] 1) Use the Tiangen DNA Extraction Kit to extract bacterial DNA from the target strain according to the manufacturer's instructions.

[0067] 2) PCR amplification was performed using 16S rDNA sequence primers 27F and 1492R. The nucleotide sequence of 27F is shown in SEQ ID NO:2, and the nucleotide sequence of 1492R is shown in SEQ ID NO:3. PCR amplification was performed in a 20 μL reaction system: 1 μL template DNA, 10 μL 2×Mix, 1 μL primer 27F, 1 μL primer 1492R, and 7 μL ddH2O. The PCR amplification conditions included: a pre-denaturation program of 95℃ for 4 min; denaturation at 95℃ for 1 min, annealing at 55℃ for 1 min, extension at 72℃ for 1 min, for 35 cycles; and a final extension at 72℃ for 10 min, followed by storage at 4℃.

[0068] 3) The amplification products were subjected to electrophoresis and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The obtained sequences were compared with existing sequences in the NCBI database using BLAST analysis, and strains with similar homology were selected to construct a phylogenetic tree using MEGA 11 software.

[0069] The results showed that PEI LAB LA-17 belonged to the same clade as *Brevundimonas huaxiensis*, and its 16S rDNA sequence showed 99.41% similarity to *Brevundimonas huaxiensis* strain 090558 (NR_181184.1). Based on colony morphology, physiological and biochemical characteristics, and 16S rDNA sequence analysis, a phylogenetic tree was constructed to determine its species classification, and it was preliminarily identified as *Brevundimonas huaxiensis*. The phylogenetic tree is as follows: Figure 3 As shown in SEQ ID NO:1. The nucleotide sequence of the 16S rDNA of *Syntrophus spp.* is shown in SEQ ID NO:1.

[0070] Example 2. Degradation effect of PEI LAB LA-17 on polyethylene plastic film

[0071] To evaluate the application of PEI LAB LA-17 in the biodegradation of polyethylene plastics, this example uses common polyethylene plastic films for degradation effect determination. PEI LAB LA-17 was inoculated into LB medium and cultured until the logarithmic growth phase. The polyethylene film was cut into 2×2cm pieces. 2 After weighing, the samples were sterilized with 75% ethanol for 2 hours, and then the ethanol on the surface was evaporated using a sterile airflow in a laminar flow hood to complete surface sterilization. 100 ml of carbon-free mineral salt liquid culture medium was added to an Erlenmeyer flask, along with three pieces of surface-sterilized polyethylene film, and the aforementioned PEI LAB LA-17 culture solution was inoculated. The Erlenmeyer flasks were placed in a constant temperature shaking incubator at 30℃ and 150 r / min for 30 days. During this period, the growth curve of PEI LAB LA-17 with polyethylene (PE) plastic as the sole carbon source was measured. After 90 days, samples were taken to determine the colonization ability of PEI LAB LA-17 on polyethylene film and bacterial viability (live / dead bacteria staining kit), as well as the changes in surface morphology and functional groups of polyethylene film caused by PEI LAB LA-17. Details are as follows:

[0072] (1) Growth of PEI LAB LA-17 strain in polyethylene plastic

[0073] Methods: Sterilized polyethylene plastic and PEI LAB LA-17 bacterial solution were added to 50 mL of carbon-free mineral salt liquid culture medium. The control group was not treated with polyethylene film. The OD600 nm of the bacterial solution was recorded at 1d, 3d, 5d, 7d, 14d, 21d, and 30d.

[0074] Result: As Figure 4As shown, the PEI LAB LA-17 strain exhibited significant growth in polyethylene plastic medium. From day 3 to day 7, the OD600 increased from 0.057 to 0.071, reaching its maximum OD600. From day 14 to day 30, the bacterial growth rate decreased, and the OD600 dropped from 0.067 to 0.061, indicating that the strain had completed its exponential growth phase and had just entered the stationary phase.

[0075] (2) Bacterial viability detection

[0076] Methods: PEI LAB LA-17 strain was cultured for 90 days in a medium with polyethylene plastic as the sole carbon source. Bacterial viability was assessed by staining the polyethylene plastic with two dyes. NucGreen stained bacteria green, representing live bacteria, while EthD-III stained bacteria red, representing dead bacteria.

[0077] Result: As Figure 5 As shown, the vast majority of the PEI LAB LA-17 strain on polyethylene plastic remained active after 90 days of cultivation.

[0078] (3) Determination of bacterial biofilm on polyethylene plastic surface

[0079] Methods: Excess culture medium on the surface of polyethylene plastic was rinsed off with sterile water, fixed with 2.5% glutaraldehyde for 2 h, then dehydrated with a series of ethanol concentrations for 15 min each time, and then replaced with tert-butanol twice for 30 min each time. After fixation and gold sputtering, the microstructure characteristics were observed under a scanning electron microscope.

[0080] Result: As Figure 6 As shown, after 90 days, the surface of the untreated polyethylene plastic (control CK) was smooth and showed no microbial adhesion. After the addition of PEI LAB LA-17 strain, the polyethylene film exhibited adhesiveness, and bacterial colonization was observed. These results indicate that the PEI LAB LA-17 strain can adapt to the environment using polyethylene as a carbon source and can grow normally.

[0081] (4) Measurement of surface changes in polyethylene plastic

[0082] Methods: First, biofilm was removed by washing polyethylene plastic with 2% SDS solution with shaking for 2 hours, followed by sonication for 15 minutes to remove the surface biofilm. The plastic was then washed three times with sterile water and dried in a desiccator for 48 hours. The surface morphology changes of the treated samples were observed using an optical microscope. The effects of PEI LAB LA-17 strain on the surface morphology of polyethylene plastic were also observed under a scanning electron microscope.

[0083] Result: As Figure 7As shown, after 90 days of cultivation, under a scanning electron microscope, the polyethylene plastic in the PEI LAB LA-17 strain treatment group had a rougher surface and showed clear microscopic morphology such as cracks compared to the control group, while the control group had a smoother surface without cracks.

[0084] (5) Determination of functional groups on the surface of polyethylene plastic

[0085] Methods: After the polyethylene film with biofilm removed was naturally dried, Fourier transform infrared spectroscopy was used for measurement, with a scanning wavelength range of 500-4000 cm⁻¹. -1 4cm resolution -1 The number of scans was 32.

[0086] Result: As Figure 8 As shown, in the range of 500–4000 cm -1 Fourier transform infrared spectra between 1000 and 1500 cm⁻¹ showed that, compared with the control group, the PEI LAB LA-17 strain improved the performance of polyethylene film at 1000–1500 cm⁻¹. -1 A new CO characteristic peak was generated at 1500–1670 cm⁻¹. -1 A new C=C characteristic peak was generated in the range of 1650–1750 cm⁻¹. -1 A new C=O characteristic peak was generated in the range of 3200–3600 cm⁻¹. -1 A new -OH characteristic peak was generated. Studies have shown that the appearance of carbon groups, hydroxyl groups, and carboxylic acid groups is considered a preliminary and indispensable step in the oxidation and biodegradation process of plastics. The appearance of CO, C=O, and -OH is a basic indicator of the biodegradation of microplastics, indicating that the microplastics have undergone oxidative degradation.

[0087] In summary, this invention has isolated and screened a polyethylene plastic-degrading bacterium from insects that gnaw on polyethylene plastic, naming it *Brevundimonas huaxiensis* PEI LAB LA-17. This bacterium can utilize polyethylene plastic as its sole carbon source for growth. The PEI LAB LA-17 strain induces hydrolysis on polyethylene plastic, leading to molecular chain breakage. This bacterium exhibits good degradation activity on polyethylene films, demonstrating significant application potential in degrading polyethylene plastics in the environment and providing new resources and approaches for the treatment of microplastic waste in the environment.

Claims

1. A strain capable of degrading polyethylene plastic, characterized in that, The strain was PEI LAB LA-17, and its classification name was *Syntropha spp.* (West China shortwave monoclonal antibody). Brevundimonas huaxiensis It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 26, 2024, with accession number CGMCC No. 32083.

2. The strain according to claim 1, characterized in that, The nucleotide sequence of the 16S rDNA of the strain is shown in SEQ ID NO:

1.

3. A formulation product for degrading polyethylene plastic, characterized in that, The formulation product uses the strain described in any one of claims 1 to 2 as the active ingredient.

4. A method for degrading polyethylene plastic, characterized in that, The strain described in any one of claims 1 to 2 is co-cultured with polyethylene plastic to degrade the polyethylene plastic; the strain grows using polyethylene as the sole carbon source.

5. The method according to claim 4, characterized in that, The strain was cultured together with polyethylene plastic in an inorganic salt culture medium.

6. The method according to claim 4, characterized in that, During degradation, polyethylene plastic undergoes chemical structural changes, resulting in the appearance of carbon groups, hydroxyl groups, and carboxylic acid groups.

7. The use of the strain according to any one of claims 1 to 2 and / or the formulation product according to claim 3 in the preparation of a degrading agent for degrading polyethylene plastics.

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