Fibricated cellulosimicrobium sp. yh-1 and application thereof
By screening fibrous microorganism YH-1 from landfills, the problem of efficiently degrading polyethylene terephthalate (PET) plastics has been solved, achieving effective degradation and environmentally friendly treatment of plastics. The degradation products are obvious and the cost is low.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGSU OCEAN UNIV
- Filing Date
- 2024-10-15
- Publication Date
- 2026-04-28
AI Technical Summary
The scarcity of microorganisms that can efficiently degrade polyethylene terephthalate (PET) plastics in existing technologies leads to severe plastic waste pollution. Traditional treatment methods such as landfill and incineration pose problems of land occupation and secondary pollution.
A fibrous microbacterium named Cellulosimicrobium cellulans YH-1 was isolated and screened from a landfill and used to degrade polyethylene terephthalate (PET) plastics. The degradation effect was observed and the degradation products were analyzed by preparing a bacterial solution and degrading it in a liquid culture medium.
It achieves effective degradation of polyethylene terephthalate plastic, making the surface rough and uneven, producing typical degradation products, with significant degradation effect, green and environmentally friendly, and low cost.
Smart Images
Figure CN119193406B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial degradation of plastics technology, specifically relating to a fibrous microbacterium YH-1 and its application in degrading polyethylene terephthalate plastics. Background Technology
[0002] Synthetic plastics are a class of polymeric compounds formed from monomers through addition or condensation polymerization reactions. They possess excellent properties such as low price, good stability, superior elasticity, high crystallinity, and stable physicochemical properties. Polyethylene terephthalate (PET) is the largest category of synthetic plastics, accounting for over 70% of the total. Due to its good wear resistance, fatigue resistance, high-temperature resistance, and gas barrier properties, it is commonly used as packaging material in the food and beverage industry. Statistics show that approximately 27 million tons of PET are used annually in China, with about 85% used to manufacture plastic bottles. In recent years, with the increasing demand for plastics, the pollution problem caused by plastic waste has become increasingly prominent. PET accounts for 60% of plastic waste, resulting in a large amount of PET waste being indiscriminately dumped into soil and water bodies every year, causing serious environmental pollution problems.
[0003] Methods for treating polyethylene terephthalate (PET) waste mainly include landfill, incineration, and biodegradation. Landfill requires land resources, has a long processing time, and pollutes the environment; incineration produces toxic gases such as dioxins and carbon monoxide, and the resulting smoke and dust can cause secondary pollution. Microbial degradation of PET has advantages such as low pollution and no secondary pollution, making it a green, environmentally friendly, and promising method for solving the problem of plastic degradation. However, highly efficient microorganisms for degrading plastics are still scarce. Summary of the Invention
[0004] To overcome the shortcomings of the prior art, this invention isolates and screens a fibrotic microbacterium from a landfill, naming it fibrotic microbacterium (Fibriophyta). Cellulosimicrobium cellulans YH-1, this strain can effectively reduce polyethylene terephthalate.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0006] The first objective of this invention is to provide a fibrotic microbacterium ( Cellulosimicrobium cellulans YH-1, the fibrotic microorganism YH-1, is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 65134 and deposit date of September 13, 2024.
[0007] The fibrotic microbes ( Cellulosimicrobium cellulans The 16S rDNA sequence of YH-1:
[0008] SEQ ID No: 1:
[0009]
[0010] A second objective of this invention is to provide the application of the fibrous microorganism YH-1 in the degradation of polyethylene terephthalate plastics.
[0011] Furthermore, the polyethylene terephthalate plastic includes polyethylene terephthalate film or polyethylene terephthalate granules.
[0012] A third objective of this invention is to provide a method for degrading polyethylene terephthalate plastics, firstly by introducing fibrous microorganisms (… Cellulosimicrobium cellulans The YH-1 strain was prepared into a bacterial suspension, which was then added to a liquid culture medium containing polyethylene terephthalate plastic to degrade the microplastics.
[0013] Six days after degradation, the plastic was analyzed for weight loss, scanning electron microscopy (SEM), and high-performance liquid chromatography (HPLC). Weight loss of polyethylene terephthalate (PET) plastic was observed. SEM analysis showed that the surfaces of both the PET film and particles became noticeably rough and uneven. HPLC analysis revealed three typical PET degradation products: mono-(2-hydroxyethyl) terephthalate (MHET), bishydroxyethyl terephthalate (BHET), and terephthalic acid (TPA), indicating that strain YH-1 has a good degradation effect on PET plastic.
[0014] Furthermore, the liquid culture medium is a liquid inorganic salt culture medium comprising 0.7 g / L K₂HPO₄, 0.7 g / L KH₂PO₄, 0.7 g / L MgSO₄·7H₂O, 1.0 g / L NH₄NO₃, 0.005 g / L NaCl, 0.002 g / L FeSO₄·7H₂O, 0.002 g / L ZnSO₄·7H₂O, 0.001 g / L MnSO₄·H₂O, prepared with ultrapure water, pH 7.
[0015] Furthermore, fibrotic microbes ( Cellulosimicrobium cellulans The method for preparing bacterial culture of strain YH-1 is as follows: Fibrous microorganisms (… Cellulosimicrobium cellulansAfter culturing strain YH-1 in LB medium for 12-18 hours, the cells were collected by centrifugation, then washed with inorganic salt medium to remove residual LB medium, and finally resuspended in inorganic salt medium containing sterile polyethylene terephthalate plastic. The inorganic salt medium consisted of K2HPO4 0.7 g / L, KH2PO4 0.7 g / L, MgSO4·7H2O 0.7 g / L, NH4NO3 1.0 g / L, NaCl 0.005 g / L, FeSO4·7H2O 0.002 g / L, ZnSO4·7H2O 0.002 g / L, MnSO4·H2O 0.001 g / L, prepared with ultrapure water, pH 7.
[0016] Furthermore, the volume ratio of the bacterial solution to the liquid culture medium is 1:5-10.
[0017] A fourth objective of this invention is to provide a microbial agent for degrading polyethylene terephthalate plastics, said microbial agent being fibrous microorganisms (… Cellulosimicrobium cellulans YH-1 strain is used as the main active ingredient.
[0018] The above technical solution can achieve the following beneficial effects:
[0019] This invention isolates and screens a microplastic-degrading bacterial strain from the ocean, named Cellulosimicrobium cellulans YH-1. This strain exhibits excellent degradation effects on polyethylene terephthalate (PET) plastics, showing good degradation capabilities for both PET films and PET particles. It can cause mass loss in PET plastics. Scanning electron microscopy analysis shows that the surfaces of both PET films and particles become noticeably rough and uneven. High-performance liquid chromatography (HPLC) analysis reveals three typical PET degradation products: mono-(2-hydroxyethyl) terephthalate (MHET), bishydroxyethyl terephthalate (BHET), and terephthalic acid (TPA).
[0020] The strain provided by this invention can be applied to the biodegradation of polyethylene terephthalate (PET) plastics. The method of degrading PET using this strain has the advantages of being green, environmentally friendly, and low-cost, and is beneficial to the treatment of microplastic waste in the environment. Attached Figure Description
[0021] Figure 1This is a graph showing the weight loss of polyethylene terephthalate film after treatment with strain YH-1;
[0022] Figure 2 This is a colony morphology diagram of strain YH-1;
[0023] Figure 3 This is a scanning electron microscope image of strain YH-1;
[0024] Figure 4 This is a 16S electrophoresis image of strain YH-1;
[0025] Figure 5 This is a phylogenetic tree diagram of strain YH-1;
[0026] Figure 6 The effects of temperature, pH, and NaCl concentration on the growth of strain YH-1, along with growth curves;
[0027] Figure 7 This is a scanning electron microscope image of a polyethylene terephthalate film treated with YH-1.
[0028] As shown in Figures (a)-(c), the film surface is smooth. The surface morphology of the PET film after YH-1 treatment, as shown in Figures (d)-(f), shows cracks and obvious erosion marks on the PET film surface, with the surface changing from smooth to rough.
[0029] Figure 8 This is a scanning electron microscope image of polyethylene terephthalate particles after YH-1 treatment.
[0030] In the figure, it can be seen that in (a)-(c), the surface is relatively smooth and the edges are prominent. However, after YH-1 treatment, as in (d)-(f), the surface is severely eroded and becomes rough and uneven.
[0031] Figure 9 This is a high-performance liquid chromatogram of polyethylene terephthalate particles after YH-1 treatment. Detailed Implementation
[0032] The invention will be further described below with reference to the accompanying drawings:
[0033] The present invention relates to the development of a fibrotic microbacterium ( Cellulosimicrobium cellulans YH-1, the fibrotic microorganism YH-1, is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 65134, deposit date of September 13, 2024, and deposit address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou.
[0034] Example 1: Isolation, screening, and identification of polyethylene terephthalate degrading bacteria strain YH-1
[0035] 1. Isolation and screening of strain YH-1
[0036] Initial screening: 10 g of soil sample from the Lianyungang landfill was placed in 100 ml of inorganic salt medium containing polyethylene terephthalate (PET) particles, with three parallel groups, and incubated at 35℃ and 180 rpm for 6 days. After 6 days, 10 ml of the enriched solution was transferred to fresh 100 ml of inorganic salt medium containing PET particles, and this process was repeated 5 times. The final enriched solution was serially diluted with sterile water to a final concentration of 10. -1 10 -2 10 -3 10 -4 10 -5 and 10 -6 Use a pipette to transfer 200 μL of the culture medium onto an inorganic salt solid medium. Spread the medium evenly with a spreader and incubate at 37 ℃ for 3-6 days before observation. Based on the size of the colonies, select several vigorous strains, isolate and purify them on inorganic salt solid medium, and preserve them.
[0037] Secondary screening: Several strains obtained from the initial screening were cultured on LB medium in a shaker (35 ℃, 180 rpm) for 12-18 h. They were then washed three times with sterile 0.9% NaCl solution in a centrifuge (8000 rpm, 10 min) to obtain a bacterial suspension with an OD600 of 1. 10-25% of this bacterial suspension was inoculated into 100 ml of inorganic salt medium containing sterile polyethylene terephthalate (PET) film. Each experimental group was set up in triplicate. The control group was kept under the same conditions but without bacterial inoculation. After culturing at 35 ℃ and 180 rpm for 6 days, the mass loss of the PET film was analyzed. For example... Figure 1 After 6 days of cultivation, polyethylene terephthalate (PET) showed a significant loss of mass under the influence of YH-1.
[0038] The preparation process of polyethylene terephthalate plastic is as follows:
[0039] (1) Preparation of polyethylene terephthalate granules: Purchase 200-mesh polyethylene terephthalate granules from Mingyan Plastic Flagship Store on Taobao, soak them in 75% ethanol for 10 minutes, then rinse them 2-3 times with sterile ultrapure water, and air dry them in a clean bench for later use.
[0040] (2) Preparation of polyethylene terephthalate film: polyethylene terephthalate film purchased from Taobao High Temperature Insulation Materials Flagship Store was evenly cut into small pieces of 10×10mm, soaked in 75% ethanol for 10min, then rinsed with ultrapure water 2-3 times, and dried in a clean bench for later use.
[0041] (3) The LB liquid culture medium used was prepared with 10 g / L peptone, 5 g / L yeast, 10 g / L sodium chloride, and ultrapure water, pH 7.0.
[0042] (4) The liquid inorganic salt culture medium used includes 0.7 g / L K2HPO4, 0.7 g / L KH2PO4, 0.7 g / L MgSO4·7H2O, 1.0 g / L NH4NO3, 0.005 g / L NaCl, 0.002 g / L FeSO4·7H2O, 0.002 g / L ZnSO4·7H2O, and 0.001 g / L MnSO4·H2O, prepared with ultrapure water at pH 7. Adding 20 g / L agar completes the inorganic salt solid culture medium.
[0043] 2. Morphological characteristics of strain YH-1
[0044] The bacterial strain was inoculated onto LB liquid medium and cultured at 35 ℃ for 12-18 h. Then, it was transferred to LB solid medium using an inoculation loop and cultured at 37 ℃ for 24 h. The morphological characteristics of the strain were observed, such as... Figure 2 As shown, YH-1 colonies are small and yellow; the colony surface is shiny and the edges are smooth and flat. Figure 3 This is a scanning electron microscope image of strain YH-1.
[0045] The LB liquid medium used was prepared with 10 g / L peptone, 5 g / L yeast, 10 g / L sodium chloride, and ultrapure water, at pH 7.0. 20 g / L agar was added to the above LB liquid medium to form LB solid medium.
[0046] 3. Molecular biological identification of strain YH-1
[0047] Take 2 mL of bacterial culture that has been cultured for 18 h and add it to a 2 mL EP tube. Centrifuge at 8000 rpm for 2 min. Extract the gene of strain YH-1 according to the bacterial DNA extraction kit and amplify it using PCR technology.
[0048] (1) Genome: The genome of strain YH-1 was extracted using a bacterial DNA extraction kit (Tiangen Biotech Co., Ltd.).
[0049] (2) Primers: 27F and 1492R (Sangon Biotech Co., Ltd.)
[0050] (3) PCR reaction system (50 μL): Taq PCR Master Mix 25 μL, DNA template 1 μL, 27F2 μL, 1492R 2 μL, ddH2O 20 μL.
[0051] (4) Reaction procedure: denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 90 s, 34 cycles; extension at 72 °C for 10 min; storage at 4 °C.
[0052] (5) Agarose gel electrophoresis: The products obtained from PCR amplification are electrophoresed on a 1% agarose gel until a single bright band appears. Figure 4 The sample was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.
[0053] (6) Phylogenetic Tree: The bacterial 16S rRNA sequences from the sequencing results were compared with the NCBI database for homology. A phylogenetic tree of strain YH-1 was constructed using MEGA7 software. It was found that this strain had the highest similarity (100%) to *Microbes fibroticus*. Figure 5 As shown.
[0054] (7) Fiber-forming microbes ( Cellulosimicrobium cellulans
[0055] Example 2: Growth characteristics of strain YH-1
[0056] 1. Effect of temperature on the growth of strain YH-1
[0057] Using a controlled variable method, the growth of strain YH-1 was measured after culturing in LB liquid medium at different temperatures (20, 25, 30, 35, 40, and 45℃) for 24 h. The OD value at absorbance 600 was measured every 2 h to compare the growth of strain YH-1 under different culture temperature conditions. Figure 6 As shown, the results indicate that 35℃ is the optimal temperature for the growth of strain YH-1.
[0058] 2. Effect of pH on the growth of strain YH-1
[0059] Using a controlled variable method, the OD value of strain YH-1 was measured every 2 hours after culturing in LB liquid medium at different pH values (3, 4, 5, 6, 7, 8, 9, 10, and 11) for 24 h. The growth of strain YH-1 under different pH conditions was compared. Figure 7 As shown, the results indicate that pH 7 is the most suitable pH for the growth of strain YH-1.
[0060] 3. Effect of NaCl concentration on the growth of strain YH-1
[0061] Using a controlled variable method, the growth of strain YH-1 was measured after culturing in LB liquid medium with different NaCl concentrations of 1%, 2%, 3%, 4%, 5%, 10%, 15%, and 20% for 24 h. The OD value at an absorbance of 600 was measured every 2 h to compare the growth of strain YH-1 at different NaCl concentrations. Figure 8 As shown, the results indicate that a 1% NaCl concentration is most suitable for the growth of strain YH-1.
[0062] 4. Growth curve of strain YH-1 under optimal conditions
[0063] The growth curve of strain YH-1 was determined based on the optimal growth conditions obtained above. The strain was cultured in LB liquid medium for 24 h, and the OD value was measured every 2 h at an absorbance of 600. The resulting growth curve is shown below. Figure 9 .
[0064] The LB liquid culture medium used was prepared with 10 g / L peptone, 5 g / L yeast, 10 g / L sodium chloride, and ultrapure water at pH 7.0.
[0065] The inoculation volume of the bacterial solution each time is 1%.
[0066] Example 3: Degradation experiment of strain YH-1 on polyethylene terephthalate plastic
[0067] 1. The preparation process of polyethylene terephthalate plastic is as follows:
[0068] (1) Preparation of polyethylene terephthalate granules: Purchase 200-mesh polyethylene terephthalate granules from Mingyan Plastic Flagship Store on Taobao, soak them in 75% ethanol for 10 minutes, then rinse them 2-3 times with sterile ultrapure water, and air dry them in a clean bench for later use.
[0069] (2) Preparation of polyethylene terephthalate film: polyethylene terephthalate film purchased from Taobao High Temperature Insulation Materials Flagship Store was evenly cut into small pieces of 10×10mm, soaked in 75% ethanol for 10min, then rinsed with ultrapure water 2-3 times, and dried in a clean bench for later use.
[0070] (3) The LB liquid culture medium used was prepared with 10 g / L peptone, 5 g / L yeast, 10 g / L sodium chloride, and ultrapure water, pH 7.0.
[0071] (4) The liquid inorganic salt culture medium used includes 0.7 g / L K2HPO4, 0.7 g / L KH2PO4, 0.7 g / L MgSO4·7H2O, 1.0 g / L NH4NO3, 0.005 g / L NaCl, 0.002 g / L FeSO4·7H2O, 0.002 g / L ZnSO4·7H2O, and 0.001 g / L MnSO4·H2O, prepared with ultrapure water, with a pH value of 7.
[0072] 2. Experimental methods:
[0073] After culturing strain YH-1 in LB medium on a shaker (35 ℃, 180 rpm) for 12-18 h, the culture was washed three times with sterile 0.9% NaCl solution in a centrifuge (8000 rpm, 10 min) to obtain a bacterial suspension with an OD600 of 1. 10-25% of this bacterial suspension was inoculated into 100 ml of inorganic salt medium containing sterile polyethylene terephthalate (PET) film. Each experimental group was set up in triplicate. The control group was kept under the same conditions but without bacterial inoculation. After culturing at 35 ℃ and 180 rpm on a shaker for 3-6 days, the PET plastic was analyzed by scanning electron microscopy (SEM) and high-performance liquid chromatography (HPLC).
[0074] 3. Analytical methods for polyethylene terephthalate plastics:
[0075] (1) The polyethylene terephthalate film treated with strain YH-1 was repeatedly washed with deionized water 3-5 times, washed and soaked with 1% SDS solution, placed in an ultrasonic cleaner for 30 min, then washed several times with 20% anhydrous ethanol, and finally dried in an oven at 60℃. After fixing and sputtering gold, the polyethylene terephthalate film was observed under a scanning electron microscope to observe its micromorphological changes.
[0076] (2) After treatment with strain YH-1, polyethylene terephthalate particles were repeatedly washed with deionized water 3-5 times, washed and soaked with 1% SDS solution, placed in an ultrasonic cleaner for 30 min, then washed several times with 20% anhydrous ethanol, and finally dried in an oven at 60℃. After fixing and sputtering gold, the polyethylene terephthalate particles were observed under a scanning electron microscope to observe their micromorphological changes.
[0077] (3) On days 3 and 6, inorganic salt culture medium (100 ml) of polyethylene terephthalate particles treated with strain YH-1 and inorganic salt culture medium (100 ml) of uninoculated strain YH-1 were collected and centrifuged (8000 rpm, 10 min, 4 ℃) to remove cells. The supernatant was adjusted to pH 2.0 with 2.0 M HCl and extracted once with 100 mL ethyl acetate. The ethyl acetate layer was recovered and evaporated on a rotary evaporator. The residue was dissolved in 2.0 ml methanol, filtered through a 0.22 μm pore size filter, and analyzed by high performance liquid chromatography (HPLC). The analytical column was an Agilent TC-C18(2) (250 × 4.6 mm). The mobile phase was 60% methanol: 40% 20 mM potassium dihydrogen phosphate solution, the column temperature was 35 ℃, the flow rate was 0.5 ml / min, and the eluent was monitored at a wavelength of 240 nm. The degradation products of polyethylene terephthalate (monohydroxyethyl terephthalate (MHET), bishydroxyethyl terephthalate (BHET), and terephthalic acid (TPA)) were dissolved in dimethyl sulfoxide and used as standards.
[0078] 4. Experimental Results:
[0079] pass Figure 7 and Figure 8 It can be seen that after being treated with strain YH-1 for 6 days, the surface of polyethylene terephthalate plastic became rough and showed obvious signs of erosion.
[0080] pass Figure 9It was found that three typical degradation products were present in the inorganic salt culture medium of polyethylene terephthalate particles treated with strain YH-1: mono-(2-hydroxyethyl) terephthalate (MHET), bishydroxyethyl terephthalate (BHET), and terephthalic acid (TPA), indicating that significant degradation had occurred.
[0081] The above descriptions are all preferred embodiments of the present invention. For those skilled in the art, any modifications to the present invention in various equivalent forms without departing from the principle of the present invention shall fall within the protection scope of the appended claims.
Claims
1. A strain of fibrotic microbe ( Cellulosimicrobium cellulans YH-1, characterized in that: The fibrotic microorganism YH-1 is deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC NO: 65134, and the deposit date is September 13, 2024. Cellulosimicrobium cellulans The 16S rDNA sequence of YH-1 is shown in SEQ ID No:
1.
2. The fibrotic microbacterium according to claim 1 ( Cellulosimicrobium cellulans Application of YH-1 in degradable polyethylene terephthalate plastics.
3. The fibrotic microbacterium according to claim 2 ( Cellulosimicrobium cellulans The application of YH-1 in degradable plastics is characterized by: The polyethylene terephthalate plastic includes polyethylene terephthalate film or polyethylene terephthalate granules.
4. A method for degrading polyethylene terephthalate plastic, characterized in that: First, the fibrous microorganisms described in claim 1 ( Cellulosimicrobium cellulans The YH-1 strain was prepared into a bacterial suspension, which was then added to a liquid culture medium containing polyethylene terephthalate plastic to degrade the microplastics.
5. The method for degrading polyethylene terephthalate plastic according to claim 4, characterized in that: The liquid culture medium is a liquid inorganic salt culture medium, comprising 0.7 g / L K₂HPO₄, 0.7 g / L KH₂PO₄, 0.7 g / L MgSO₄·7H₂O, 1.0 g / L NH₄NO₃, 0.005 g / L NaCl, 0.002 g / L FeSO₄·7H₂O, 0.002 g / L ZnSO₄·7H₂O, and 0.001 g / L MnSO₄·H₂O, prepared with ultrapure water, with a pH of 7.
6. The method for degrading polyethylene terephthalate plastic according to claim 4, characterized in that: Fiber-forming microbes ( Cellulosimicrobium cellulans The method for preparing bacterial culture of strain YH-1 is as follows: Fibrous microorganisms (… Cellulosimicrobium cellulans After culturing strain YH-1 in LB medium for 12-18 hours, the cells were collected by centrifugation, then washed with inorganic salt medium to remove residual LB medium, and finally resuspended in inorganic salt medium containing sterile polyethylene terephthalate plastic. The inorganic salt medium consisted of K2HPO4 0.7 g / L, KH2PO4 0.7 g / L, MgSO4·7H2O 0.7 g / L, NH4NO3 1.0 g / L, NaCl 0.005 g / L, FeSO4·7H2O 0.002 g / L, ZnSO4·7H2O 0.002 g / L, MnSO4·H2O 0.001 g / L, prepared with ultrapure water, and the pH value was 7.
7. The method for degrading polyethylene terephthalate plastic according to claim 4, characterized in that: The volume ratio of the bacterial solution to the liquid culture medium is 1:5-10.
8. A microbial agent for degrading polyethylene terephthalate plastics, characterized in that: The microbial agent is the fibrotic microorganism described in claim 1 ( Cellulosimicrobium cellulans YH-1 strain is used as the main active ingredient.