Cellulomicrobium fibrosis YH-1 and application thereof

Abstract

The invention discloses a Cellulosimicrobium cellulans YH-1, which is characterized in that the Cellulosimicrobium cellulans YH-1 is preserved in the Guangdong Microbiological Culture Collection Center, the preservation number is GDMCC NO: 65134, the preservation date is September 13, 2024, and the 16S rDNA sequence of the Cellulosimicrobium cellulans YH-1 is shown as SEQ ID No: 1, and the Cellulosimicrobium cellulans YH-1 is preserved in the Guangdong Microbiological Culture Collection Center, the preservation number is GDMCC NO: 65134, the preservation date is September 13, 2024, and the 16S rDNA sequence of the Cellulosimicrobium cellulans YH-1 is shown as SEQ ID No: 1. The strain provided by the invention can be applied to biodegradation of polyethylene glycol terephthalate plastics, and a method for degrading polyethylene glycol terephthalate by using the strain has the advantages of greenness, environmental protection, low cost and the like, and is beneficial to treatment of micro-plastic wastes in the environment.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial degradation of plastics, and specifically relates to a fibrous fiber microbacterium YH-1 and application thereof in degradation of polyethylene terephthalate plastics. Background Art

[0002] Synthetic plastics are a type of high molecular compound formed by the addition or condensation reaction of monomers. They have excellent properties such as low price, good stability, excellent elasticity, high crystallinity, and stable physical and chemical properties. Polyethylene terephthalate (PET) is the largest variety of synthetic plastics, accounting for more than 70% of the total amount of synthetic plastics. Due to its good wear resistance, fatigue resistance, high temperature resistance, gas barrier properties and other properties, it is often used as a packaging material in the food and beverage industry. According to statistics, about 27 million tons of polyethylene terephthalate are used in the country every year, of which about 85% are used to make plastic bottles.

[0003] The main methods for treating polyethylene terephthalate waste include landfill, incineration and biodegradation. Landfill requires land resources, takes a long time to process, and pollutes the environment; incineration produces toxic gases and smoke such as dioxins and carbon monoxide, which can cause secondary pollution. Microbial degradation of polyethylene terephthalate has the advantages of low pollution and no secondary pollution. It is a green and environmentally friendly method with good prospects to solve the problem of plastic degradation. However, microorganisms that can efficiently degrade plastics are still scarce. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the present invention isolates and screens a fibrogenic microbacterium from a landfill and names it fibrogenic microbacterium ( Cellulosimicrobium cellulans ) YH-1, which can effectively degrade polyethylene terephthalate.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The first object of the present invention is to provide a fibrous fiber microbacterium ( Cellulosimicrobium cellulans ) YH-1, the fibrous fiber microbacterium YH-1 is preserved in Guangdong Provincial Microbiological Culture Collection Center, the preservation number is GDMCC NO: 65134, and the preservation date is September 13, 2024.

[0006] The fibrous fiber microbacteria ( Cellulosimicrobium cellulans ) 16S rDNA sequence of YH-1: SEQ ID No: 1: The second object of the present invention is to provide the use of fibrous fiber microbacteria YH-1 in degrading polyethylene terephthalate plastics.

[0007] Furthermore, the polyethylene terephthalate plastic includes polyethylene terephthalate film or polyethylene terephthalate particles.

[0008] The third object of the present invention is to provide a method for degrading polyethylene terephthalate plastics, firstly, the fibrous fiber microbacteria ( Cellulosimicrobium cellulans ) The YH-1 strain was prepared into a bacterial liquid, which was then put into a liquid culture medium containing polyethylene terephthalate plastic to degrade microplastics.

[0009] After 6 days, the degraded plastics were analyzed by weight loss, scanning electron microscopy (SEM), and high-performance liquid chromatography (HPLC). It was observed that the polyethylene terephthalate plastic had a mass loss. By analyzing the scanning electron microscope, the surface of the polyethylene terephthalate film and particles became obviously rough and uneven; by high-performance liquid chromatography analysis, three typical polyethylene terephthalate degradation products (mono-(2-hydroxyethyl) terephthalate (MHET), bishydroxyethyl terephthalate (BHET) and terephthalic acid (TPA)) were produced, indicating that strain YH-1 has a good degradation effect on polyethylene terephthalate plastics.

[0010] Furthermore, the liquid culture medium is a liquid inorganic salt culture medium, including 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, ultrapure water, pH 7.

[0011] Furthermore, fibroblasts ( Cellulosimicrobium cellulans ) The method for preparing the bacterial solution of YH-1 strain is as follows: Fibroblastic microorganisms ( Cellulosimicrobium cellulans) After the YH-1 strain was cultured in LB medium for 12-18 hours, the cells were collected by centrifugation, and then the residual LB medium was washed with inorganic salt medium to remove the residual LB medium, and finally the cells were resuspended in inorganic salt medium containing sterile polyethylene terephthalate plastic. The inorganic salt medium included K2HPO40.7 g / L, KH2PO40.7 g / L, MgSO4·7H2O 0.7 g / L, NH4NO31.0 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.

[0012] Furthermore, the volume ratio of the bacterial liquid to the liquid culture medium is 1:5-10.

[0013] The fourth object of the present invention is to provide a bacterial agent for degrading polyethylene terephthalate plastics, wherein the bacterial agent is a fibrogenic fiber microbacterium ( Cellulosimicrobium cellulans ) YH-1 strain as the main active ingredient.

[0014] The following beneficial effects can be obtained through the above technical solution: The present invention isolates and screens a microplastic degradation bacterial strain from the ocean, and names it Cellulosimicrobium cellulans YH-1. The strain has a good degradation effect on polyethylene terephthalate plastics, and the bacteria has a good degradation effect on polyethylene terephthalate films and polyethylene terephthalate particles. The polyethylene terephthalate plastics can be mass-lost. By analyzing the scanning electron microscope image, the surfaces of the polyethylene terephthalate film and particles are obviously rough and uneven; by high performance liquid chromatography analysis, three typical polyethylene terephthalate degradation products (mono-(2-hydroxyethyl) terephthalate (MHET), bishydroxyethyl terephthalate polymer (BHET) and terephthalic acid (TPA)) are produced.

[0015] The strain provided by the present invention can be applied to biodegradation of polyethylene terephthalate plastics. The method of degrading polyethylene terephthalate using the strain has the advantages of being green, environmentally friendly, low cost, etc., and is conducive to the treatment of microplastic waste in the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1is the weight loss graph of polyethylene terephthalate film after treatment with strain YH-1; Figure 2 This is the colony morphology of strain YH-1; Figure 3 is a scanning electron micrograph of strain YH-1; Figure 4 is the 16s electrophoresis pattern of strain YH-1; Figure 5 is the phylogenetic tree of strain YH-1; Figure 6 The effects of temperature, pH, and NaCl concentration on the growth of strain YH-1 and the growth curve diagram; Figure 7 This is a scanning electron microscope image of the polyethylene terephthalate film after YH-1 treatment. In the figure, as shown in Figures (a)-(c), the surface of the film is smooth. The surface morphology of the PET film after being treated with YH-1, as shown in Figures (d)-(f), cracks appeared on the surface of the PET film, and there were obvious signs of erosion, and the surface changed from smooth to rough; Figure 8 This is a scanning electron microscope image of polyethylene terephthalate particles treated with YH-1. In the figure, it can be seen that in (a)-(c), the surface is relatively smooth with prominent edges and corners, while after YH-1 treatment, as in (d)-(f), it can be seen that the surface is severely eroded and becomes rough and uneven; Fig. 9 It is the HPLC chromatogram of polyethylene terephthalate particles treated with YH-1. DETAILED DESCRIPTION

[0017] The present invention will be further described below in conjunction with the accompanying drawings: The present invention proposes a fibrogenic fiber microbacterium ( Cellulosimicrobium cellulans ) YH-1, the fibrous fiber microbacterium YH-1 is preserved in the Guangdong Provincial Microbial Culture Collection Center, the preservation number is GDMCC NO: 65134, the preservation date is September 13, 2024, and the preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0018] Example 1 Isolation, screening and identification of polyethylene terephthalate-degrading bacteria YH-1 strain 1. Isolation and screening of strain YH-1 Initial screening: 10 g of soil sample from Lianyungang landfill was placed in 100 ml of inorganic salt medium containing polyethylene terephthalate particles, and three parallel groups were set up for 6 days at 35°C and 180 rpm. After 6 days, 10 ml of the enriched solution was placed in a new 100 ml inorganic salt medium containing polyethylene terephthalate particles, and this was repeated 5 times. The enriched solution was diluted to 10 with sterile water. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 , use a pipette to draw 200 μL onto the inorganic salt solid culture medium, spread it evenly with a coating rod, and place it in a constant temperature incubator at 37 ℃ for 3-6 days before observation. According to the size of the colony, select several strains with better growth, separate and purify them on the inorganic salt solid culture medium, and store them.

[0019] Rescreening: After culturing the several strains obtained from the initial screening in LB medium in a shaker (35°C, 180 rpm) for 12-18h, wash them three times with sterile 0.9% NaCl solution in a centrifuge (8000 rpm, 10 min) to obtain a bacterial solution with an OD600 of 1. Inoculate 10-25% of the above bacterial solution into 100 ml of inorganic salt culture medium containing sterile polyethylene terephthalate film. Each experimental group has 3 parallel groups. The control bacteria are set to control the other conditions in the same way, but are not inoculated with bacterial solution. After being cultured in a shaker at 35°C and 180 rpm for 6 days, the mass loss of the polyethylene terephthalate film is analyzed. Figure 1 , After 6 days of cultivation, polyethylene terephthalate produced obvious mass loss under the action of YH-1.

[0020] Among them, the preparation process of polyethylene terephthalate plastic is as follows: (1) Preparation of polyethylene terephthalate particles: 200-mesh polyethylene terephthalate particles were purchased from Taobao Mingyan Plastic Flagship Store, soaked in 75% ethanol for 10 min, then rinsed with sterile ultrapure water 2-3 times, and dried in a clean bench for later use.

[0021] (2) Preparation of polyethylene terephthalate film: The polyethylene terephthalate film purchased from Taobao's flagship store of high temperature resistant insulation materials was evenly cut into small pieces of 10×10 mm, immersed in 75% ethanol for 10 min, then rinsed with ultrapure water 2-3 times, and dried on a clean bench for later use.

[0022] (3) The LB liquid medium used was prepared with 10 g / L peptone, 5 g / L yeast, 10 g / L sodium chloride, and ultrapure water, pH 7.0.

[0023] (4) The liquid inorganic salt culture medium used includes 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. Add 20 g / L agar to obtain the inorganic salt solid culture medium.

[0024] 2. Morphological characteristics of strain YH-1 Inoculate the strain into LB liquid medium and culture at 35 ℃ for 12-18 h. Use an inoculation loop to inoculate into LB solid medium and culture in a 37 ℃ incubator for 24 h. Observe the morphological characteristics of the strain, such as Figure 2 As shown, the colonies of YH-1 are smaller and yellow in color; the surface of the colonies is shiny and the edges are smooth and flat. Figure 3 This is a scanning electron micrograph of strain YH-1.

[0025] The LB liquid medium used was prepared with 10 g / L peptone, 5 g / L yeast, 10 g / L sodium chloride, ultrapure water, pH 7.0. 20 g / L agar was added to the above LB liquid medium to form LB solid medium.

[0026] 3. Molecular biological identification of strain YH-1 2 mL of bacterial solution cultured for 18 h was added to a 2 mL EP tube and centrifuged at 8000 rpm for 2 min. The gene of strain YH-1 was extracted according to the bacterial DNA extraction kit and amplified using PCR technology.

[0027] (1) Genome: The genome of strain YH-1 was extracted using a bacterial DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.).

[0028] (2) Primers: 27F and 1492R (Sangon Biotechnology Co., Ltd.) (3) PCR reaction system (50 μL): Taq PCR Master Mix 25 μL, DNA template 1 μL, 27F2 μL, 1492R 2 μL, ddH2O 20 μL.

[0029] (4) Reaction procedure: 34 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 90 s; extension at 72 °C for 10 min; and storage at 4 °C.

[0030] (5) Agarose gel electrophoresis: The PCR amplified product was electrophoresed on a 1% agarose gel until a single bright band appeared. Figure 4 , and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing.

[0031] (6) Phylogenetic tree: The bacterial 16S rRNA sequences of the sequencing results were compared with the homology of the NCBI database, and the phylogenetic tree of strain YH-1 was drawn using MEGA7 software. It was found that the strain had the highest similarity with F. cellulosum, which was 100%, such as Figure 5 shown.

[0032] (7) Fibroblastic microorganisms ( Cellulosimicrobium cellulans Example 2 Growth characteristics of strain YH-1 1. Effect of temperature on the growth of strain YH-1 Using the controlled variable method, the strain was cultured in LB liquid culture medium at different temperatures of 20, 25, 30, 35, 40, and 45°C for 24 h. The OD value at an absorbance of 600 was measured every 2 h to compare the growth potential of strain YH-1 under each culture temperature condition. Figure 6 As shown, the results showed that 35℃ was the most suitable temperature for the growth of strain YH-1.

[0033] 2. Effect of pH on the growth of strain YH-1 Using the controlled variable method, the strain was cultured in LB liquid medium with different pH values ​​of 3, 4, 5, 6, 7, 8, 9, 10, and 11 for 24 h. The OD value at an absorbance of 600 was measured every 2 h to compare the growth potential of strain YH-1 under different pH conditions. Figure 7 As shown, the results showed that pH 7 was most suitable for the growth of strain YH-1.

[0034] 3. Effect of NaCl concentration on the growth of strain YH-1 Using the controlled variable method, the strain was cultured 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 potential of strain YH-1 under different NaCl concentrations. Figure 8 As shown, the results showed that 1% NaCl concentration was most suitable for the growth of strain YH-1.

[0035] 4. Growth curve of strain YH-1 under optimal conditions The growth curve of strain YH-1 was determined according to the optimal growth conditions. The strain was cultured in LB liquid medium for 24 h, and the OD value at an absorbance of 600 was measured every 2 h. The growth curve was as follows: Fig. 9 .

[0036] The LB liquid culture medium used was prepared with 10 g / L peptone, 5 g / L yeast, 10 g / L sodium chloride, ultrapure water, and pH 7.0.

[0037] The inoculation amount of bacterial solution each time was 1%.

[0038] Example 3 Degradation experiment of polyethylene terephthalate plastic by strain YH-1 1. The preparation process of polyethylene terephthalate plastic is as follows: (1) Preparation of polyethylene terephthalate particles: 200-mesh polyethylene terephthalate particles were purchased from Taobao Mingyan Plastic Flagship Store, soaked in 75% ethanol for 10 min, then rinsed with sterile ultrapure water 2-3 times, and dried in a clean bench for later use.

[0039] (2) Preparation of polyethylene terephthalate film: The polyethylene terephthalate film purchased from Taobao's flagship store of high temperature resistant insulation materials was evenly cut into small pieces of 10×10 mm, immersed in 75% ethanol for 10 min, then rinsed with ultrapure water 2-3 times, and dried on a clean bench for later use.

[0040] (3) The LB liquid medium used was prepared with 10 g / L peptone, 5 g / L yeast, 10 g / L sodium chloride, and ultrapure water, pH 7.0.

[0041] (4) The liquid inorganic salt culture medium used included 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, and MnSO4·H2O 0.001 g / L, prepared with ultrapure water, with a pH value of 7.

[0042] 2. Experimental methods: After strain YH-1 was cultured in LB medium in a shaker (35 ℃, 180 rpm) for 12-18 h, it was washed three times with sterile 0.9% NaCl solution in a centrifuge (8000 rpm, 10 min) to obtain a bacterial solution with an OD600 of 1. 10-25% of the above bacterial solution was inoculated into 100 ml of inorganic salt medium containing sterile polyethylene terephthalate film. Three parallel groups were set up for each experimental group. The control group was set up to control other conditions in the same way, but without bacterial solution. After being cultured in a shaker at 35 ℃ and 180 rpm for 3-6 days, the polyethylene terephthalate plastic was analyzed by scanning electron microscopy (SEM) and high performance liquid chromatography (HPLC).

[0043] 3. Analysis method of polyethylene terephthalate plastic: (1) The polyethylene terephthalate film treated with strain YH-1 was repeatedly washed with deionized water for 3-5 times, washed and soaked with 1% SDS solution, placed in an ultrasonic cleaner for 30 min, and then washed several times with 20% anhydrous ethanol. Finally, it was placed in an oven for drying at 60°C. The polyethylene terephthalate film was fixed and sprayed with gold, and its microscopic morphology was observed under a scanning electron microscope.

[0044] (2) The polyethylene terephthalate particles treated with strain YH-1 were repeatedly washed with deionized water for 3-5 times, washed and soaked with 1% SDS solution, placed in an ultrasonic cleaner for 30 min, and then washed several times with 20% anhydrous ethanol. Finally, they were dried in an oven at 60°C. The polyethylene terephthalate particles were fixed and sprayed with gold, and their microscopic morphology changes were observed under a scanning electron microscope.

[0045] (3) The inorganic salt culture of polyethylene terephthalate particles treated with strain YH-1 and the inorganic salt culture medium (100 ml) of strain YH-1 not inoculated were collected at 3 days and 6 days, respectively, and centrifuged (8000 rpm, 10 min, 4 °C) to remove cells. The supernatant was adjusted to pH 2.0 using 2.0 M HCl and extracted once with 100 mL of ethyl acetate. The ethyl acetate layer was recovered and evaporated on a rotary evaporator. The residue was dissolved in 2.0 ml of methanol, filtered through a 0.22 μm pore size filter, and analyzed by high performance liquid chromatography (HPLC). The analytical column was 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 °C, the flow rate was 0.5 ml / min, and the effluent was monitored at a wavelength of 240 nm. Polyethylene terephthalate degradation products (monohydroxyethyl terephthalate (MHET), bishydroxyethyl terephthalate (BHET), and terephthalic acid (TPA)) dissolved in dimethyl sulfoxide were used as standards.

[0046] 4. Experimental results: pass Figure 7 and Figure 8 It can be seen that after 6 days of treatment with strain YH-1, the surface of polyethylene terephthalate plastic became rough and had obvious signs of erosion.

[0047] pass Fig. 9 It can be seen that there are three typical degradation products in the inorganic salt culture medium of polyethylene terephthalate particles treated with strain YH-1, namely (mono-(2-hydroxyethyl) terephthalate (MHET), bishydroxyethyl terephthalate (BHET) and terephthalic acid (TPA)), which indicates that it has undergone obvious degradation.

[0048] 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 fibroblastic microorganisms ( Cellulosimicrobium cellulans )YH-1, characterized by: The fibrous fiber microbacterium YH-1 is deposited in Guangdong Microbial Culture Collection Center with a deposit number of GDMCC NO: 65134 and a deposit date of September 13, 2024. Cellulosimicrobium cellulans ) The 16S rDNA sequence of YH-1 is shown in SEQ ID No:

1.

2. A fibrogenic fiber microbacterium according to claim 1 ( Cellulosimicrobium cellulans )Application of YH-1 in the degradation of polyethylene terephthalate plastics.

3. A fibrogenic fiber microbacterium according to claim 2 ( Cellulosimicrobium cellulans )The application of YH-1 in degradable plastics is characterized by: The polyethylene terephthalate plastic includes a polyethylene terephthalate film or polyethylene terephthalate particles.

4. A method for degrading polyethylene terephthalate plastic, characterized in that: First, the fibrous fiber microbacteria ( Cellulosimicrobium cellulans ) The YH-1 strain was prepared into a bacterial liquid, which was then put into a liquid culture medium containing polyethylene terephthalate plastic to degrade microplastics.

5. A method for degrading polyethylene terephthalate plastic according to claim 4, characterized in that: The liquid culture medium is a liquid inorganic salt culture medium, including K2HPO4 0.7 g / L, KH2PO4 0.7 g / L, MgSO4·7H2O0.7 g / L, NH4NO3 1.0 g / L, NaCl 0.005 g / L, FeSO4·7H2O 0.002 g / L, ZnSO4·7H2O 0.002g / L, MnSO4·H2O 0.001 g / L, ultrapure water, and a pH value of 7.

6. A method for degrading polyethylene terephthalate plastic according to claim 4, characterized in that: Fibroblastic microorganisms ( Cellulosimicrobium cellulans ) The method for preparing the bacterial solution of YH-1 strain is as follows: Fibroblastic microorganisms ( Cellulosimicrobium cellulans ) After the YH-1 strain was cultured in LB medium for 12-18 hours, the cells were collected by centrifugation, and then the residual LB medium was washed with inorganic salt medium to remove the residual LB medium, and finally the cells were resuspended in inorganic salt medium containing sterile polyethylene terephthalate plastic. The inorganic salt medium included K2HPO4 0.7 g / L, KH2PO40.7 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. A method for degrading polyethylene terephthalate plastic according to claim 4, characterized in that: The volume ratio of the bacterial liquid to the liquid culture medium is 1:5-10.

8. A bacterial agent for degrading polyethylene terephthalate plastic, characterized in that: The bacterial agent is the fibrous fiber microbacteria ( Cellulosimicrobium cellulans ) YH-1 strain as the main active ingredient.

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