Trichoderma PBW1 and application thereof in plastic degradation
By using Trichoderma asperellum PBW1 and its fermentation broth for biodegradation, the problem of difficult degradation of PET plastics is solved, and an efficient and environmentally friendly plastic degradation effect is achieved.
Patent Information
- Application Number
- CN202411887734.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-20
- Publication Date
- 2025-03-07
AI Technical Summary
The prior art is difficult to effectively degrade plastics, especially PET plastics, and traditional recycling methods are often accompanied by environmental pollution.
PET plastic particles were degraded by biodegradation method using Trichoderma asperellum PBW1 and its fermentation broth.
The metabolites of Trichoderma PBW1 significantly promote the degradation of PET plastics, which is twice as effective as traditional degraded bacteria and avoid environmental pollution.
Smart Images
Figure CN119570638A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of microbial technology, and in particular to Trichoderma PBW1 and application thereof in plastic degradation. Background Art
[0003] Plastic recycling methods are mainly divided into physical recycling, chemical recycling and biological recycling. Although physical and chemical recycling methods can process plastics to a certain extent, they are often accompanied by damage to the environment. Therefore, it is particularly important to find an economical and environmentally friendly method to manage polyester-based plastic waste. As a sustainable recycling method, biological recycling is centered on biodegradation, which decomposes plastics through the action of microorganisms and enzymes and converts them into oligomers or monomers that are harmless to the environment.
[0004] Biosurfactants are substances produced by microorganisms. They have the advantages of high biodegradability, low environmental impact, low toxicity, high specificity, high stability and activity under extreme conditions. In addition, they have structural diversity. Due to their properties of emulsification, dispersion, solubilization, mobilization, wetting, reduction of surface tension, formation of micelles and foams, biosurfactants have great potential application value in the field of environmental remediation and are considered to be one of the most promising and widely used types of process chemicals.
[0005] Therefore, developing a microbial strain that can effectively degrade plastics, especially PET plastics, is of great significance for environmental protection and resource recycling. Summary of the invention
[0006] In view of this, the present invention proposes a Trichoderma PBW1 and application thereof in plastic degradation, aiming to provide an efficient and environmentally friendly plastic degradation method.
[0007] The invention provides a Trichoderma sp. The Trichoderma sp. is Trichoderma asperellum PBW1, and the preservation number of the Trichoderma sp. in China Center for Type Culture Collection is CCTCC NO: M 20242612.
[0008] The present invention also provides a bacterial agent, characterized in that the bacterial agent contains the Trichoderma described in claim 1.
[0009] The present invention also provides a fermentation broth, characterized in that the fermentation broth is obtained by fermenting the Trichoderma according to claim 1.
[0010] The present invention also provides a composition, characterized in that the composition comprises the Trichoderma and / or the bacterial agent and / or the fermentation liquid according to claim 3.
[0011] The present invention also proposes an application of the Trichoderma in any of the following:
[0012] A1) Use in the production of biological surfactants and / or in the preparation of products produced by the production of biological surfactants;
[0013] A2) Application in the degradation of PET and / or application in the preparation of degraded PET products.
[0014] The present invention also proposes an application of the bacterial agent in any of the following:
[0015] A1) Use in the production of biological surfactants and / or in the preparation of products produced by the production of biological surfactants;
[0016] A2) Application in degrading PET and / or application in preparing degraded PET products
[0017] The present invention also proposes an application of the composition in any of the following:
[0018] A1) Use in the production of biological surfactants and / or in the preparation of products produced by the production of biological surfactants;
[0019] A2) Application in the degradation of PET and / or application in the preparation of degraded PET products.
[0020] According to any of the applications, the activity detection method of the biosurfactant is a collapse method, a Parafilm M method, an oil diffusion method, an emulsification index method or a surface tension test method.
[0021] The present invention also provides a method for preparing a degradation product, which comprises the step of reacting PET with the Trichoderma described in claim 1.
[0022] The invention also provides a method for preparing a biological surfactant, which comprises the steps of fermenting the Trichoderma to obtain a fermentation product.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] The present invention discloses a Trichoderma asperellum PBW1, which can be easily cultured and fermented on an artificial culture medium, which is essential for waste treatment strains and is convenient for factory production and wide promotion. The metabolites of Trichoderma asperellum PBW1 significantly promote the degradation of PET plastic particles, and the effect is twice that of traditional degradation bacteria. Through the biotechnology application of the present invention, the degradation process of PET plastic particles is effectively accelerated, and compared with chemical agents and physical methods, it avoids problems such as environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:
[0026] Figure 1 The plate strain is Trichoderma asperellum PBW1;
[0027] Figure 2 This is the microstructure of Trichoderma asperellum PBW1;
[0028] Figure 3 The emulsification index of Trichoderma asperellum PBW1 is measured;
[0029] Figure 4 After 30 days of degradation experiment, the fermentation broth was used to detect the presence of degradation product terephthalic acid by HPLC, wherein A was an inorganic salt culture medium, and B was a cell-free culture medium of Trichoderma asperellum PBW1. DETAILED DESCRIPTION
[0030] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features described in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.
[0031] The culture medium formula adopted by the present invention is as follows:
[0032] PDA medium: 200 g / L potato; 2% glucose; 2% agar; and appropriate amount of deionized water.
[0033] PDB culture medium formula: 200 g / L potato; 2% glucose; add appropriate amount of deionized water.
[0034] Fermentation liquid medium formula: contains 2% canola oil, 0.5% yeast extract, and the pH is adjusted to 7.
[0035] Inorganic salt liquid culture medium formula (g / L): 1.0 potassium dihydrogen phosphate, 1.5 disodium hydrogen phosphate, 2.0 ammonium chloride, 0.1 calcium chloride dihydrate, 0.15 potassium chloride, 0.2 magnesium sulfate heptahydrate, 0.01 zinc sulfate heptahydrate, 0.001 manganese sulfate monohydrate.
[0036] Seed liquid culture medium formula: 2% glucose; 1% yeast extract; 2% peptone; and an appropriate amount of deionized water.
[0037] Example 1 Isolation of Trichoderma PBW1 strain
[0038] The strain provided by the present invention is Trichoderma asperellum PBW1, which is derived from sewage samples from mangrove garbage pollution areas in Malaysia. The isolation process of Trichoderma asperellum PBW1 is as follows:
[0039] 1) Put 800 ml of sewage sample into a 1000 ml bottle with a blue cap and bring it back to the laboratory; 2) Use an ultrafiltration device to filter the collected water sample, using a 0.22 micron water filter membrane for every 100 ml of water sample; 3) Add a filtered filter membrane to 250 μl of 100 mg / ml chloramphenicol solution, place it in a 30°C environment, and resuscitate it at 180 rpm for 1-3 hours; 4) Take 100 μl of the pre-culture suspension and evenly spread it on the PDA solid culture medium containing chloramphenicol; 5) Place the culture dish in a biochemical incubator at 30°C and culture for 2-3 days. After the colony grows to an appropriate size, select the filamentous fungal colonies of different colors and shapes for numbering and recording; 6) Use a puncher to select the obvious and different-shaped single colonies on the culture medium, transfer them to a new PDA solid culture medium for secondary or multiple purifications for subsequent identification and biological activity testing. Through the above steps, the filamentous fungus PBW1 of the present invention, namely Trichoderma asperellum PBW1, was successfully isolated.
[0040] The microstructure of Trichoderma asperellum PBW1 is shown as follows. The strain exhibits the following biological characteristics: On PDA medium, the colonies appear fluffy or felt-like, with colors ranging from green to dark green and neat edges. Initially, the colonies are white, and with the formation of spores, the color gradually changes to green. Through sequence gene determination based on the internal transcribed spacer (ITS) and nucleic acid sequence homology comparison with other similar sequences included in the GenBank database, the results showed that PBW1 had the highest similarity with the Trichoderma asperellum strain (reaching 100%). Trichoderma asperellum PBW1 has been deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on November 21, 2024, with a deposit number of CCTCC NO: M 20242612. The biological surfactant produced by this strain has the function of promoting the degradation of PET plastic particles.
[0041] Example 2
[0042] 1. Cultivation and fermentation process of Trichoderma PBW1 strain
[0043] A sample was taken from the purified Trichoderma PBW1 strain using a cork punch, inoculated on a PDA medium, and cultured in a biochemical incubator at 30°C for 3 to 5 days to obtain a plate strain. Figure 2 The plate strain of filamentous fungus PBW1 is shown in Figure 1. Subsequently, the cork punch was used to take samples again, and the strain was transferred to PDB medium and cultured on a shaker at 30°C and 180 rpm for 3 days to prepare seed strains. The seed strains were transferred to the fermentation medium at an inoculation ratio of 0.5%, and cultured at 30°C and 180 rpm for 10 days to obtain seed liquid.
[0044] 2. Biosurfactant production test
[0045] Preparation of cell-free culture fluid: Place the seed solution in a high-speed centrifuge, centrifuge at 12,000 rpm for 20 minutes, collect the supernatant, and filter through a 0.22 μm filter membrane to obtain a cell-free culture fluid. Using the cell-free culture fluid, perform experiments according to the hierarchical screening procedure for surfactant-producing strains.
[0046] 1. Collapse method
[0047] 100 μL of crude oil was transferred to a 96-well microplate and allowed to equilibrate for 24 hours. 20 μL of cell-free culture medium was placed on the center of the oil and the droplet size was measured after 1 minute. When the droplet was larger than the diameter of the sterile MSM, the result was considered favorable for the production of biosurfactants.
[0048] Preparation of cell-free culture medium: Place the above seed solution in a high-speed centrifuge and centrifuge at 12,000 rpm for 20 minutes, collect the supernatant, and filter using a 0.22 μm filter membrane to obtain the cell-free culture medium.
[0049] 2. Parafilm M method
[0050] 15 μL of cell-free culture fluid was spotted on Parafilm M membrane and the droplet diameter was measured with a ruler. Uninoculated culture medium and distilled water were used as negative controls, and 1% sodium dodecyl sulfate (SDS) was used as positive controls. When the droplet diameter was larger than that of uninoculated sterile culture medium and distilled water, the result was considered positive for biosurfactant production.
[0051] 3. Oil diffusion method
[0052] Place 25 mL of distilled water in a 9 cm diameter polystyrene dish. Then, deposit 500 microliters of crude oil on the water surface in the center of the dish. After 2 minutes, place 100 microliters of supernatant on the oil surface. If a halo is observed in the cell-free supernatant, it indicates the presence of surfactant compounds. 1% SDS and distilled water are used as positive and negative controls, respectively. Measure the diameter of the exclusion halo zone with a ruler.
[0053] 4. Emulsification index (E24)
[0054] Mix 2 mL of cell-free culture medium with 3 mL of crude oil in a test tube. Shake the mixture vigorously on a vortex shaker for 2 minutes. After 24 hours, compare the ratio of emulsified oil to total volume. The emulsification index E24 is defined as the percentage of the height of the emulsified layer (mm) divided by the total height of the liquid column (mm). 1% (w / v) SDS and sterile uninoculated medium were used as positive and negative controls, respectively.
[0055] 5. Surface tension test
[0056] The surface tension was measured using a maximum bubble pressure surface tension meter.
[0057] Formula: σ=K*Δρ;
[0058] Where K is the capillary coefficient.
[0059] 3. PET particle degradation experiment
[0060] The obtained fermentation broth was used for PET degradation experiment.
[0061] 1. PET pretreatment
[0062] After weighing, the PET particles were immersed in 75% anhydrous ethanol for 36 hours, then transferred to 20% anhydrous ethanol and ultrasonically cleaned for 30 minutes using an ultrasonic cleaner, repeated three times. Then, they were washed three times with double distilled water and irradiated with ultraviolet light for 48 hours in a clean bench to ensure adequate sterilization.
[0063] 2. Obtaining Trichoderma asperellum PBW1
[0064] The seed liquid of Trichoderma asperellum PBW1 capable of degrading PET was collected, centrifuged at 10,000 revolutions per minute for 10 minutes, and washed three times with double distilled water to obtain Trichoderma asperellum PBW1 bacterial bodies.
[0065] 3. Obtaining cell-free culture medium from fermentation broth
[0066] The fermentation broth of Trichoderma asperellum PBW1 was collected and centrifuged at 12,000 revolutions per minute for 20 minutes. The supernatant was collected and then filtered using a 0.22 micron filter membrane to obtain a fermentation cell-free culture broth.
[0067] 4. Implementation of PET particle degradation experiment
[0068] 0.5 g of Trichoderma asperellum PBW1 cells were transferred to an inorganic salt liquid culture medium, and a PET plastic particle was added, and the culture was carried out in a constant temperature oscillator at 30° C. and 180 rpm for 30 days.
[0069] 0.5 g of Trichoderma asperellum PBW1 cells were transferred to the fermentation cell-free culture medium, and a PET plastic particle was added, and the culture was placed in a constant temperature oscillator at 30° C. and 180 rpm for 30 days.
[0070] 5. Detection of degradation products in culture medium after degradation experiment
[0071] Analysis was performed by high performance liquid chromatography (HPLC).
[0072] Chromatographic column: Use C18 reverse phase column, specification is 250mm×4.6mm, 5μm;
[0073] Mobile phase: formic acid (0.1%) / water / acetonitrile (50:30:20, v / v / v);
[0074] Flow rate: 0.8-1.0mL / min;
[0075] Detector: Use UV-Vis Detector. TPA has maximum absorption at 240-270nm, and 245nm is selected as the detection wavelength;
[0076] Column temperature: 25°C;
[0077] Injection volume: Take 20-50 μL of sample and inject it into the HPLC system;
[0078] Running time: 10-15 minutes.
[0079] 4. Test results
[0080] Table 1 shows the test results of various indicators of Trichoderma asperellum PBW1. Figure 3 This is the result of measuring the emulsification index of filamentous fungus PBW1.
[0081] Table 1
[0082] Name Collapse Parafilm M method Oil diffusion method E24(％) σ(mN / m) Culture medium \ 0.35CM 0.28CM 0.6CM - 50.32 SDS (1%) \ + + +++ 69.23 36.74 PBW1 Trichoderma asperellum + + +++ 66 37.2
[0083] As can be seen from Table 1, the biosurfactant produced by the PBW1 strain exhibited similar performance to the standard surfactant SDS (1%) in the collapse method, Parafilm M method and oil diffusion method. Specifically, in the collapse method, the E24 value of the PBW1 strain was 66, which is close to 69.23 of SDS, indicating that it has good surface activity. In the Parafilm M method, the collapse diameter of the PBW1 strain was 0.35 cm, compared with 0.28 cm of SDS, showing similar surface activity. In the oil diffusion method, the diffusion diameter of the PBW1 strain was 0.6 cm, which is consistent with the +++ level of SDS, indicating that it has strong oil-water interfacial activity. In addition, the surface tension b value of the PBW1 strain is 37.2 mN / m, which is close to 36.74 mN / m of SDS, further confirming the activity of its surfactant.
[0084] In the PET particle degradation experiment, the types and contents of degradation products in the culture medium can be detected by HPLC analysis. Figure 4 As shown. Specific peaks related to PET degradation can be observed on the HPLC chromatogram, indicating that the fermentation cell-free culture fluid of the PBW1 strain can promote the degradation of PET particles. The appearance and changes of these peaks can be used as a basis for evaluating the biological activity of the PBW1 strain in PET degradation.
[0085] In summary, the Trichoderma PBW1 strain can not only produce substances with surface activity, but also show potential biodegradation ability in the degradation experiment of PET particles. These results indicate that the PBW1 strain has potential application value in the fields of environmental remediation and biodegradable plastics.
[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.
Claims
1. A Trichoderma sp., characterized in that The Trichoderma is Trichoderma asperellum PBW1, and its deposit number in China Center for Type Culture Collection is CCTCC NO: M20242612.
2. A bacterial agent, characterized in that The bacterial agent contains the Trichoderma according to claim 1.
3. A fermentation broth, characterized in that: The fermentation broth is obtained by fermenting the Trichoderma according to claim 1.
4. A composition, characterized in that The composition comprises the Trichoderma described in claim 1 and / or the bacterial agent described in claim 2 and / or the fermentation broth described in claim 3.
5. Use of the Trichoderma according to claim 1 in any of the following: A1) Use in the production of biological surfactants and / or in the preparation of products produced by the production of biological surfactants; A2) Application in the degradation of PET and / or application in the preparation of degraded PET products.
6. Use of the bacterial agent according to claim 2 in any of the following: A1) Use in the production of biological surfactants and / or in the preparation of products produced by the production of biological surfactants; A2) Application in the degradation of PET and / or application in the preparation of degraded PET products.
7. Use of the composition according to claim 4 in any of the following: A1) Use in the production of biological surfactants and / or in the preparation of products produced by the production of biological surfactants; A2) Application in the degradation of PET and / or application in the preparation of degraded PET products.
8. The use according to any one of claims 5 to 7, characterized in that: The activity detection method of the biological surfactant is a collapse method, a Parafilm M method, an oil diffusion method, an emulsification index method or a surface tension test method.
9. A method for preparing a degradation product, characterized in that: The method comprises the step of reacting PET with the Trichoderma described in claim 1.
10. A method for preparing a biological surfactant, characterized in that: The method comprises the steps of fermenting the Trichoderma according to claim 1 to obtain a fermentation product.