Method for degrading plastic film by using white-rot fungi

Abstract

The invention relates to the technical field of solid waste treatment, and particularly discloses a method for degrading a plastic film by using white-rot fungi, which comprises the following steps: inoculating the white-rot fungi into a solid matrix, then adding the plastic film to be degraded, and carrying out solid state fermentation at 30-37 DEG C for 40-50 days. After solid-state fermentation, white-rot fungi are effectively planted on the surface of the plastic film, and oxidative depolymerization and mineralization of the plastic film are promoted, so that degradation of the plastic film is realized.

Description

Technical Field

[0001] This invention relates to the field of solid waste treatment technology, and specifically to a method for degrading plastic film using white-rot fungi. Background Technology

[0002] Currently, biodegradable plastic films, such as polylactic acid (PLA) and polybutylene adipate / terephthalate (PET), are gradually being used in agricultural covering and packaging materials to replace traditional polyethylene plastic films. Although these materials can degrade under certain conditions, their degradation efficiency remains low in actual use and in the natural environment, often resulting in excessively long degradation cycles, residue accumulation, and poor environmental adaptability. This can potentially lead to higher concentrations of microplastics within the same timeframe, thereby exacerbating microplastic pollution and its related hazards. Existing methods for degrading plastic films mainly include physical treatment, chemical oxidation, and microbial degradation. Among these, microbial degradation offers advantages such as environmental friendliness and resource recycling; however, existing microbial systems have limited degradation efficiency and exhibit instability in actual composting or soil environments, making it difficult to meet the needs of efficient treatment of agricultural waste films. Therefore, it is necessary to provide a new method for degrading plastic films. Summary of the Invention

[0003] To develop a method for degrading plastic films, this invention provides a method for degrading plastic films using white-rot fungi. After solid-state fermentation, this invention effectively colonizes the white-rot fungi on the surface of the plastic film, promoting the oxidative depolymerization and mineralization of the plastic film, thereby achieving its degradation.

[0004] This invention provides a method for degrading plastic film using white-rot fungi. The white-rot fungi are inoculated into a solid substrate, and then the plastic film to be degraded is added. Anaerobic solid-state fermentation is carried out at 30℃~37℃ for 40 to 50 days.

[0005] After solid-state fermentation, the present invention effectively colonizes white-rot fungi on the surface of plastic film, promoting the oxidation, depolymerization, and mineralization of plastic film, thereby achieving the degradation of plastic film.

[0006] Furthermore, the white-rot fungus is *Phanerochaete chrysosporium* (…). Phanerochaetechrysosporium ).

[0007] Furthermore, before inoculation with the white-rot fungus, a bacterial solution is prepared: the white-rot fungal cells are suspended in sterile water, and the turbidity is adjusted to 80 NTU to 100 NTU using a turbidimeter to obtain the white-rot fungal bacterial solution.

[0008] Furthermore, during the inoculation of white-rot fungi into the solid substrate, the ratio of white-rot fungal inoculum to fermentation substrate is 2 mL to 5 mL: 5 g to 10 g.

[0009] Furthermore, the fermentation substrate is one or a mixture of corn cobs, straw and grain husks.

[0010] Furthermore, the plastic film to be degraded needs to be pretreated before being added to the solid matrix: soaked and rinsed in deionized water, and then dried.

[0011] Furthermore, the soaking time in deionized water is 1.5h to 2h.

[0012] Furthermore, the drying temperature is 40℃~45℃.

[0013] Furthermore, the plastic film comprises one or both of polybutylene adipate and polylactic acid.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: Solid-state fermentation technology utilizes fungi to ferment on solid substrates lacking free water, such as waste, agricultural byproducts, and lignocellulose. Its advantages include: fungi can form high-density mycelia in solid substrates, significantly improving the production efficiency of the target product; simultaneously, it can fully utilize inexpensive raw materials such as waste and agricultural byproducts as substrates, greatly reducing production costs. More importantly, utilizing these unavoidable and inedible abundant agricultural and food wastes and residues for material conversion and recycling is crucial for promoting the development of a circular bioeconomy and practicing sustainable development principles. Applying white-rot fungal solid-state fermentation technology to the treatment of biodegradable plastic films can effectively accelerate the oxidation and molecular chain breakage of the plastic surface, increasing its degradation rate and solving the problems of low degradation efficiency and insufficient stability in existing technologies.

[0015] This invention achieves efficient degradation of plastics by inoculating a white-rot fungal culture into a solid matrix and co-fermenting the plastic film with the fungus. Electron microscopy revealed that the surface of the untreated plastic was generally rough before biodegradation; however, after 40-50 days of co-cultivation, the compostable plastic developed pores and was covered with a large number of white-rot fungal hyphae. Simultaneously, the self-made plastic sample, after 40-50 days of solid-state fermentation, showed obvious cracks and lysis on its surface. Film weight loss analysis showed a gradual decrease in plastic mass at 2, 4, and 6 weeks, indicating effective degradation of the plastic molecules. Infrared spectroscopy revealed changes in the functional groups of the plastic. Further gel permeation chromatography showed a significant decrease in molecular weight. Therefore, this invention significantly improves the degradation efficiency of plastics by white-rot fungi, with a simple, feasible, and low-cost process, effectively achieving the harmless treatment of plastic pollutants and holding significant importance for environmental protection. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is an experimental diagram showing the colonization of fungi on the plastic surface after solid-state fermentation of plastic film and Proteobacterium chrysosporium in this invention. In the figure, A is an experimental diagram of fungal colonization on the surface of compostable biodegradable plastic film; B is an experimental diagram showing the colonization of fungi on the surface of self-degrading plastic film.

[0018] Figure 2 This is a diagram of the solid-state fermentation experiment of the compostable biodegradable plastic film of the present invention with *Procambarus chrysosporus*. In the diagram, A represents a solid-state fermentation experiment. B represents the biodegradation product.

[0019] Figure 3 This is an experimental diagram of the co-culture of the compostable biodegradable plastic film of the present invention with *Procambarus chrysosporus* (without solid-state fermentation). Figure A shows the co-culture experiment. B represents the biodegradation product.

[0020] Figure 4 This is a diagram of the co-culture experiment of self-degradable plastic film and *Phanerochaete chrysosporium* in this invention; In the diagram, A represents a solid-state fermentation experiment. B represents the biodegradation product.

[0021] Figure 5 These are electron microscope images of the compostable biodegradable plastic film before and after biodegradation in this invention. In the diagram, A represents the area before biodegradation; B represents the biodegradation product; C is a magnified view of a portion after biodegradation.

[0022] Figure 6 These are electron microscope images of the self-degradable plastic film before and after biodegradation in this invention; In the diagram, A represents the area before biodegradation; B represents the biodegradation product; C is a magnified view of a portion after biodegradation.

[0023] Figure 7 This is a graph showing the weight loss rate of Sample 1 and Sample 2 during the biodegradation process in this invention; wherein, Sample 1 is a compostable biodegradable plastic film; and Sample 2 is a self-degradable plastic film.

[0024] Figure 8 These are the infrared spectra of Sample 1 and Sample 2 before and after biodegradation in this invention; wherein, Sample 1-Before represents Sample 1 before degradation; Sample 1-After represents Sample 1 after degradation; Sample 2-Before represents Sample 2 before degradation; and Sample 2-After represents Sample 2 after degradation. Detailed Implementation

[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0026] Example 1: A method for promoting the colonization of white-rot fungi and the oxidation, depolymerization, and mineralization of reinforced plastic films through solid-state fermentation.

[0027] 1. Experimental materials The white-rot fungus is *Phanerochaete chrysosporium* (… P. chrysosporium BKM-F1767 was purchased from the China Center for Type Culture Collection, strain accession number: CCTCC AF96007.

[0028] 2. Experimental steps: (1) Pretreatment of plastic film The compostable biodegradable plastic film used in this invention is taken from waste plastic film generated from beverage shop takeout packaging, referred to as Sample 1. The main components of Sample 1 are polybutylene adipate terephthalate (PEG) and polylactic acid (PLA). Sample 1 pretreatment: Soaking in deionized water for 1.5 hours, rinsing three times with deionized water, and then drying in a 40°C oven for 24 hours. After pretreatment, Sample 1 appears as sheet-like plastic.

[0029] (2) Experimental method for solid-state fermentation degradation of plastics by white-rot fungi Weigh 8g of corn cob as the fermentation substrate and place it in an Erlenmeyer flask. Add deionized water to adjust the moisture content of the fermentation substrate to 55%, and sterilize it in a high-pressure steam sterilizer at 121℃ for 30 minutes to obtain a solid substrate. After cooling, it is ready for use.

[0030] *Phanerochaete chrysosporium* cells were scraped using an inoculation loop, suspended in sterile water, and the turbidity was adjusted to 90 NTU using a turbidimeter to obtain a bacterial suspension. 3 mL of the bacterial suspension was inoculated onto the surface of the solid substrate, along with the pretreated sample 1 to be degraded. The conical flask was sealed with a breathable sealing film, wrapped with aluminum foil, and incubated at 37°C for 45 days to obtain the degraded sample. Samples were taken and tested at weeks 2, 4, and 6.

[0031] Comparative Example 1: A method for directly degrading plastics by co-culturing white-rot fungi with plastic film.

[0032] Comparative Example 1 did not undergo solid-state fermentation; instead, compostable biodegradable plastic film was directly co-cultured with *Phanerochaete chrysosporium*. The specific steps were as follows: Sample 1 after pretreatment was obtained according to the steps of Example 1.

[0033] In a sterile environment, *Phanerochaete chrysosporium* was inoculated onto a sterilized culture medium consisting of 200 g / L potato extract, 20 g / L glucose, 1.5 g / L magnesium sulfate heptahydrate, 3 g / L potassium dihydrogen phosphate, and 20 g / L agar powder, with a pH of 7.5. The medium was incubated at 37°C for 3 days to reach the vegetative growth stage, at which point the biomass of *Phanerochaete chrysosporium* was at its peak, yielding a *Phanerochaete chrysosporium* culture. Pretreated sample 1 was then co-cultured with the obtained *Phanerochaete chrysosporium* culture for 45 days to obtain the degraded sample.

[0034] like Figure 3 As shown, in Comparative Example 1 without solid-state fermentation, the degradation effect was poor, the colonization effect of fungi was poor, and the change in film weight loss rate was not obvious, so no further tests were conducted.

[0035] Example 2: A method for promoting the colonization of white-rot fungi and the oxidation, depolymerization, and mineralization of reinforced plastic films through solid-state fermentation.

[0036] The specific experimental steps are basically the same as in Example 1, except that: Sample 1 in Example 1 is replaced with "Sample 2", which is a self-made polylactic acid plastic film.

[0037] The specific preparation steps of the self-made polylactic acid (PLA) plastic film are as follows: PLA powder and oxalic acid are mixed, dissolved in dichloromethane, and stirred evenly to obtain a mixture. The mass ratio of PLA powder, oxalic acid, and dichloromethane is 5:1:94. The obtained mixture is poured onto a glass plate, stretched, cooled, and separated to obtain a self-degradable plastic film, hereinafter referred to as "Sample 2". The obtained self-degradable plastic film is soaked in deionized water for 1.5 hours and repeatedly rinsed, then placed in a 40°C oven and dried for 24 hours to obtain the pretreated Sample 2. The pretreated Sample 2 is subjected to degradation experiments according to the solid-state fermentation method of Example 1.

[0038] The polylactic acid powder conforms to the American ASTM D6400 standard; the oxalic acid supplier is "Aladdin", and its purity reaches the "analytical grade" level.

[0039] The colonization and degradation of *Phanerochaete chrysosporium* on the plastic film surface in Examples 1 and 2 were investigated. Specific methods and results are as follows: I. Experimental Methods 1. Determination of film weight loss Samples were collected at weeks 2, 4, and 6 of solid-state fermentation to observe damage. The resulting plastic fragments were first soaked in a 2% (w / v) sodium dodecyl sulfate solution for 4 hours. The solid residue that could not be dissolved by the sodium dodecyl sulfate solution, i.e., the plastic solid fragments left after degradation, was collected and filtered through a vacuum pump. The collected residue was then suspended in 20 mL of saturated brine, stirred for 15 minutes to ensure thorough mixing, and allowed to stand for 2 hours. The supernatant was then vacuum filtered through a 0.25 μm glass fiber membrane to collect microplastics with a particle size ≥0.25 μm. The collection process was repeated 5 times to ensure data stability. The film was dried in an oven at 40 °C until constant weight, and the weight loss rate was calculated after weighing.

[0040] 2. Observation using a scanning electron microscope At the end of the solid-state fermentation plastic film degradation experiment, all samples were washed to remove residual microbial components from the film surface before analysis and characterization. The specific steps were as follows: First, the samples were washed with distilled water, suspended in 25 mL of distilled water containing 0.5 g sodium dodecyl sulfate, and stirred at 120 rpm for 2 hours to remove any biofilm. They were then washed twice with distilled water and vacuum filtered for recovery. Finally, all samples were vacuum dried overnight in a vacuum drying oven. The morphology of the plastic films and the colonization process of *Phanerochaete chrysosporium* on the plastic films were observed using thermal field emission scanning electron microscopy. Untreated plastic films were first removed with phosphate buffer, fixed by immersion in 2.5% glutaraldehyde, dehydrated using a gradient of 25%, 50%, 75%, and 100% ethanol for 10 min, and then washed with distilled water. Finally, gold was sputtered onto the samples, and they were observed using thermal field emission scanning electron microscopy at 2 kV.

[0041] 3. Infrared spectroscopy detection The infrared spectroscopy of the thin film was performed using attenuated total reflectance infrared spectroscopy. The specific procedure was as follows: After wiping the thin film sample clean with alcohol, it was placed on the platform's detection window. The probe was aligned with the sample, and the sample was pressed firmly by rotating clockwise. The sample scanning range was 400 cm⁻¹. -1 ~4000cm -1 The resolution is set to 4cm. -1 The number of scans was 32.

[0042] 4. Gel permeation chromatography detection The average relative molecular mass of the plastics before and after treatment was determined by high-temperature gel permeation chromatography, including the weight average molecular weight (Mw) and number average molecular weight (Mn). Samples 1 and 2 were dissolved in tetrahydrofuran, filtered through a 0.22 µm organic phase microporous membrane, and placed in 1 mL liquid chromatography vials for analysis. Tetrahydrofuran was used as the solvent, and the flow rate was 1.0 mL. -1 The injection volume was 200 μL.

[0043] 5. Gas chromatography-mass spectrometry detection The biodegradation products of sample 2 were characterized using gas chromatography-mass spectrometry (GC-MS). Degraded plastic fragments and bacterial cells were collected. Organic matter in the filtrate was extracted with ethyl acetate, and the organic phase was collected in a 20 mL amber glass bottle. The aqueous phase was discarded. The sample was stored at room temperature in preparation for GC-MS.

[0044] II. Experimental Results 1. Determination of film weight loss rate in Examples 1 and 2 like Figure 1 As shown, in the solid-state fermentation system, white-rot fungi have a good colonization effect on the surface of the plastic film. Figure 2 and Figure 4 The figures show the colonization effect of white-rot fungi during the degradation process of Sample 1 and Sample 2, and the final degradation effect of the plastic film after washing off the surface biofilm. Compared with Sample 1, Sample 2 showed pyrolysis after degradation. The weight loss rate of both films gradually increased with the extension of cultivation time, as shown in the figures. Figure 7 As shown, the weight loss rate of sample 2 was slightly higher than that of sample 1. The results indicate that the weight loss rates of both plastic films exceeded 15%, demonstrating that plastics can be gradually degraded in the solid-state fermentation system of white-rot fungi.

[0045] 2. Observation using a scanning electron microscope like Figure 5 As shown in Figure A, sample 1 has a relatively rough surface before biodegradation; Figure 5 As shown in B, after co-culturing with *Phanerochaete chrysosporium* in a solid-state fermentation system for 42 days, its surface tended to be smooth and some pores were produced. (See Figure B.) Figure 5 As shown in C, a magnified view shows that *Proteus xanthosporium* can adhere to the plastic surface and cause degradation.

[0046] like Figure 6 As shown in Figure A, sample 2 also had a relatively rough surface before degradation; as Figure 6 As shown in B, after 42 days of co-cultivation, its surface became smooth and cracks appeared; as Figure 6 As shown in C, a magnified view of the part shows that pores have been generated on the plastic surface, indicating that sample 2 has undergone significant biodegradation.

[0047] 3. Infrared spectroscopy detection like Figure 8 As shown in the infrared spectrum, the surface functional groups of the two plastic films changed significantly before and after degradation.

[0048] Compared with sample 1, a new group was generated on sample 1 after solid-state fermentation culture. This group was located at 1621±5 cm⁻¹. -1 The highest absorbance is found at the COO2 level. - Antisymmetric stretching, the broad absorption peak in this region is related to the formation of carboxylate groups. Under the action of microorganisms or enzymes, plastics first generate low-molecular-weight hydroxy acids or dicarboxylic acids. These low-molecular-weight organic acids are easily further ionized in the fermentation system to form COO2. - Groups. Also observed at 1411±10 cm. -1 The weakening of the –CH2– absorption peak indicates that during solid-state fermentation degradation, enzymes and free radical oxidation reactions cause the breakage of the molecular backbone or side chains. The reduction of –CH2– is accompanied by the appearance of new carboxyl groups, suggesting that the plastic is transformed from aliphatic segments to polar small molecules.

[0049] Compared to sample 2, after co-solid-state fermentation, a new R–OH absorption peak appeared in the infrared spectrum of biodegraded sample 2. Oxalic acid, being acidic, may directly participate in the reaction during degradation, or it may promote the formation of hydroxyl terminal groups and small molecule products. After degradation, at 1017 cm⁻¹... -1 The enhanced C–O–C stretching symmetry of fatty acid esters and lactones indicates that polylactic acid (PLA) undergoes ester bond cleavage under the action of oxalic acid, accompanied by the formation of lactones or low-molecular-weight esters. The accumulation of low-molecular-weight fatty acid esters in the degradation products leads to a more pronounced C–O–C vibration peak.

[0050] 4. Gel permeation chromatography detection Table 1 lists the changes in weight-average molecular weight and number-average molecular weight of Sample 1 and Sample 2 before and after biodegradation. The results show that after solid-state fermentation treatment with white-rot fungi, the weight-average and number-average molecular weights of both plastic samples decreased significantly, indicating that the metabolites produced during solid-state fermentation can promote the breakage of plastic molecular chains. Comparing the molecular weights of the samples before and after degradation reveals that *Phanerochaete chrysosporium* attaches to the plastic surface and acts on the molecular chain structure, leading to a decrease in molecular weight.

[0051] Table 1. Changes in Mw and Mn before and after biodegradation in Samples 1 and 2.

[0052] Compared to sample 1, sample 2 showed a greater change in molecular weight, with its Mw decreasing by approximately 46.5%. This result is consistent with... Figure 4The plastic degradation phenomenon shown is consistent with that observed in the sample 2. This difference is related to the addition of oxalic acid in sample 2. Oxalic acid, as a provider of an acidic environment, can accelerate the breakage of the polylactic acid backbone and promote the formation and transformation of lactone structures, thereby enhancing the degradation effect of the plastic.

[0053] 5. Gas chromatography-mass spectrometry detection results As shown in Table 2, gas chromatography-mass spectrometry results indicate that polylactic acid (PLA) plastics containing oxalic acid undergo degradation, producing degradation products consisting of ethers, acids, and esters. These products include C2H6O, C2H4O2, C4H8O2, and C5H... 10 O2, C5H 10 O2 and C4H 10 O corresponds to Figure 8 This is consistent with the previous infrared spectroscopy results. Polylactic acid (PLA) is first hydrolyzed into lactic acid. The –CH– and –CH(CH3)– bonds in the PLA chain are easily oxidized. The degradation process involves the breaking of –C–O– bonds. The intermediate products are further transformed in the solid-state fermentation system. Through oxidative decarboxylation, acidic substances such as acetic acid and propionic acid are produced. Propyl acetate and ethyl propionate in the products are secondary ester products in the oxidative degradation of PLA, mainly originating from the esterification / lactoneation reaction of lactic acid or oligolactic acid.

[0054] Table 2 Biodegradation products of Sample 2

[0055] In summary, after treatment with white-rot fungi solid-state fermentation technology, the weight of the plastic film changed, and its surface morphology also showed significant alterations. Infrared spectroscopy results showed that the –CH2– absorption peak in sample 1 weakened, while a new carboxyl group, “–COOH / COO,” appeared. - The characteristic peaks indicate that the plastic gradually transforms from aliphatic chains into polar small molecules. Simultaneously, the enhanced R-OH absorption peak in sample 2, along with the enhanced C-O-C symmetric stretching vibration signals of fatty acid esters and lactones, suggests that polylactic acid undergoes ester bond cleavage under the action of oxalic acid, accompanied by the formation of lactones or low-molecular-weight esters and the accumulation of low-molecular-weight fatty acid esters. Gas chromatography-mass spectrometry results show that oxalic acid-containing polylactic acid undergoes hydrolysis, oxidative chain scission, and decarboxylation reactions during degradation, generating small molecule products such as ethers, acids, and esters. Propyl acetate and ethyl propionate are formed by the esterification / lactoneation reaction of lactic acid or oligolactic acid, consistent with the infrared spectroscopy results. Further gel permeation chromatography results indicate that the plastic molecular chains break down and the molecular weight decreases, proving that the solid-state fermentation technology of white-rot fungi can accelerate the biodegradation process of plastics.

[0056] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0057] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for degrading plastic film using white-rot fungi, characterized in that, White-rot fungi were inoculated into a solid substrate, and then a plastic film to be degraded was added. Anaerobic solid-state fermentation was carried out at 30℃~37℃ for 40 to 50 days.

2. The method for degrading plastic film using white-rot fungi according to claim 1, characterized in that, The white-rot fungus is *Phanerochaete chrysosporium* (…). Phanerochaetechrysosporium ).

3. The method for degrading plastic film using white-rot fungi according to claim 1, characterized in that, Before inoculation, the white-rot fungus is prepared into a bacterial solution: the white-rot fungus cells are suspended in sterile water, and the turbidity is adjusted to 80 NTU to 100 NTU using a turbidimeter to obtain the white-rot fungus bacterial solution.

4. The method for degrading plastic film using white-rot fungi according to claim 3, characterized in that, The solid substrate is obtained by high-pressure sterilization of the fermentation substrate.

5. The method for degrading plastic film using white-rot fungi according to claim 4, characterized in that, The ratio of white rot fungal inoculum to solid substrate is 2 mL to 5 mL: 5 g to 10 g.

6. The method for degrading plastic film using white-rot fungi according to claim 4, characterized in that, The fermentation substrate is one or a mixture of corn cobs, straw and grain husks.

7. The method for degrading plastic film using white-rot fungi according to claim 1, characterized in that, Before the plastic film to be degraded is added to the solid matrix, it needs to be pretreated: soaked and rinsed in deionized water, and then dried.

8. The method for degrading plastic film using white-rot fungi according to claim 7, characterized in that, Soaking time in deionized water is 1.5h to 2h.

9. The method for degrading plastic film using white-rot fungi according to claim 7, characterized in that, The drying temperature is 40℃~45℃.

10. The method for degrading plastic film using white-rot fungi according to claim 1, characterized in that, The plastic film is composed of one or both of polybutylene adipate and polylactic acid.

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