Fungus for degrading polystyrene plastic and application thereof

Abstract

The invention discloses a fungus for degrading polystyrene plastic and application of the fungus, and relates to the field of environmental microorganism technology and plastic pollution biological treatment. The polystyrene degrading fungus is basidiomycete WYK-PD01, the basidiomycete WYK-PD01 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.35989, and the preservation date is September 26, 2025, and the preservation number is CGMCC No.35989. The strain disclosed by the invention grows by taking PS as a unique carbon source and energy, and in a 28-day degradation experiment, the degradation rate of a PS film reaches 3.5%; according to the co-metabolism process of degrading PS in a low-carbon potato glucose culture medium, the degradation rate of a PS film on the 15th day reaches 9.87%. And FTIR (Fourier transform infrared spectroscopy) and GC-MS (gas chromatography-mass spectrometry) analysis prove the breakage of PS molecular chains and the generation of oxidation products. The strain and the preparation thereof provided by the invention can be used for biodegradation of PS plastic pollutants in the environment and environmental remediation, and have wide application prospects.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology technology and biological remediation of plastic pollution, specifically to a fungus for degrading polystyrene plastic and its application. Background Technology

[0002] Plastic pollution, especially "white pollution" represented by single-use plastic products, has become a major environmental problem urgently needing to be addressed globally. Polystyrene (PS), a widely used general-purpose plastic, is commonly found in packaging materials, cushioning foam, and disposable tableware. Due to its stable benzene rings and carbon-carbon backbone in its molecular structure, PS is chemically inert and highly hydrophobic, making it extremely difficult for microorganisms to utilize and decompose it under natural conditions, allowing it to persist for hundreds of years. Large quantities of PS waste entering the environment not only damage the visual landscape but also gradually break down into microplastics, which infiltrate soil and water bodies, posing a long-term and far-reaching threat to the survival of plants and animals and even human health.

[0003] Currently, the disposal of PS waste still relies primarily on traditional methods, including landfill and incineration. Landfill not only consumes a large amount of land resources, but PS is also difficult to degrade, posing a long-term risk of potential leakage pollution. While incineration can reduce volume and quantity, improper control can easily release toxic and harmful substances such as dioxins and styrene monomers, along with large amounts of carbon dioxide emissions, exacerbating the greenhouse effect. Both methods are "end-of-pipe treatments," failing to fundamentally solve the PS pollution problem and incurring drawbacks such as secondary pollution, high energy consumption, and non-recyclable resources. Therefore, developing green, sustainable, and low-cost PS biodegradation technologies has become an urgent need in the fields of environmental governance and the circular economy.

[0004] In recent years, researchers both domestically and internationally have increasingly focused on the potential of microorganisms in plastic degradation. Reports have shown that certain types of bacteria (such as *Pseudomonas* spp. and *Bacillus* spp.) and fungi (such as *Aspergillus* spp. and *Engyodontium album*) can utilize PS as a carbon source for growth and disrupt its polymer structure through enzymatic processes. However, most existing degrading strains still have significant limitations, such as slow degradation rates (typically less than 5% degradation over several months), stringent environmental requirements (such as specific pH, temperature, or external carbon source induction), and poor stability in practical applications. These factors severely restrict the progress of microbial degradation technology from the laboratory to engineering applications.

[0005] Against this backdrop, isolating and screening highly efficient degrading strains from special environments rich in plastic pollutants has become an effective strategy to overcome current technological bottlenecks. Habitats such as landfills, waste plastic accumulation areas, and long-term contaminated soils may harbor microbial communities with adaptive evolutionary characteristics, potentially containing new species or strains with highly efficient plastic degradation capabilities. Through targeted enrichment culture, multiple rounds of screening, and molecular identification, it is hoped that candidate strains with excellent degradation performance and strong environmental adaptability can be obtained, providing a material basis for the development of novel biological treatment agents. Summary of the Invention

[0006] In order to solve the problem of PS pollution, this invention provides a fungus for degrading polystyrene plastic and its application. The strain is a fungal strain derived from landfill soil that can efficiently degrade polystyrene (PS) plastic. Another objective of this invention is to provide the application of the above-mentioned fungal strain in degrading PS plastic waste.

[0007] This invention discloses a fungus for degrading polystyrene plastic, characterized in that the polystyrene-degrading fungus is Cystobasidium minutum WYK-PD01, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35989 and deposit date of September 26, 2025.

[0008] Cells and / or fermentation broth of Cystobasidium minutum strain WYK-PD01 containing the fungus described in this invention.

[0009] Furthermore, the microbial preparation is a liquid fermentation agent, a solid microbial agent, or a lyophilized powder.

[0010] The application of the above-mentioned fungi in the degradation of polystyrene plastics.

[0011] Furthermore, the application of the fungus in the treatment of polystyrene plastic waste degradation.

[0012] Furthermore, the polystyrene plastic waste includes waste polystyrene plastic packaging, polystyrene food containers, and polystyrene foam.

[0013] Furthermore, the fungus is used for the bioremediation of soil and water bodies contaminated with polystyrene.

[0014] Furthermore, the fungal strain or the microbial preparation is subjected to contact culture with the polystyrene plastic waste to be treated.

[0015] Furthermore, the contact culture conditions are: temperature 20~35°C, pH 5.0~8.0, and treatment time 7~180 days.

[0016] The *Cystobasidium minutum* WYK-PD01 strain exhibits the following microbiological characteristics: After culturing on PDA solid medium at 30°C for 4 days, the colonies are round, with neat edges, smooth surfaces, moist, and mucous-like. The colonies are a bright pink color due to the production of carotenoids. Under an optical microscope (×400x magnification), the vegetative cells are spherical, with a cell diameter of approximately 3–5 μm. This strain reproduces asexually via polygonal budding and does not form pseudohyphae or fungal hyphae. The ITS gene sequence of the strain (using primers ITS1 / ITS4) was amplified and sequenced. The obtained raw sequencing sequence underwent quality control and trimming to remove low-quality bases, and the high-quality sequence was then assembled to obtain the complete ITS sequence. The sequence was compared with known sequences in the NCBI database using BLAST (Basic Local Alignment Search Tool). The results showed that the ITS sequence of the strain of this invention had a similarity of more than 99% with that of Cystobasidium minutum (accession number: HQ326999.1).

[0017] Advantages of this invention:

[0018] The strain of this invention grows using PS as the sole carbon and energy source. In a 28-day degradation experiment, it achieved a degradation rate of 3.5% for PS film. In a low-carbon potato dextrose medium, the strain exhibited a co-metabolic degradation process of PS, achieving a degradation rate of 9.87% on day 15. FTIR and GC-MS analyses confirmed the breakage of PS molecular chains and the formation of oxidation products. The strain and its formulation provided by this invention can be used for the biodegradation and remediation of PS plastic pollutants in the environment, showing broad application prospects.

[0019] The fungal strain described in this invention was sampled from soil surrounding a landfill in Daxing District, Beijing. After enrichment culture, isolation, purification, and multiple rounds of degradation verification, a fungal strain with significant polystyrene degradation capabilities was successfully screened. This strain not only grows using PS as the sole carbon source but also exhibits a significant degradation effect in a short period. Analysis using a combination of techniques confirmed its degradation products and mechanisms. The fungal strain (Cystobasidium minutum) WYK-PD01 described in this invention has excellent effects on the biodegradation of PS plastics. Using the strain of this invention for plastic degradation has advantages such as being environmentally friendly, low-cost, and easy to operate.

[0020] The strain of this invention exhibits excellent degradation characteristics, providing a new resource and approach for the bioremediation of PS waste in the environment, with broad application prospects. This invention provides a new microbial resource and application pathway for solving the PS pollution problem, overcoming the shortcomings of existing biodegradation technologies such as low efficiency and poor applicability, and has significant scientific research value and broad application prospects.

[0021] The polystyrene-degrading fungus of this invention is Cystobasidium minutum WYK-PD01, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35989 and deposit date of September 26, 2025. Attached Figure Description

[0022] Figure 1 Photographs showing the colony morphology of strain WYK-PD01 on PDA plates;

[0023] Figure 2 Microscopic morphology photograph of strain WYK-PD01 (×400x).

[0024] Figure 3 This is a scanning electron microscope image of the PS film surface after treatment with strain WYK-PD01 for 28 days;

[0025] Figure 4 Comparison of FTIR spectra of degraded PS films (PS: before degradation; Cys+PS: after degradation);

[0026] Figure 5 This is a mass spectrum of key intermediates in PS degradation detected by GC-MS. Detailed Implementation

[0027] To clearly and completely describe the specific implementation methods and technical solutions of the present invention, the following description will be provided in conjunction with the accompanying drawings of the embodiments of the present invention. The embodiments described below are some, but not all, embodiments of the present invention. All embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0028] Example 1: Isolation, screening, and identification of Cystobasidium minutum WYK-PD01, a fungus that efficiently degrades polystyrene according to the present invention.

[0029] 1. Culture medium: The inorganic salt culture medium consists of 0.7 g KH2PO4, 0.7 g K2HPO4, 0.7 g MgSO4·7H2O, 1.0 g NH4NO3, 0.005 g NaCl, 0.002 g FeSO4·7H2O, 0.002 g ZnSO4·7H2O and 0.001 g MnSO4·H2O per 1000 mL, with the pH adjusted to 6.0-6.5; the solid culture medium is prepared by adding 20 g / L agar.

[0030] 2. In December 2024, soil samples were collected from the area surrounding a landfill in Daxing District, Beijing. Using a quincunx sampling method, one sample of the 0-10 cm surface layer was taken. Immediately after sampling, the samples were inoculated into an inorganic salt culture medium with PS microplastics as the sole carbon source for enrichment culture. The PS microplastics were pre-washed with 75% alcohol and sterilized under ultraviolet light. The culture system was cultured at 30°C and 150 rpm on a rotating shaker. After 28 days, the culture solution was serially diluted and separated using the streak plate method to obtain PS-degrading bacteria, which were then numbered and stored.

[0031] 3. Isolation and purification: The enriched culture was serially diluted and spread on PDA plates and incubated at 30°C for 4 days. Single colonies were picked according to the differences in colony morphology, and the pure culture was obtained after multiple purifications.

[0032] 4. Initial screening: Spot the purified strain onto a plate with PS film as the sole carbon source and observe whether a clear zone or film deformation appears around the colony.

[0033] 5. Secondary screening: The positive strains obtained from the initial screening were inoculated into liquid culture medium with PS as the sole carbon source for shake-flask fermentation experiments, and the degradation rate was determined by the weight loss method. Finally, the strain with the best degradation effect, WYK-PD01, was selected.

[0034] 6. Identification: Through the above isolation and screening work, and multiple isolation and purification processes, a strain WYK-PD01 that efficiently degrades polystyrene using PS was obtained, and the microbiological characteristics of strain WYK-PD01 were identified.

[0035] 6.1 When strain WYK-PD01 was inoculated onto PDA solid medium and cultured at 30°C for 4 days, the colonies were round, with neat edges, smooth surfaces, moist, and mucous-like. The colonies were a bright pink color due to the production of carotenoids. Under an optical microscope (×400x magnification), the vegetative cells were spherical, with a cell diameter of approximately 3–5 μm. This strain reproduces asexually via polygonal budding and does not form pseudohyphae or fungal hyphae. Figure 1 This is a photograph of the colony morphology of strain WYK-PD01 on a PDA plate. Figure 2Microscopic morphology photograph of strain WYK-PD01 (×400x magnification).

[0036] 6.2 The ITS gene sequence of strain WYK-PD01 (using primers ITS1 / ITS4) was amplified and sequenced. The obtained raw sequence was quality controlled and pruned to remove low-quality bases, and then assembled to obtain a complete ITS sequence. This sequence was compared with known sequences in the NCBI database using BLAST (Basic Local Alignment Search Tool). The results showed that the ITS sequence of strain WYK-PD01 had a similarity of over 99% with that of Cystobasidium minutum (accession number: HQ326999.1), identifying strain WYK-PD01 as Cystobasidium minutum. The endogenous transcribed spacer region in the ribosomal RNA gene cluster was selected as the standard DNA barcode for species identification, and PCR amplification was performed using fungal primers (ITS1: TCCGTAGGTGAACCTGCGG1 and ITS4: TCGMCGCTTATTGATATGC). PCR amplification system (20 µL): template DNA 5 µL, Taq enzyme 10 µL, forward primer (ITS1) 1 µL, reverse primer (ITS4) 1 µL, ddH2O 3 µL. PCR reaction conditions were: 95℃ for 3 min; 95℃ for 30 s, annealing for 30 s, 72℃ for 45 s, 30 cycles; 72℃ for 10 min; 10℃ for 10 min.

[0037] Example 2: Validation experiment on PS degradation efficiency of strain Cystobasidium_minutum WYK-PD01

[0038] Strain preparation: Inoculate strain Cystobasidium minutumWYK-PD01 into yeast extract peptone glucose medium until the logarithmic growth phase, wash three times with the above inorganic salt medium to remove the surface medium, and finally resuspend in inorganic salt medium.

[0039] Degradation experiment: In an Erlenmeyer flask containing inorganic salt medium, add a 100 mg pre-weighed (initial weight W0) PS film (1 cm × 1 cm) (inorganic salt liquid medium containing 0.1% PS film) sterilized under UV light. Inoculate with a 2% (v / v) suspension of Cystobasidium_minutum WYK-PD01 to make the cell concentration in the medium approximately 2 × 10⁻⁶ cells / mL. 7The concentration of cells / ml was determined by using uninoculated culture medium as a blank control. Each treatment was performed in triplicate. The cells were incubated at 30°C and 150 rpm in the dark for 28 days. Samples were taken periodically to analyze the morphological changes, functional group changes, mass loss, and degradation products on the surface of the PS membrane.

[0040] Figure 3 The images show scanning electron microscope (SEM) images of PS films after 28 days of treatment with strain WYK-PD01. (a) is an original PS plastic SEM image, (b) is a PS SEM image after 28 days of shaking culture in an inorganic salt medium, and (c) is a PS SEM image after 28 days of degradation by Cystobasidium minutum. Figure 3 The surface morphology of PS before and after degradation can be seen. After 28 days of treatment with strain WYK-PD01, the surface of the PS film showed obvious erosion and pores.

[0041] Example 3: Validation experiment on PS co-metabolism degradation efficiency of strain Cystobasidium_minutum WYK-PD01

[0042] Inoculum preparation: Inoculate strain Cystobasidium minutum WYK-PD01 into low-carbon potato glucose medium until the logarithmic growth phase. The medium composition is: 5g glucose, 3g KH2PO4, 1.5g MgSO4, and 200g potato per 1000mL, boiled and filtered.

[0043] Degradation experiment: In an Erlenmeyer flask containing low-carbon potato dextrose medium, a 100 mg pre-weighed (initial weight W0) PS film (1 cm × 1 cm) sterilized under UV light (low-carbon potato dextrose medium containing 0.1% PS film) was added. A 2% (v / v) suspension of Cystobasidium minutum WYK-PD01 was then inoculated to bring the cell concentration in the medium to approximately 2 × 10⁻⁶ cells / mL. 7 The concentration of cells / ml was determined by using uninoculated culture medium as a blank control. Each treatment was performed in triplicate. The cells were incubated at 30°C and 150 rpm in the dark for 15 days, and samples were taken periodically to analyze the mass loss of the PS membrane.

[0044] The results were tested:

[0045] Degradation rate: After cultivation, the PS film was removed and washed with 2% SDS solution for 4 h to completely remove the biofilm. After drying, it was accurately weighed (final weight W1). Degradation rate (%) = (W0 ~ W1) / W0 × 100%. The calculated result was 3.5% in inorganic salt medium and 9.87% in low-carbon potato dextrose medium, indicating that the addition of a small amount of exogenous carbon source can significantly promote the degradation of PS by the strain.

[0046] FTIR analysis: FTIR analysis was performed on the films before and after degradation. Figure 4 The image shows a comparison of the FTIR spectra of the degraded PS films (PS: before degradation; Cys+PS: after degradation). Figure 4 As shown, in PS treated with Cystobasidium minutum, the FTIR peak intensity at 650–1000 cm⁻¹ (ring bending vibration) is much weaker than that in untreated PS samples. Meanwhile, the characteristic peaks representing the PS benzene ring (C=C stretching vibration, 1550–1610 cm⁻¹ and 1800–2000 cm⁻¹) are weakened, providing evidence of ring breakage. At the same time, the CH stretching vibration at 2800–3100 cm⁻¹ is weakened, indicating that its amorphous structure is fragmented.

[0047] GC-MS analysis: The culture medium at 28 days was extracted with ethyl acetate and derivatized before GC-MS analysis (some substances in the figure below are products obtained after derivatization and reverse derivatization). Figure 5 The mass spectra of key intermediates in PS degradation detected by GC-MS are shown below. Figure 5 As can be seen, benzyl alcohol, terephthalaldehyde, and o-hydroxyacetone, which contain benzene rings, were detected, indicating oxidative modification of branched fragments. Medium- and long-chain fatty acid products, such as n-pentadecanol, dodecanol / acid, and octanoic acid, were also detected, indicating that the enzyme attacked and broke the PS backbone, producing long-chain alkane fragments. These fragments are oxidized at the terminal to form alcohols, then further oxidized to acids, and enter the standard β-oxidation pathway. In addition, the products also contain short-chain fatty acids / alcohols such as adipic acid, glutaric acid, and propylene glycol, indicating that Cystobasidium minutum opens the benzene ring, followed by hydrogenation and β-oxidation, gradually shortening the carbon chain, and finally entering the tricarboxylic acid cycle, converting it into CO2, water, and energy, thus confirming the biodegradation process of PS.

Claims

1. A fungus for degrading polystyrene plastic, characterized in that, The fungus for degrading polystyrene is Cystobasidium minutum WYK-PD01, which is preserved in China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No. 35989 and the preservation date of September 26, 2025.

2. A microbial preparation containing the fungus according to claim 1, characterized in that, The microbial preparation comprises cells and / or fermentation liquor of the Cystobasidium minutum WYK-PD01 strain of claim 1.

3. The microbial preparation according to claim 2, characterized in that: The microbial preparation is a liquid starter, a solid bacterial agent or a freeze-dried powder.

4. The use of the fungus of claim 1 in degrading polystyrene plastic.

5. Use of a fungus according to claim 4 for degrading polystyrene plastic, characterized in that, The use of the fungus in degrading polystyrene plastic waste.

6. Use of a fungus according to claim 5 for degrading polystyrene plastic, characterized in that, The polystyrene plastic waste includes waste polystyrene plastic packaging, polystyrene food containers and polystyrene foam.

7. Use of a fungus according to claim 4 for degrading polystyrene plastic, characterized in that, The fungus is used for bioremediation of soil and water bodies contaminated by polystyrene.

8. A method for degrading polystyrene plastic waste using the fungus described in claim 1, characterized in that, The fungus strain of claim 1 or the microbial preparation of claim 2 is cultured in contact with polystyrene plastic waste to be treated.

9. The method of degrading polystyrene plastic waste by fungi according to claim 8, wherein, The culture condition is that the temperature is 20-35°C, the pH is 5.0-8.0 and the treatment time is 7-180 days.

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