Biodegradation of low-density polyethylene by mixed fungi composed of 
Plastics are widely used in production and daily life due to their advantages of low cost, corrosion resistance, and great plasticity . Since 1950, there has been a significant increase in plastic production every year . In recent years, the production of plastics has reached nearly 400 million tons per year, and it is inferred that until 2050, the annual production of plastics will reach 600 million tons . When disposing of discarded plastic waste, 40% is incinerated, releasing toxins into the air that harm organisms and the environment . Additionally, some plastic waste is landfilled or leaked into the natural environment, while only a small portion is recycled. Due to their nonbiodegradable properties, plastics need to be buried underground for nearly a decade before mineralization occurs . It is worth noting that burning plastic emits large amounts of toxic and harmful gases . Buried plastic may be transformed into small plastic particles through weathering or other decomposition processes, and inhaling small plastic particles into organisms can seriously harm their health , while other debris will float in the air and combine with organic pollutants before falling back into the soil and ocean, contaminating them . Bioremediation is a low-cost, ecologically friendly technology that is commonly regarded as the most effective means of eliminating a variety of environmental pollutants. In contrast to traditional approaches, the use of microorganisms can effectively degrade refractory compounds, such as polymers, polyaromatic hydrocarbons, phenols, pesticides, and so on. Many microorganisms have been well-studied in plastic biodegradation , and some fungi, such as Trichoderma spp., Penicillium spp., Paecilomyces spp., Aspergillus spp., and Aureobasidium spp., have been reported to degrade LDPE . Fungi, unlike bacteria, grow faster in the soil and may reach deeper positions through the growth expansion and penetration of mycelia, making them more suitable for polyethylene degradation. In addition to the well-studied filamentous fungi mentioned above, certain Alternaria sp. have also been found to be capable of degrading plastics. Among them, the marine fungus Alternaria alternata has been shown to have polyethylene-degrading capabilities, and its molecular mechanisms for plastic degradation, including laccase and peroxidase, have been determined via transcriptomics . Moreover, the degradation ability of Alternaria sp. is not restricted to industrially produced polyethylene products, as it is also capable of degrading oil that pollutes the ocean and, to a significant extent, extra-heavy crude oil (EHCO) . These findings highlight the critical involvement of the genus Alternaria in the breakdown of polythene and other marine pollutants. Recent studies have shown that Cladosporium sp. has the ability to degrade High-Density Polyethylene (HDPE)  and Low-Density Polyethylene (LDPE) . Furthermore, according to Birolli et al. . , the genus Cladosporium sp. is capable of breaking down a range of polycyclic aromatic hydrocarbons and polyurethanes. Because of these properties, Cladosporium spp. are recognized as decomposers of environmental pollutants, and they are particularly good candidates for degrading polyethylene materials. Although Trametes sp. has been reported less frequently in the biodegradation of polyethylene, it has been successfully used to degrade phenanthrenes, which are toxic and hazardous to the environment , chlorocellulosic acid , pesticides—organophosphorus malathion, and other environmental pollutants . Trametes sp. has also been found to contribute significantly to the degradation of lignin . Conventional biodegradation methods typically rely on a single strain, but recently, the use of microbial mixed culture has emerged as an efficient strategy for enhancing microbial performance . In the biomes formed by microbial mixed culture, different strains collaborate to fully utilize their unique advantages and capabilities . At present, microbial co-cultivation has become a prominent topic in various fields such as industry, agriculture, pharmaceuticals, and environmental protection; it has also become an important means to boost production or discover new substances . When applied to LDPE degradation, the degradation capacity of the combined microorganisms may also exceed that of a single strain. Studies have shown that fungal microbiota may contain enzymes that catalyze polymer degradation through oxidation. These enzymes can disrupt polymer chains, release oligomers and monomers, and attack the end chains of high molecular weight materials. Microbial communities can also attack polymers through co-metabolic processes where members of the microbial community grow on substrates prone to oxidation, further oxidizing substances that are difficult to use as sole carbon and energy sources . In this study, three LDPE-degrading fungi (CPEF-1, CPEF-6, and PE2F-4) were isolated from landfills, and these highly efficient degrading fungi were used for LDPE degradation experiments individually or in pairs. Our goal was to identify individual strains or combinations that can degrade environmental pollutants, such as polyethylene, more effectively. We hypothesized that combined fungal culture should have greater degradation capacity than single strain treatment due to the additive effect of the respective advantages between different strains. As a result, the combined fungi (H-3 group) composed of Alternaria sp. CPEF-1 and Trametes sp. PE2F-4 exhibited the greatest degradation effect on the heat-treated LDPE (T-LDPE) membrane. The degradation performance of the T-LDPE films by the combined fungal culture was further investigated. 

Soil landfill experiments Soil samples from various sources were collected and properly mixed, and then fragments of polyethylene cling film and plastic sealed bags, which are widely available in the market, were added to the combined soil samples for enrichment. Proper flipping and spraying were used to simulate the natural temperature and humidity. After 100 days of enrichment, plastic debris with more adhesive soil was selected for subsequent experimental manipulations. Preparation of LDPE samples for degradation experiments A certain amount of purchased LDPE Microplastic Particles (Shanghai McLin Biochemical, Shanghai, China) with a particle size of approximately 1000 mesh and a melt index of 20–30 g /10 min were weighed, and placed in a Petri dish, and then exposed to ultraviolet irradiation on a sterile workbench for 12 h. The LDPE transparent open bags (Aladdin Industrial Co., Ltd., Shanghai, China) with a thickness of 0.038 mm and uniform texture were cut into small pieces of 3 cm × 3 cm and weighed. For the preparation of untreated sheets (U-LDPE), the sheets were soaked and sterilized in 75% ethanol for 3 h on a sterile workbench, followed by rinsing with sterile water multiple times. The sheets were then air-dried and sterilized with ultraviolet light for an additional 12 h. For the preparation of heat-treated material (T-LDPE), the sheets were subjected to heating in a hot air oven under dry and dark conditions at 75 °C for 7 days to induce changes in the structure of LDPE. The subsequent UV treatment method was identical to that used for U-LDPE . Media and culture conditions Minimal salt medium (MSM) (g/L): 1.0 g/L KH 2 PO 4 , 1.5 g/L Na 2 HPO 4 , 2.0 g/L NH 4 Cl, 0.1 g/L CaCl 2 ·2H 2 O, 0.15 g/L KCl, 0.2 g/L MgSO 4 ·7H 2 O, 0.01 g/L FeSO 4 ·7H 2 O, 0.01 g/L ZnSO 4 ·7H 2 O, and 0.001 g/L MnSO 4 ·H 2 O. Micro carbon source medium (MCM) (g/L): 0.5 g/L yeast extract, 2.0 g/L (NH 4 ) 2 SO 4 , 1.0 g/L FeSO 4 ·7H 2 O, 1.0 g/L MgSO 4 ·7H 2 O, 0.1 g/L CuSO 4 ·5H 2 O, 0.1 g/L MnSO 4 ·H 2 O, and 0.1 g/L ZnSO 4 ·7H 2 O. The above liquid media can be prepared into agar plates by adding agar at a concentration of 15 g/L. Potato dextrose agar medium (PDA) (g/L): 6.0 g/L potato powder soaking, 20.0 g/L glucose, and 15.0 g/L agar. PDA plate was used to maintain the growth of the isolated fungi, MSM medium added LDPE sheets as the sole carbon source to study the degradation of LDPE by fungi, and MCM medium contained trace amounts of carbon sources, which was employed for pre-domestication of fungi in formal experiments. All liquid cultures were performed at 30℃ with a rotation speed of 150 rpm. Isolation, screening, and identification of LDPE-degrading fungi A series of diluted soil solutions were inoculated into 100 ml of MCM liquid media for domestication, in which 1 g of LDPE microplastic particles were added. The LDPE particles were taken out after 30 days of culture and then transferred to 100 ml of MSM medium for another 30 days. The enriched solution was collected and coated on the MSM agar media using LDPE sheets as the sole carbon source. If necessary, 1% streptomycin can be added to the plate culture medium to inhibit the growth of bacteria. Subsequently, single colonies were selected and purified on a sterile PDA plate to obtain a pure fungal culture. To further obtain fungi with strong degradation performance, the isolates were cultured in MSM-LDPE (U-LDPE sheets) media for 30 days. By observing fungal growth and adhesion on LDPE sheets, the biodegradation capability of LDPE was preliminarily investigated. Molecular identification of LDPE-degrading isolates The genomic DNA of LDPE-degrading isolates was extracted using a simple approach , and the universal fungal primers ITS4 and ITS5  were used for PCR amplification and molecular identification. The DNA sequences acquired by sequencing were compared to BLAST in the NCBI database. Clustal X was used to compare the resulting ITS gene sequences, and Bio-Edit for gene editing. The Neighbor-Joining method  was used to construct phylogenetic trees, with 1000 repetitions determining the bootstrap value in MEGA7. Biodegradation of the LDPE sheets The status of the LDPE sheets was utilized to further assess the biodegradability of the tested strains. 500 µl of spore suspension was added into 100 ml MSM liquid medium, containing sterilized LDPE sheets as the sole carbon source, and incubated at 150 rpm on a shaking table at 30 °C for 30 days. Samples without spore dispersion were utilized as controls, and each experiment was repeated three times. Determination of weight loss The weight loss method is commonly used to measure the rate of biodegradation . First, after 30 days of fungal treatment, LDPE sheets were collected and washed overnight with 2% (v/v) SDS to eliminate the biofilm. Then, the LDPE sheet was soaked and sterilized with 75% ethanol, rinsed with distilled water three to four times, and dried in an oven. Finally, the dried LDPE sheets were weighed, and the weight loss percentage  was determined according to the following formula. 1 [12pt]{minimal}
				
				 
				 
				 
				
				
				
				
				$$\:\:\:\\=\:)\:-\:(\:)}{\:}\:100$$ Surface morphology of LDPE sheets The surface morphologies of untreated LDPE (U-LDPE) and heat-treated LDPE (T-LDPE) sheets were observed by an FEI Quanta 200 environmental scanning electron microscope (FEI Company, Hillsboro, OR, USA). The samples were dehydrated, dried, mounted, and then sputter-coated with gold for ESEM observation. FTIR spectroscopic analysis Fourier transformed infrared spectroscopy (Bruker VERTEX 80 V; Bruker Corporation, Bremen, Germany) was used for comparative analysis of the chemical structure of biodegraded LDPE sheets . Determination of laccase activity The activity of laccase secreted by isolated fungi during the degradation of T-LDPE sheets was detected using the ABTS (2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) method . After 0, 2, and 4 weeks of culture, 5 mL of fungal culture medium was collected and centrifuged at 4 ℃ for 10 min at a rotational speed of 4000 rpm. Then, 1 mL of the supernatant was used for enzyme activity determination. A total of 0.5 mM ABTS solution 1.0 mL was mixed with 1.0 mL of 0.01 mol/L acetic acid-sodium acetate buffer (pH 4.5), and 1 mL of culture supernatant was added to start the reaction at 37 ℃ as the experimental group. In the same experiment, 1.0 mL of pure water was added instead of culture supernatant to serve as a blank control group. After incubation for 10 min, the samples from the blank control group and experimental group were analyzed with a Nano-Drop 2000c spectrophotometer to record changes in absorbance at a wavelength of 420 nm. The enzyme activity unit (U) is defined as the amount of enzyme required to increase the OD value by 0.001 per minute at 37 ℃, which is one enzyme activity unit. The enzyme activity was calculated using the following formula (U/L). 2 [12pt]{minimal}
				
				 
				 
				 
				
				
				
				
				$$\:U()=\:420\:\:\:106\:\:\:}{\:{\:}\:}$$ (n: coefficient of dilution; V: volume of culture medium (mL)) Statistical analysis SPSS software was used to perform one-way ANOVA for statistical analysis. The resulting data are expressed as the mean ± standard deviation (SD). A p value < 0.05 indicated that the difference was statistically significant. All experiments were done in triplicates. 

Soil samples from various sources were collected and properly mixed, and then fragments of polyethylene cling film and plastic sealed bags, which are widely available in the market, were added to the combined soil samples for enrichment. Proper flipping and spraying were used to simulate the natural temperature and humidity. After 100 days of enrichment, plastic debris with more adhesive soil was selected for subsequent experimental manipulations. 

A certain amount of purchased LDPE Microplastic Particles (Shanghai McLin Biochemical, Shanghai, China) with a particle size of approximately 1000 mesh and a melt index of 20–30 g /10 min were weighed, and placed in a Petri dish, and then exposed to ultraviolet irradiation on a sterile workbench for 12 h. The LDPE transparent open bags (Aladdin Industrial Co., Ltd., Shanghai, China) with a thickness of 0.038 mm and uniform texture were cut into small pieces of 3 cm × 3 cm and weighed. For the preparation of untreated sheets (U-LDPE), the sheets were soaked and sterilized in 75% ethanol for 3 h on a sterile workbench, followed by rinsing with sterile water multiple times. The sheets were then air-dried and sterilized with ultraviolet light for an additional 12 h. For the preparation of heat-treated material (T-LDPE), the sheets were subjected to heating in a hot air oven under dry and dark conditions at 75 °C for 7 days to induce changes in the structure of LDPE. The subsequent UV treatment method was identical to that used for U-LDPE . 

Minimal salt medium (MSM) (g/L): 1.0 g/L KH 2 PO 4 , 1.5 g/L Na 2 HPO 4 , 2.0 g/L NH 4 Cl, 0.1 g/L CaCl 2 ·2H 2 O, 0.15 g/L KCl, 0.2 g/L MgSO 4 ·7H 2 O, 0.01 g/L FeSO 4 ·7H 2 O, 0.01 g/L ZnSO 4 ·7H 2 O, and 0.001 g/L MnSO 4 ·H 2 O. Micro carbon source medium (MCM) (g/L): 0.5 g/L yeast extract, 2.0 g/L (NH 4 ) 2 SO 4 , 1.0 g/L FeSO 4 ·7H 2 O, 1.0 g/L MgSO 4 ·7H 2 O, 0.1 g/L CuSO 4 ·5H 2 O, 0.1 g/L MnSO 4 ·H 2 O, and 0.1 g/L ZnSO 4 ·7H 2 O. The above liquid media can be prepared into agar plates by adding agar at a concentration of 15 g/L. Potato dextrose agar medium (PDA) (g/L): 6.0 g/L potato powder soaking, 20.0 g/L glucose, and 15.0 g/L agar. PDA plate was used to maintain the growth of the isolated fungi, MSM medium added LDPE sheets as the sole carbon source to study the degradation of LDPE by fungi, and MCM medium contained trace amounts of carbon sources, which was employed for pre-domestication of fungi in formal experiments. All liquid cultures were performed at 30℃ with a rotation speed of 150 rpm. 

A series of diluted soil solutions were inoculated into 100 ml of MCM liquid media for domestication, in which 1 g of LDPE microplastic particles were added. The LDPE particles were taken out after 30 days of culture and then transferred to 100 ml of MSM medium for another 30 days. The enriched solution was collected and coated on the MSM agar media using LDPE sheets as the sole carbon source. If necessary, 1% streptomycin can be added to the plate culture medium to inhibit the growth of bacteria. Subsequently, single colonies were selected and purified on a sterile PDA plate to obtain a pure fungal culture. To further obtain fungi with strong degradation performance, the isolates were cultured in MSM-LDPE (U-LDPE sheets) media for 30 days. By observing fungal growth and adhesion on LDPE sheets, the biodegradation capability of LDPE was preliminarily investigated. 

The genomic DNA of LDPE-degrading isolates was extracted using a simple approach , and the universal fungal primers ITS4 and ITS5  were used for PCR amplification and molecular identification. The DNA sequences acquired by sequencing were compared to BLAST in the NCBI database. Clustal X was used to compare the resulting ITS gene sequences, and Bio-Edit for gene editing. The Neighbor-Joining method  was used to construct phylogenetic trees, with 1000 repetitions determining the bootstrap value in MEGA7. 

The status of the LDPE sheets was utilized to further assess the biodegradability of the tested strains. 500 µl of spore suspension was added into 100 ml MSM liquid medium, containing sterilized LDPE sheets as the sole carbon source, and incubated at 150 rpm on a shaking table at 30 °C for 30 days. Samples without spore dispersion were utilized as controls, and each experiment was repeated three times. Determination of weight loss The weight loss method is commonly used to measure the rate of biodegradation . First, after 30 days of fungal treatment, LDPE sheets were collected and washed overnight with 2% (v/v) SDS to eliminate the biofilm. Then, the LDPE sheet was soaked and sterilized with 75% ethanol, rinsed with distilled water three to four times, and dried in an oven. Finally, the dried LDPE sheets were weighed, and the weight loss percentage  was determined according to the following formula. 1 [12pt]{minimal}
				
				 
				 
				 
				
				
				
				
				$$\:\:\:\\=\:)\:-\:(\:)}{\:}\:100$$ Surface morphology of LDPE sheets The surface morphologies of untreated LDPE (U-LDPE) and heat-treated LDPE (T-LDPE) sheets were observed by an FEI Quanta 200 environmental scanning electron microscope (FEI Company, Hillsboro, OR, USA). The samples were dehydrated, dried, mounted, and then sputter-coated with gold for ESEM observation. FTIR spectroscopic analysis Fourier transformed infrared spectroscopy (Bruker VERTEX 80 V; Bruker Corporation, Bremen, Germany) was used for comparative analysis of the chemical structure of biodegraded LDPE sheets . 

The weight loss method is commonly used to measure the rate of biodegradation . First, after 30 days of fungal treatment, LDPE sheets were collected and washed overnight with 2% (v/v) SDS to eliminate the biofilm. Then, the LDPE sheet was soaked and sterilized with 75% ethanol, rinsed with distilled water three to four times, and dried in an oven. Finally, the dried LDPE sheets were weighed, and the weight loss percentage  was determined according to the following formula. 1 [12pt]{minimal}
				
				 
				 
				 
				
				
				
				
				$$\:\:\:\\=\:)\:-\:(\:)}{\:}\:100$$ 

The surface morphologies of untreated LDPE (U-LDPE) and heat-treated LDPE (T-LDPE) sheets were observed by an FEI Quanta 200 environmental scanning electron microscope (FEI Company, Hillsboro, OR, USA). The samples were dehydrated, dried, mounted, and then sputter-coated with gold for ESEM observation. 

Fourier transformed infrared spectroscopy (Bruker VERTEX 80 V; Bruker Corporation, Bremen, Germany) was used for comparative analysis of the chemical structure of biodegraded LDPE sheets . 

The activity of laccase secreted by isolated fungi during the degradation of T-LDPE sheets was detected using the ABTS (2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) method . After 0, 2, and 4 weeks of culture, 5 mL of fungal culture medium was collected and centrifuged at 4 ℃ for 10 min at a rotational speed of 4000 rpm. Then, 1 mL of the supernatant was used for enzyme activity determination. A total of 0.5 mM ABTS solution 1.0 mL was mixed with 1.0 mL of 0.01 mol/L acetic acid-sodium acetate buffer (pH 4.5), and 1 mL of culture supernatant was added to start the reaction at 37 ℃ as the experimental group. In the same experiment, 1.0 mL of pure water was added instead of culture supernatant to serve as a blank control group. After incubation for 10 min, the samples from the blank control group and experimental group were analyzed with a Nano-Drop 2000c spectrophotometer to record changes in absorbance at a wavelength of 420 nm. The enzyme activity unit (U) is defined as the amount of enzyme required to increase the OD value by 0.001 per minute at 37 ℃, which is one enzyme activity unit. The enzyme activity was calculated using the following formula (U/L). 2 [12pt]{minimal}
				
				 
				 
				 
				
				
				
				
				$$\:U()=\:420\:\:\:106\:\:\:}{\:{\:}\:}$$ (n: coefficient of dilution; V: volume of culture medium (mL)) 

SPSS software was used to perform one-way ANOVA for statistical analysis. The resulting data are expressed as the mean ± standard deviation (SD). A p value < 0.05 indicated that the difference was statistically significant. All experiments were done in triplicates. 

Screening of highly effective LDPE-degrading fungi Following the secondary enrichment of MSM-LDPE medium, a number of fungi capable of degrading LDPE were isolated. The biodegradation process of polyethylene begins with microbial adhesion to the surface of the substrate material. Therefore, these isolates were cultured in MSM-LDPE media for 30 days, after which mycelial adherence to LDPE sheets and fungal growth were observed (Table ) for further screening of fungi with high LDPE biodegradability. The results indicated that strains CPEF-6, CPEF-7, and PE2F-4 had the most robust growth status, followed by CPEF-1, PE1F-13, and PE2F-7. On LDPE sheets, CPEF-6 and PE2F-4 exhibited the greatest mycelial adhesion, followed by CPEF-1 and PE2F-7. Based on these findings, three fungi—CPEF-1, CPEF-6, and PE2F-4—were chosen as highly efficient LDPE-degrading fungi for further investigation. Morphological and phylogenetic analysis of screened LDPE-degrading fungi Cultured on PDA plates at 30 °C for 7 days, the colony diameter of strain CPEF-1 reached 57–62 mm. On PDA plates, the mycelia of CPEF-1 grew on the surface and were immersed, and the colonies were flocculent or villous, with abundant aerial hyphae, hairy yellow edges, gray-brown surface, black to grayish-brown reverse side, flat and loosely flocculent center, and ovate or oval spores (Fig. ). According to the description of colony and spore morphology, this fungus is similar to Alternaria sp. Similarly, the colonies and microscopic morphology of strains CPEF-6 and PE2F-4 were observed. In a previous study, the morphology of CPEF-6 was described; for example, CPEF-6 had a diameter of 25–27 mm after 7 days of cultivation at 30℃ on PDA, colonies were greenish olivaceous to grey-olivaceous, and spores were abundant  (Fig. ). PE2F-4 exhibited a single colony diameter of 65–69 mm after being cultured on PDA plates at 30 °C for 5 days. The colonies appeared white and uniform with dense mycelia, a flat felt-like or cotton-like surface, rounded edges and abundant aerial hyphae (Fig. ). The ITS regions of strains CPEF-1, CPEF-6, and PE2F-4 amplified by PCR were sequenced and uploaded to NCBI with accession numbers OQ921460, OQ651281 and OQ921462, respectively, and their phylogenetic trees were constructed using the neighbor-joining method. Based on morphological characteristics as well as phylogenetic tree analysis, CPEF-1, CPEF-6, and PE2F-4 were identified as Alternaria angustiovoidea , Cladosporium basi-inflatum , and Trametes hirsuta , respectively (Table - ; Fig. - ). Weight loss measurement of LDPE sheets The three LDPE-degrading fungi CPEF-1, CPEF-6 and PE2F-4 described above were employed for LDPE degradation studies either alone or in pairs. Table illustrates the combination approach, where three groups of combined microorganisms, H-1, H-2 and H-3, were acquired (Table ). To verify the difference in the degradation ability of LDPE films between individual and combined fungi, we measured the dry weight of residual LDPE consumables to calculate the weight loss of the films (Fig. ). The results showed that after 30 days of treatment, fungi CPEF-1, CPEF-6, and PE2F-4 reduced the weight of the U-LDPE films by 0.36 ± 0.15%, 0.30 ± 0.06%, and 0.37 ± 0.11%, respectively. The combined strains H-1, H-2, and H-3 reduced the weight of the U-LDPE films by 0.37 ± 0.05%, 0.31 ± 0.01%, and 0.26 ± 0.11%, respectively, with no significant difference compared to the single-strain treatment (Fig. ). To change the structure of LDPE and make it easier to degrade, the LDPE sheets were subjected to heat treatment (T-LDPE). After 30 days of biodegradation, the weights of the T-LDPE films co-cultured with CPEF-1, CPEF-6, and PE2F-4 decreased by 0.28 ± 0.13%, 0.43 ± 0.01%, and 0.38 ± 0.06%, respectively. In addition, compared with U-LDPE, in the combined microbial treatment, only group H-1 had a reduced degradability in the T-LDPE film, while the weight loss rates of the T-LDPE films treated with groups H-2 and H-3 increased by approximately 58% and 154%, respectively (Fig. ). The weight loss rate of the T-LDPE films treated in the H-3 group reached 0.66 ± 0.06%, which was significantly higher than that of the H-1 group, showing no increase in weight loss rate, and the H-2 group, with a weight loss rate of 0.49 ± 0.06%. Interestingly, group H-3 did not perform well in degrading U-LDPE films, but showed the highest capacity in degrading T-LDPE films. The findings indicated that most of the combined fungal treatments could more effectively degrade heat-treated LDPE films. Observation of the surface morphology of LDPE sheets The morphological changes on the surface of LDPE films after 30 days of incubation with fungi were observed using ESEM. The surface of the U-LDPE film uninoculated with fungi remained smooth and intact without any changes (Fig. A). In contrast, the surfaces of the U-LDPE films treated by fungal inoculation became rough and uneven, showing obvious corrosion and erosion holes on the surface, clear cracks and pits, as well as the formation of granular materials (Fig. B-G). Among them, in the CPEF-6 inoculated samples, mycelia were clearly attached to the surface of the U-LDPE film (Fig. D-E). Notably, the PE2F-4 strain caused extensive morphological changes on the sheet surface (Fig. B-C). These findings suggest that these fungi can degrade U-LDPE, cause certain damage to its surface, and grow on the surface of LDPE sheets. Furthermore, ESEM was used to observe the morphological changes of the T-LDPE surface after 30 days of fungal culture. The surface of the T-LDPE films without fungal inoculation had slight streaks, but remained smooth and intact overall (Fig. A). In contrast, the surface of the fungus-inoculated T-LDPE sheets became rougher and more uneven, with extensive surface corrosion and numerous erosion holes, visible cracks, and deep pits (Fig. B-M). Particularly, in the samples inoculated with CPEF- 6 or PE2F-4 alone, the surfaces of the T-LDPE sheets exhibited irregular folds and obvious delamination (Fig. D-M). Interestingly, the surface of the heat-treated LDPE sheets inoculated with the combination fungi (Fig. H-M) exhibited a rough and wrinkled texture, along with extensive surface corrosion and erosion holes, as well as visible cracks, debris, and granular material formation, some of these features almost penetrated the LDPE film. In comparison to other treatments, the edges of the T-LDPE sheets degraded by group H-2 combined strains displayed distinct traces of fungal erosion, forming deep pits (Fig. J-K), while the T-LDPE sheets degraded by combined fungi in group H-3 showed large depressions (Fig. L-M). These observations suggest that these fungi possess the ability to colonize LDPE sheets and degrade LDPE materials, resulting in extensive damage to the LDPE surface as they grow on the surface of LDPE film and utilize it as a nutrient source for their growth. FTIR analysis of the degraded LDPE film surface The changes in the surface structural functional groups of T-LDPE after inoculation with different fungi were analyzed using FTIR spectroscopy within the frequency range of 400 to 4000 cm − 1 . For the fungus-inoculated T-LDPE films, as depicted in Fig. , the peaks at 2914 cm − 1 , 2847 cm − 1 , 1462 cm − 1 , 1375 cm − 1 , and 719 cm − 1 in the spectra showed significant alterations, which represented significant changes in the stretching vibration of the -CH functional group on the surface of the T-LDPE films after biodegradation. In particular, after 30 days of CPEF-6 or PE2F-4 culture, a new peak at 2395 to 2281 cm − 1 (corresponding to C ≡ C stretching and O = C = O stretching) appeared on the surface of the T-LDPE film, and unlike other spectral data, CPEF-6 treatment resulted in more pronounced changes in the C ≡ C and O = C = O functional groups. Only strain PE2F-4 treatment caused a noticeable change in the T-LDPE films, with a new peak between 1301 and 1215 cm − 1 (corresponding to -O-H stretching). The T-LDPE films degraded by combined fungal groups H-1 and H-2 also showed a new peak at 2395 to 2281 cm − 1 , among which the peak attributed to the H-1 group was the most pronounced. In addition, compared with the control, peak changes were observed in all fungus-treated T-LDPE films at 1140 to 994 cm − 1 (corresponding to C-O stretching), with more pronounced fluctuations in H-1 treated samples. The FTIR data revealed a more diverse variation in the chemical structure on the surface of LDPE films treated with PE2F-4 single or H-1 group, as opposed to the weight loss result (Fig. ). Determination and comparison of laccase activity Microorganisms can release a large number of extracellular enzymes into the environment for various complex metabolic activities , among which laccase is one of the principal fungal extracellular enzymes implicated in the biodegradation of polyethylene . The activity of laccase in the MSM-LDPE culture supernatant was determined using the ABTS method during the degradation of T-LDPE films by fungi. Co-culturing CPEF-1, CPEF-6, and PE2F-4 with T-LDPE films, respectively, resulted in detectable laccase production, and laccase activity gradually increased over time. By the fourth week of culture, PE2F-4 exhibited the highest laccase production, and its laccase activity reached 11,400 ± 2862 U/L, followed by CPEF-6 at 2700 ± 520 U/L, and CPEF-1 at 2400 ± 300 U/L (Fig. ). 

Following the secondary enrichment of MSM-LDPE medium, a number of fungi capable of degrading LDPE were isolated. The biodegradation process of polyethylene begins with microbial adhesion to the surface of the substrate material. Therefore, these isolates were cultured in MSM-LDPE media for 30 days, after which mycelial adherence to LDPE sheets and fungal growth were observed (Table ) for further screening of fungi with high LDPE biodegradability. The results indicated that strains CPEF-6, CPEF-7, and PE2F-4 had the most robust growth status, followed by CPEF-1, PE1F-13, and PE2F-7. On LDPE sheets, CPEF-6 and PE2F-4 exhibited the greatest mycelial adhesion, followed by CPEF-1 and PE2F-7. Based on these findings, three fungi—CPEF-1, CPEF-6, and PE2F-4—were chosen as highly efficient LDPE-degrading fungi for further investigation. 

Cultured on PDA plates at 30 °C for 7 days, the colony diameter of strain CPEF-1 reached 57–62 mm. On PDA plates, the mycelia of CPEF-1 grew on the surface and were immersed, and the colonies were flocculent or villous, with abundant aerial hyphae, hairy yellow edges, gray-brown surface, black to grayish-brown reverse side, flat and loosely flocculent center, and ovate or oval spores (Fig. ). According to the description of colony and spore morphology, this fungus is similar to Alternaria sp. Similarly, the colonies and microscopic morphology of strains CPEF-6 and PE2F-4 were observed. In a previous study, the morphology of CPEF-6 was described; for example, CPEF-6 had a diameter of 25–27 mm after 7 days of cultivation at 30℃ on PDA, colonies were greenish olivaceous to grey-olivaceous, and spores were abundant  (Fig. ). PE2F-4 exhibited a single colony diameter of 65–69 mm after being cultured on PDA plates at 30 °C for 5 days. The colonies appeared white and uniform with dense mycelia, a flat felt-like or cotton-like surface, rounded edges and abundant aerial hyphae (Fig. ). The ITS regions of strains CPEF-1, CPEF-6, and PE2F-4 amplified by PCR were sequenced and uploaded to NCBI with accession numbers OQ921460, OQ651281 and OQ921462, respectively, and their phylogenetic trees were constructed using the neighbor-joining method. Based on morphological characteristics as well as phylogenetic tree analysis, CPEF-1, CPEF-6, and PE2F-4 were identified as Alternaria angustiovoidea , Cladosporium basi-inflatum , and Trametes hirsuta , respectively (Table - ; Fig. - ). 

The three LDPE-degrading fungi CPEF-1, CPEF-6 and PE2F-4 described above were employed for LDPE degradation studies either alone or in pairs. Table illustrates the combination approach, where three groups of combined microorganisms, H-1, H-2 and H-3, were acquired (Table ). To verify the difference in the degradation ability of LDPE films between individual and combined fungi, we measured the dry weight of residual LDPE consumables to calculate the weight loss of the films (Fig. ). The results showed that after 30 days of treatment, fungi CPEF-1, CPEF-6, and PE2F-4 reduced the weight of the U-LDPE films by 0.36 ± 0.15%, 0.30 ± 0.06%, and 0.37 ± 0.11%, respectively. The combined strains H-1, H-2, and H-3 reduced the weight of the U-LDPE films by 0.37 ± 0.05%, 0.31 ± 0.01%, and 0.26 ± 0.11%, respectively, with no significant difference compared to the single-strain treatment (Fig. ). To change the structure of LDPE and make it easier to degrade, the LDPE sheets were subjected to heat treatment (T-LDPE). After 30 days of biodegradation, the weights of the T-LDPE films co-cultured with CPEF-1, CPEF-6, and PE2F-4 decreased by 0.28 ± 0.13%, 0.43 ± 0.01%, and 0.38 ± 0.06%, respectively. In addition, compared with U-LDPE, in the combined microbial treatment, only group H-1 had a reduced degradability in the T-LDPE film, while the weight loss rates of the T-LDPE films treated with groups H-2 and H-3 increased by approximately 58% and 154%, respectively (Fig. ). The weight loss rate of the T-LDPE films treated in the H-3 group reached 0.66 ± 0.06%, which was significantly higher than that of the H-1 group, showing no increase in weight loss rate, and the H-2 group, with a weight loss rate of 0.49 ± 0.06%. Interestingly, group H-3 did not perform well in degrading U-LDPE films, but showed the highest capacity in degrading T-LDPE films. The findings indicated that most of the combined fungal treatments could more effectively degrade heat-treated LDPE films. 

The morphological changes on the surface of LDPE films after 30 days of incubation with fungi were observed using ESEM. The surface of the U-LDPE film uninoculated with fungi remained smooth and intact without any changes (Fig. A). In contrast, the surfaces of the U-LDPE films treated by fungal inoculation became rough and uneven, showing obvious corrosion and erosion holes on the surface, clear cracks and pits, as well as the formation of granular materials (Fig. B-G). Among them, in the CPEF-6 inoculated samples, mycelia were clearly attached to the surface of the U-LDPE film (Fig. D-E). Notably, the PE2F-4 strain caused extensive morphological changes on the sheet surface (Fig. B-C). These findings suggest that these fungi can degrade U-LDPE, cause certain damage to its surface, and grow on the surface of LDPE sheets. Furthermore, ESEM was used to observe the morphological changes of the T-LDPE surface after 30 days of fungal culture. The surface of the T-LDPE films without fungal inoculation had slight streaks, but remained smooth and intact overall (Fig. A). In contrast, the surface of the fungus-inoculated T-LDPE sheets became rougher and more uneven, with extensive surface corrosion and numerous erosion holes, visible cracks, and deep pits (Fig. B-M). Particularly, in the samples inoculated with CPEF- 6 or PE2F-4 alone, the surfaces of the T-LDPE sheets exhibited irregular folds and obvious delamination (Fig. D-M). Interestingly, the surface of the heat-treated LDPE sheets inoculated with the combination fungi (Fig. H-M) exhibited a rough and wrinkled texture, along with extensive surface corrosion and erosion holes, as well as visible cracks, debris, and granular material formation, some of these features almost penetrated the LDPE film. In comparison to other treatments, the edges of the T-LDPE sheets degraded by group H-2 combined strains displayed distinct traces of fungal erosion, forming deep pits (Fig. J-K), while the T-LDPE sheets degraded by combined fungi in group H-3 showed large depressions (Fig. L-M). These observations suggest that these fungi possess the ability to colonize LDPE sheets and degrade LDPE materials, resulting in extensive damage to the LDPE surface as they grow on the surface of LDPE film and utilize it as a nutrient source for their growth. 

The changes in the surface structural functional groups of T-LDPE after inoculation with different fungi were analyzed using FTIR spectroscopy within the frequency range of 400 to 4000 cm − 1 . For the fungus-inoculated T-LDPE films, as depicted in Fig. , the peaks at 2914 cm − 1 , 2847 cm − 1 , 1462 cm − 1 , 1375 cm − 1 , and 719 cm − 1 in the spectra showed significant alterations, which represented significant changes in the stretching vibration of the -CH functional group on the surface of the T-LDPE films after biodegradation. In particular, after 30 days of CPEF-6 or PE2F-4 culture, a new peak at 2395 to 2281 cm − 1 (corresponding to C ≡ C stretching and O = C = O stretching) appeared on the surface of the T-LDPE film, and unlike other spectral data, CPEF-6 treatment resulted in more pronounced changes in the C ≡ C and O = C = O functional groups. Only strain PE2F-4 treatment caused a noticeable change in the T-LDPE films, with a new peak between 1301 and 1215 cm − 1 (corresponding to -O-H stretching). The T-LDPE films degraded by combined fungal groups H-1 and H-2 also showed a new peak at 2395 to 2281 cm − 1 , among which the peak attributed to the H-1 group was the most pronounced. In addition, compared with the control, peak changes were observed in all fungus-treated T-LDPE films at 1140 to 994 cm − 1 (corresponding to C-O stretching), with more pronounced fluctuations in H-1 treated samples. The FTIR data revealed a more diverse variation in the chemical structure on the surface of LDPE films treated with PE2F-4 single or H-1 group, as opposed to the weight loss result (Fig. ). 

Microorganisms can release a large number of extracellular enzymes into the environment for various complex metabolic activities , among which laccase is one of the principal fungal extracellular enzymes implicated in the biodegradation of polyethylene . The activity of laccase in the MSM-LDPE culture supernatant was determined using the ABTS method during the degradation of T-LDPE films by fungi. Co-culturing CPEF-1, CPEF-6, and PE2F-4 with T-LDPE films, respectively, resulted in detectable laccase production, and laccase activity gradually increased over time. By the fourth week of culture, PE2F-4 exhibited the highest laccase production, and its laccase activity reached 11,400 ± 2862 U/L, followed by CPEF-6 at 2700 ± 520 U/L, and CPEF-1 at 2400 ± 300 U/L (Fig. ). 

Plastic pollution has become one of the major environmental issues of global concern , and as a result, several countries and regions have implemented policies to change this critical pollution phenomenon . The primary strategies for addressing this problem involve reducing the generation of plastic-based pollutants at the source  and managing existing plastic pollutants, with biodegradation being an undeniably safe and effective approach . The mechanisms by which fungi degrade plastic have been extensively studied, as they can attach to and colonize plastic surfaces , and secrete various powerful extracellular enzymes capable of degrading plastics . Here, we preliminarily verified the biodegradation advantage of mixed cultures in addressing plastic pollution based on the fact that treatment with combined flora H-3 resulted in a greater weight loss rate of T-LDPE sheets (Fig. ). Based on morphological observations and phylogenetic analyses (Figs. , ), two mixed fungi in group H-3, CPEF-1 and PE2F-4, were identified as members of Alternaria sp. and Trametes sp., respectively. Fungi of the genus Alternaria have been found to be isolated from plastic pollutants on land and in the ocean, with the capability to degrade polyethylene . Additionally, they have been reported to possess the ability to degrade ultra-heavy crudes  and penetrate tuffs . In addition to these, Alternaria sp. are known as common plant pathogens . In our study, it was found that Alternaria sp. CPEF-1 had relatively strong biodegradability on U-LDPE, but showed minimal degradation effect on heat-treated T-LDPE films (Fig. ). The genus Alternaria is commonly reported as a lignin-degrading fungus. Lignin is a complex aromatic heteropolymer that is challenging to utilize  but has great potential for producing commercially valuable chemicals . On the other hand, T. hirsuta has been shown to produce laccases  involved in polyethylene degradation, which aligns with our finding that PE2F-4 possesses a strong laccase-secreting capacity and that its laccase activity is much higher than that of CPEF-1 and CPEF-6 (Fig. ). In contrast, however, PE2F-4 did not show a significantly greater degradation potential for either LDPE film (U-LDPE or T-LDPE) (Fig. ). Although all three strains exhibited excellent LDPE degradation ability, the impact of a single strain was ultimately limited, and did not meet our expectations for plastic degradation. Therefore, we constructed three groups of combined fungi H-1 ∼ 3, each consisting of two fungal species. Interestingly, although neither CPEF-1 nor PE2F-4 showed greater degradation capacity individually and the H-3 group composed of them also demonstrated the lowest degradation effect on U-LDPE, the weight loss rate of the T-LDPE films treated with the H-3 group was far greater than that of the other monoculture or combined fungal treatments (Fig. ). Moreover, under treatment with combined strains in the H-3 group, the weight loss rate of T-LDPE was 154% higher than that of the U-LDPE film, which may be attributed to heat treatment altering the properties of the film at a molecular chain level . This makes it easier for H-3 colonies to adhere to the LDPE film and for their spores to colonize the membrane, thus allowing the strains to better utilize and degrade the T-LDPE film as they grow Studies have indicated that several natures such as molecular weight, crystallization rate, melt temperature and additives added to the polymer, may also influence the degradation efficiency of LDPE films . The degradation of LDPE was further confirmed through ESEM and FTIR analysis. SEM can be utilized to observe traces of fungal mycelium adhesion on the LDPE film surface and its subsequent biodegradation. Studies have shown that changes in plastic surface morphology, such as the formation of pores, cracks, and holes, can be used to evaluate degradation using SEM imaging before and after treatment . For instance, following a 90-day culture of Penicillium citrinum , SEM revealed the presence of holes and cracks on the surface of LDPE ; likewise, similar changes were observed on the LDPE surface after 90 days of Pseudomonas sp. culture . In this study, by observing the surface alterations of T-LDPE films with ESEM, we initially verified the degradation of LDPE films caused by single and combined fungi treatments. As shown in Fig. , significant alterations were observed on the surfaces of all T-LDPE films after inoculation. Notably, while the film surface treated by group H-3 did not exhibit large erosion holes like that treated by PE2F-4, the overall surface of the T-LDPE film became rough and wrinkled, with a large area of cracks and deep pits, and a large number of granular protrusions formed. These findings suggest that the combined fungal group H-3 may cause more extensive damage to the surface of T-LDPE by using LDPE as a carbon source for degradation and utilization. Fourier transform infrared spectroscopy is typically used to measure changes in functional groups on plastic surfaces after biodegradation . The chemical modifications detected by FTIR indicated that the characteristic absorbances of the T-LDPE films at 2914, 2847, 1462, and 719 cm − 1 correspond to the CH2 bending and stretching vibrations of LDPE . After 30 days of biodegradation, the FTIR spectrum of T-LDPE showed a decrease in all four peaks compared to those of the control, indicating significant changes in the surface area of the T-LDPE films caused by all fungi. These peaks represent fluctuations associated with the -CH functional group. The peak at 1301 to 1215 cm − 1 represents the formation of hydroxyl groups , and the presence of acidic O-H functional group supports the depolymerization process of LDPE under the action of microorganisms, suggesting that the fungi produced acidic substances during the degradation of T-LDPE . A distinct peak appeared between 2395 and 2281 cm − 1 , indicating an increase in the weak stretching of C ≡ C bonds and strong stretching of O = C = O bonds. Some studies have shown that the apparent peak here represents fatty acid production. Overall, the FTIR data revealed the different strategies employed by various fungi during the degradation of T-LDPE, as evidenced by differences in the wave numbers and intensities of the absorption peaks. Notably, the best-performing H-3 group did not show a clear dominant feature in the FTIR spectrum. Fungi can secrete a series of non-specific extracellular enzymes to catalyze the degradation of various pollutants , among which laccase is a polyphenol oxidase belonging to the family of blue multicopper oxidases . Research has shown that laccase can oxidize the hydrocarbon backbones of LDPE and reduce its weight and average molecular weight . Other studies have reported that Trichoderma harzianum can produce both laccase and peroxidase when participating in PE biodegradation , while Aspergillus flavus can depolymerize the long chains of HDPE by generating laccase and laccase-like multicopper oxidase . Therefore, our results, that CPEF-1, CPEF-6 and PE2F-4 produce laccase during the degradation of T-LDPE, further demonstrate that most fungi degrade polyethylene through the secretion of laccase (Fig. ). In the process of polymer biodegradation, complex polymers are initially decomposed into shorter chains or monomers by enzymes before being gradually utilized by microorganisms. Based on these findings, more complex culture strategies involving combined strains will be explored to enhance the degradation efficiency of plastic materials. 

In this study, three strains of fungi CPEF-1, CPEF-6 and PE2F-4 with strong LDPE degradation ability were isolated through enrichment experiments, and the LDPE degradation effects of single and combined strains were evaluated. The weight loss of the U-LDPE and T-LDPE films after fungal treatment revealed that the combined fungal H-3 had a significantly greater degradation effect compared to other single or combined fungal treatments. The weight loss rate of the T-LDPE film increased by 154% compared with that of the U-LDPE film. ESEM revealed morphological changes on the surface of all T-LDPE films treated with fungi, among which the overall surface of the T-LDPE films in the H-3 group became rough, wrinkled, and uneven, as did the cracks, deep pits, and surface damage, and a large number of granular processes formed. FTIR analysis showed that T-LDPE films treated with PE2F-4 formed hydroxy-OH and acidic substances. Biodegradation of T-LDPE films by the fungi CPEF-6 and PE2F-4 resulted in changes in the stretching of C ≡ C and O = C = O bonds, indicating the production of fatty acids. Overall, heat-treated LDPE films combined with different microbial cultures can result in the formation of alcohols, ethers, acids, and esters during the degradation process. The FTIR data revealed different strategies adopted by different microbial compositions for degrading T-LDPE films. In addition, PE2F-4 produced high levels of laccase (11400 ± 2862 U/L) during LDPE degradation, whereas CPEF-1 and CPEF-6 displayed relatively low laccase activity. These results indicate that the degradation of LDPE is a complex process, and further investigation will be conducted to elucidate the molecular mechanism through which combined fungal H-3 group enhances the degradation efficiency of T-LDPE. 

Below is the link to the electronic supplementary material. Supplementary Material 1 

