Mechanistic Insights into UV/H₂O₂-Aged Polystyrene Enable an Optimized Protocol for Fungal Biodegradation

Mechanistic Insights into UV/H₂O₂-Aged Polystyrene Enable an Optimized Protocol for Fungal Biodegradation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mechanistic Insights into UV/H₂O₂-Aged Polystyrene Enable an Optimized Protocol for Fungal Biodegradation Zhi Guo, Yuanyuan Zha, Xingpan Guo, Xinlei Ling, Lin Yao, Lishou Han, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8364324/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Plastic pollution is a global environmental challenge, yet plastic biodegradation remains inefficient and poorly understood. In this study, a degradation strategy for polystyrene (PS) films was developed by combining ultraviolet (UV) irradiation and hydrogen peroxide (H₂O₂) pretreatment with subsequent biodegradation by Phanerochaete chrysosporium . UV/H₂O₂ pretreatment proved optimal, resulting in a mass loss of up to 25.75% and inducing the formation of oxygen-containing functional groups, including carbonyl, hydroxyl, and carboxyl groups. These groups act as electron donors, facilitating extracellular enzymatic chain-cleavage reactions, while also promoting fungal colonization and enhancing the activities of manganese peroxidase, lignin peroxidase, and laccase. The pretreated PS was degraded through three main pathways prior to entering the tricarboxylic acid cycle: direct assimilation of carboxylated compounds, enzymatic aromatic ring cleavage, and cytochrome P450-mediated oxidation. Overall, UV/H₂O₂ pretreatment significantly improves surface oxidation, microbial activity, and enzymatic reactivity, offering an effective strategy to accelerate plastic biodegradation. Earth and environmental sciences/Environmental sciences Biological sciences/Microbiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Plastic is a man-made long-chain and/or aromatic polymer material. Owing to its excellent properties, such as light weight, high plasticity and flexibility, corrosion resistance, and low cost, it is widely used in numerous applications, including construction, electronics industry, and packaging 1 .The annual output of plastics soared to over 400 million metric tons in 2023 2 . As an agricultural application, mulch films can increase soil temperature, retain moisture, control weeds, and reduce crop diseases, thereby significantly improving the crop growth surroundings and agricultural cultivation patterns 3 . Therefore, the use of mulch films and facility-based farming has become increasingly widespread with the rapid development of agriculture 4 . However, a large amount of discarded film is not properly treated in time resulting in widespread plastic residues in the soil environment, due to the lack of a well-established recycling system, effective scheduling, and scientific management guidance 5 . These residues gradually fragment into smaller particles (< 5 mm), known as microplastics and even nanoplastics, collectively referred to as micro/nanoplastics (MNPs), under prolonged exposure to sunlight and mechanical forces 6 , 7 . MNPs can persist in the environment for hundreds or even thousands of years, leading to their gradual accumulation and widespread presence in the atmosphere, water bodies and terrestrial biosphere, and poses a serious threat to ecosystems 8 . To date, there has no optimal resolving methods for environmental residual mulch film. Conventional plastic waste treatments, including landfill and incineration, are limited by low efficiency and secondary pollution 9 . Therefore, it is imperative to develop efficient and environmentally friendly methods to mitigate both the direct and indirect impacts of plastic pollution 10 . Recent studies have identified four primary pathways for plastic degradation: biodegradation, photodegradation, chemical degradation, and thermal degradation 11 . Among them, biodegradation typically proceeds slowly and exhibits limited efficiency. In cultivated soils rich in fungi, microorganisms, and invertebrates, the degradation rate of polystyrene (PS) remains below 1% after 90 days, with no significant increase observed thereafter 12 . Similarly, after an eight-week incubation, Cephalosporium species induced a weight loss of 2.17 ± 0.16% in PS, while Mucor species resulted in a 1.81 ± 0.13% reduction 13 .Numerous investigations have demonstrated that appropriate physical or chemical pretreatment can substantially enhance the microbial degradation of plastics 14 . Such pretreatment promotes surface oxidation, decrease hydrophobicity, introduce oxygen-containing functional groups, and facilitate biofilm formation, thereby improving microbial attachment and activity 15 . For instance, exposure of polyethylene (PE) to ultraviolet (UV) radiation and thermal treatment shortens polymer chains and generates hydroxyl, carboxyl, and carbonyl groups, which significantly accelerate its biodegradation by microorganisms 16 . Moreover, thermal pretreatment has proven particularly effective in promoting the enzymatic degradation of PS, whereas alkaline or acidic pretreatment generally enhances the biodegradability of bio-based plastic wastes 17 . Despite substantial progress, the fundamental mechanisms through which pretreatments accelerate biodegradation, particularly the pathways of bond cleavage and oxygen incorporation at the polymer surface, remain largely unresolved and warrant further investigation. Currently, exclusive reliance on biological degradation faces significant challenges 18 . The strong hydrophobicity and chemically stable structure of plastics hinder effective microbial colonization and biofilm formation on their surfaces, thereby limiting degradation efficiency 19 . White-rot fungi exhibit strong oxidative decomposition capabilities through the secretion of extracellular ligninolytic enzymes such as manganese peroxidase (MnP), lignin peroxidase (LiP), and laccase (Lac) 20 . These fungi can partially oxidize and depolymerize complex polymer structures owing to their non-specific oxidative systems, exhibiting considerable potential for plastic biodegradation 21 . However, their attachment to plastic surfaces, enzyme stability, and long-term catalytic activity are often insufficient, resulting in slow and incomplete degradation 22 .Recent studies suggest that coupling biological and photocatalytic processes for plastic degradation may effectively shorten degradation time and enhance efficiency, offering a promising strategy to overcome the limitations of purely biological methods 23 . This provides an important direction for the present study, which aims to explore how pretreatment strategies can enhance the subsequent biodegradation efficiency of white-rot fungi. To further elucidate the pretreatment mechanisms of microplastics and their influence on subsequent biodegradation, this study proposes a novel strategy integrating physicochemical pretreatment with microbial degradation. The approach involves synergistic aging of plastics through combined UV irradiation and hydrogen peroxide treatment, followed by further fragmentation and mineralization by the typical white-rot fungus Phanerochaete chrysosporium ( P. chrysposporium ). The study aims that: (1) demonstrates the essential prerequisite of surface oxidation with the formation of oxygen-containing functional groups in plastic degradation; (2) illustrates the significance of oxygenated groups in enhancing microbial colonization, enzyme-mediated chain scission, and serving as electron donors that facilitate extracellular enzymatic cleavage of polymer chains; (3) integrates multi-omics analyses to identify intracellular metabolic pathways involved in PS degradation. Results and discussion Visualization process of pretreated collaborative fungi degrading plastics This study used UV radiation and H₂O₂ as a synergistic pretreatment system to induce aging in pristine plastic films. Subsequently, the pretreated plastic films exhibit enhanced colonization by P. chrysosporium , especially in the UV/H₂O₂ group (Fig. 1 A and 1 B). Upon co-cultivation with P. chrysosporium , extensive filamentous networks and dense mycelial structures were formed on the pretreated film surfaces, which facilitated the physiological fragmentation of the films (Figure S2). The degree of cracking was more evident in the UV/H₂O₂-pretreated samples. By the end of the incubation period, the plastic films have disintegrated into irregular fragments (Figure S3). The colonization of biofilms on the PS film surface largely determines its potential biodegradability. Compared with pristine PS, aged PS films exhibit a higher tendency for biofilm formation 24 . As shown in Fig. 1 C, the attached biomass of P. chrysosporium noticeably increased on pretreated films, with the UV/H₂O₂-PS group showing the strongest colonization ability. During the biodegradation process, the fungal cells maintained high viability (Fig. 1 D), with activity levels of 86.3% (P-PS), 89.4% (H₂O₂-PS), 82.3% (UV-PS), and 70.4% (UV/H₂O₂-PS). Overall, UV/H₂O₂ pretreatment may effectively modify the surface characteristics of the plastics films, enhance biofilm formation, and consequently promote the biodegradation process 25 . Compared with the P-PS and pretreatment solely, the UV/H₂O₂-PS exhibits a significantly higher weight loss throughout the biodegradation process (Fig. 1 C and 1 D). The mass of UV/H₂O₂-PS gradually decreased over 35 days, reaching a maximum weight loss of 25.75 ± 5.08%, which was significantly higher than that of the P-PS (6.95 ± 1.89%), H₂O₂-PS (7.38 ± 1.31%), and UV-PS (7.89 ± 1.76%) groups. These findings clearly demonstrate that UV/H₂O₂ pretreatment substantially enhanced the biodegradability of PS films, making them more susceptible to colonization and degradation by P. chrysosporium . The pronounced improvement in degradation efficiency is likely attributable to surface physicochemical modifications induced by UV/H₂O₂ pretreatment. Previous report has suggested that physical pretreatments, such as UV irradiation, ozonation, and plasma exposure, can disrupt the macromolecular aggregation of polymer chains, thereby increasing their accessibility to enzymatic and microbial attack 26 . In particular, UV/O₃ weathering has been reported to trigger physicochemical transformations in PS nanoparticles, including the generation of oxygen-containing functional groups and enhanced surface hydrophilicity 27 . It is therefore plausible that the combined UV/H₂O₂ pretreatment induced similar modifications on the PS surface, facilitating microbial attachment and subsequent biodegradation. Collectively, these results suggest that such oxidative pretreatment may serve as a critical prerequisite for achieving efficient biological degradation of conventional plastics 28 . P. chrysosporium deeply oxidation of PS films Surface morphology characterization was performed to evaluate the effects of pretreatment on film aging, P. chrysosporium colonization, and polymer degradation 29 . The pristine PS films and those films treated with UV or H₂O₂ alone exhibit relatively smooth surface textures without noticeable structural alterations (Fig. 2 A). In contrast, the UV/H₂O₂-PS film displays distinct cracks and pits, indicating pronounced surface erosion and oxidation. These phenomena may be attributed to the high UV sensitivity of the benzene rings in PS molecules, which enables rapid photooxidation within a short time 30 . Meanwhile, the conjugated structures formed during aging further facilitate progressive degradation of the polymer backbone 31 . Once microcracks appear on the PS surface, H₂O₂ solution can infiltrate these sites, at which, reactive oxygen species (ROS) and carbon-centered radicals are generated under UV irradiation and in the presence of oxygen. These radicals sequentially cleave the PS molecular chains, accelerating surface oxidation and eventually resulting in the formation of pits. Biodegradation experiments further confirm strong colonization of P. chrysosporium on the pretreated PS surfaces. SEM images reveal only minor wrinkles on the pristine PS films, whereas the pretreated films exhibit a roughened surface texture with visible fungal hyphae, indicating successful colonization. Notably, the UV/H₂O₂-PS show more pronounced groove-like wrinkles, larger pores, and spores embedded within the fractured layers (Fig. 2 A). Consistent with earlier findings, aged PS films exhibited a stronger tendency for biofilm formation compared with pristine PS films 32 , which is likely attributed to increased surface roughness that facilitates microbial adhesion. In general, microorganisms adhere more easily to rough microplastic surfaces, thereby promoting subsequent biofilm formation and growth 33 . These morphological and colonization results illustrate that UV/H₂O₂ synergistic aging markedly alters the surface properties of PS, eroding its originally surface structure and creating new sites for contaminant adsorption and radical attack, which in turn facilitates fungal attachment and biodegradation. To exploit the deeply molecular mechanism of PS film degradation by P. chrysosporium , the hydrophilicity was employed. Increased hydrophilicity leads to a larger contact area and more adsorption sites, thereby enhancing electrostatic interactions with the PS film 34 . The hydrophilicity of plastic surfaces influences the adhesion of biofilms, with lower surface hydrophobicity being more conducive to microbial colonization 35 . As shown in Fig. 2 B, during the pretreatment stage, all applied pretreatment methods reduced the water contact angle of the plastic films. The contact angle of untreated P-PS was 91.91 ± 0.49°, which decreased to 88.66 ± 2.23° after H₂O₂ pretreatment, 88.14 ± 0.67° after UV pretreatment, and 85.33 ± 1.28° after UV/H₂O₂ co-pretreatment. This reduction may be attributed to the introduction of hydrophilic functional groups on the PS surface during pretreatment, thereby lowering its surface hydrophobicity 36 . Generally, evolution of polar carbonyl groups on MPs’ main chain leads to increase of surface polarity and may increase the accessibility of water on the material surface 37 . In the biodegradation stage, as more clearly observed in Fig. 2 A, the contact angles of all plastic films decreased markedly. After biodegradation, the contact angle of P-PS decreased to 78.66 ± 1.71°, H₂O₂-PS to 79.81 ± 0.81°, UV-PS to 75.51 ± 0.04°, while the UV/H₂O₂-PS showed the most pronounced reduction, reaching 66.11 ± 0.88°. Comparative data revealed that the contact angle of P-PS decreased by an average of 13.25° after direct biodegradation, whereas that of the UV/H₂O₂-PS followed by biodegradation decreased by an average of 25.8°. When P. chrysosporium colonizes the plastic surface, its mycelia can penetrate the surface and spread into the matrix to absorb nutrients, enabling firm attachment to the plastic 38 . Hydrophobins, which are surfactant proteins produced by filamentous fungi, function as structural components involved in fungal growth and their interactions with the environment 39 . Acting as biosurfactants, fungal hydrophobins self-assemble into amphipathic protein membranes 40 . These amphiphilic proteins involved in the formation of aerial structures such as spores or fruiting bodies. They form chemically robust layers which can only be dissolved in strong acids. These layers adhere to different surfaces, changing their wettability, and allow the binding of other proteins 41 . These enhance the hydrophilicity of the plastic surface and promote the adhesion of the fungus to the polymer surface. Therefore, the colonization of P. chrysosporium on plastic surfaces can reduce hydrophobicity and further promote microbial attachment 42 . ATR-FTIR spectra indicating significant changes in the surface functional groups of the PS plastic films before/after pretreatment and biodegradation, are well explained contact angle changes. As shown in Fig. 2 C, changes in absorption peaks were observed at 3025 cm⁻¹ and 2922 cm⁻¹ in the PS films before and after pretreatment, attributed to C-H stretching vibrations of the aromatic rings. A carbonyl (C = O) absorption peak was observed at 1602 cm⁻¹. The appearance of the carbonyl peak suggests the introduction of polar groups into the PS backbone, leading to a transition of the plastic from hydrophobic to hydrophilic. This change facilitates the attachment of P. chrysosporium to the PS film. The increase in oxygen-containing functional groups may result from the cleavage of C-H bonds under ultraviolet light, followed by their reaction with oxygen to form peroxyl radicals (C-O•). These peroxyl radicals can abstract hydrogen atoms from the surrounding environment, forming hydroperoxide (COOH), which further decompose into other products such as carbonyl (C = O) 43 . Additionally, in the spectral region around 1100 cm⁻¹, corresponding to the stretching vibrations of oxygen-containing functional groups such as alcohols and ethers, changes in the carbon-oxygen (C-O) bond are observed. After co-cultivation with P. chrysosporium , the C = O and the C-O absorption peak in the infrared spectra of the plastic films became more pronounced, indicating the effective usage of microorganisms to disintegrate pretreatment PS films. Pretreatment induces surface oxidation of plastics, generating oxygen-containing functional groups such as carbonyl, carboxyl, and hydroxyl. Microbial metabolism further reinforces this modification, enhancing surface polarity and hydrophilicity, destabilizing polymer bonds, and accelerating degradation. Based on these sense, oxygen-containing functional groups play a critical role in the transition of plastics from physicochemical aging to biological degradation 44 . These results are also demonstrated by our previous research for P. chrysosporium attacking O-containing groups, which cause plastic crack 45 . Number average molecular weight (Mn, g/mol) and weight average molecular weight (Mw, g/mol) decrease of a polymer are important indicator of polymer degradation 46 . Changes in the average molecular weight of PS plastic films from different pretreatment groups and biodegradation groups are shown in Fig. 2 D and Fig. 2 E. It can be seen that the number average molecular weight of the P-PS sample is 161655 and the weight average molecular weight is 300642. After various pretreatments, both Mn and Mw decreased. The number average molecular weight of the directly biodegraded sample is 149757 and the weight average molecular weight is 297810, which were reduced by 7.36% and 0.94%, respectively, compared with the P-PS. In contrast, the number average molecular weight of UV/H 2 O 2 -PS after biodegradation is 77242, and the weight average molecular weight is 238639, which decreased by 52.22% and 20.62%, respectively, compared with the P-PS. This suggests the occurrence of endo-type depolymerization or random internal scission of chains in polymer macromolecules, which generated oligomeric products and mid-chain polymers 47 . Notably, the Mn and Mw of the biologically degraded samples are slightly higher than those of the pretreated samples. This can be ascribed to that P. chrysosporium preferentially consume shorter polymer chains, resulting in a relative enrichment of longer chains or arise from oxidative reactions occurring during biodegradation 48 , 49 . Since the relative atomic mass of oxygen (16.00) is higher than that of carbon (12.01), the introduction of oxygen-containing functional groups would simultaneously lead to a relative increase in the average molecular weight of the polymer 50 . Physiological status and O-containing groups triggered enzymatic degradation reaction . Extracellular activities of oxidative enzymes MnP, LiP, and Lac in P. chrysosporium exhibit significant variation across different pretreatment groups (Fig. 3 A-C). The degradation system of white-rot fungi relies on the synergistic action of ligninolytic enzymes (LMEs), organic acids, mediators, and auxiliary enzymes, characterized by a non-specific oxidation mechanism driven by free radical generation 51 . Oxygen-containing functional groups (such as C = O、C-O) induce electronic density redistribution along the polymer chain through inductive and resonance effects, weakening the stability of adjacent C-C and C-H bonds 52 . These “activated”chemical bonds are more susceptible to attack by oxidative intermediates (such as free radicals or peroxides) generated through enzyme catalysis, making them preferential sites for the cleavage of the polymer backbone 53 . Under the synergistic effect of ultraviolet radiation and hydrogen peroxide, PS forms multiple oxygen-containing functional groups, such as carbonyl and hydroxyl groups, which provide important chemical foundations for subsequent biodegradation. To further investigate the degradation mechanism of PS with extracellular enzymes secreted by P. chrysosporium , we selected the product 4,5-Dimethoxy-2-hydroxyacetophenone as a substrate for molecular docking simulations. The simulation results show that these oxygen-containing functional groups can form stable hydrogen bonds with the enzyme active sites, and the minimum hydrogen bond energies with MnP, LiP, and Lac are − 5.9, -5.0, and − 5.6, respectively (Fig. 3 D-E). These results suggest that the enzymes can be able to bind these substrates, thereby carrying out subsequent degradation. Through the synergistic action of multiple enzymes, the introduction of oxygen functional groups and the enzyme-catalyzed oxidative process form a positive feedback loop, substantially promoting the cleavage of the polymer backbone and resulting of biodegradation (Fig. 3 G) 54 . In the MnP system, the catalytic cycle is initiated by the reaction of H₂O₂ or organic peroxides with the heme center, first forming an Fe-peroxide complex 55 . This intermediate undergoes O-O bond cleavage, transferring two electrons to heme and releasing one water molecule to form Compound I, which is a Fe 4+ -oxo-porphyrin-radical complex with a highly oxidized state 56 . Compound I possesses strong oxidizing power and can abstract one electron from an electron donor, thereby being reduced to compound II (Fe 4+ -oxo-porphyrin complex). This compound is subsequently further reduced back to Fe³⁺, completing one catalytic cycle. During this process, Mn²⁺ is oxidized to Mn³⁺ and chelated with bidentate organic acids (oxalate, acetate) to form Mn³⁺-ligand complexes 57 . This complex acts as a diffusible redox mediator capable of migrating away from the enzyme molecule. It selectively oxidizes oxygen-containing functional groups or benzene ring side chains in localized regions on polystyrene (PS) surfaces via single-electron oxidation, abstracting hydrogen atoms to generate benzyl or phenoxy radicals. The generated radicals undergo resonance migration and rearrangement reactions, inducing C-C or C-H bond cleavage to form oxidation products such as carbonyl and carboxyl groups, thereby promoting polymer chain scission and degradation 58 . The presence of oxygen-containing functional groups enhances the interaction between the Mn³⁺-ligand complex and the polymer surface, improving electron transfer efficiency and further accelerating the oxidative cracking process. Similarly, the LiP catalyzed cycle also initiates with the oxidation of Fe³⁺ by H₂O₂, yielding compound I (Fe 4+ -oxo-porphyrin complex), an intermediate possessing an exceptionally high oxidation potential 59 . Compound I completes the cycle by sequentially abstracting two electrons from an aromatic substrate, forming compound II (Fe 4+ -oxo-porphyrin complex), and subsequently being reduced back to native Fe³⁺. LiP directly extracts electrons from the aromatic ring of PS to generate a radical cation. Subsequently, the radical migrates via resonance between the benzene ring and side chain, triggering C-C bond cleavage to form oligomeric products or oxidized fragments 60 . Oxygen-containing functional groups lower the substrate's ionization potential, facilitating electron extraction and enhancing LiP' s oxidative efficiency. In the Lac system, electron transfer is initiated at the T₁ copper site. The copper centers of Lac oxidize substrates and transfer the acquired electrons to molecular oxygen 61 . Electrons from oxidized substrates first transfer to this site before sequentially moving to the trinuclear copper center (T₂/T₃) 62 An oxygen molecule binds to the T₃ copper cluster plane, accepting electrons from four substrates and being reduced to two water molecules. Phenoxy or benzylic radicals generated during catalysis can diffuse from the PS surface, initiating a series of non-enzymatic radical chain reactions. The introduction of oxygen-containing functional groups significantly reduces the oxidation potential of PS, promoting electron transfer from substrates to the copper centers of Lac 63 . This accelerates radical generation and diffusion, thereby enhancing oxidative cleavage efficiency 64 . Transcriptome-level cellular degradation mechanisms and metabolic model construction This study conducted GC-MS analysis on PS subjected to UV/H₂O₂ pretreatment followed by biodegradation to characterize metabolites formed during both the physicochemical pretreatment and microbial degradation processes (Tables S1 and S2). Based on the intermediates identified by GC-MS and the transcriptomic analysis of P. chrysosporium , the proposed PS biodegradation pathway and mechanism are illustrated in Fig. 4 A. The PS backbone consists of a benzene ring and a carbon chain, with the benzene ring functioning as the primary chromophore. Upon UV irradiation, the benzene ring absorbs energy and undergoes electronic excitation, thereby decreasing the stability of the para-position C-H bond 65 . Under the synergistic action of UV and H₂O₂, hydroxyl radicals (•OH) produced from H₂O₂ photolysis further attack these activated sites, inducing hydroxylation on the aromatic ring or its para-substituent 66 . Subsequent dehydrogenation reactions may generate oxidized functional groups such as C = O 67 . As oxidation advances, the PS chains undergo consecutive photo-oxidation of side chains and backbone cleavage, producing highly reactive alkoxy and epoxy radical that drive continuous chain-scission reactions 68 . These intermediate radicals subsequently react with oxygen to form peroxides, which further decompose under UV exposure, leading to the detachment of aromatic structures from the backbone and the release of multiple low-molecular-weight aromatic organic compounds. In addition, hydroxyl and carboxyl groups formed during side-chain oxidation may undergo further esterification or condensation reactions with adjacent oxygen-containing groups, yielding oxidation derivatives such as 4,5-Dimethoxy-2-hydroxyacetophenone 69 . During the biodegradation phase, genes encoding extracellular enzymes such as MnP, LiP, and Lac were significantly upregulated (Table S4). This transcriptional pattern is consistent with the characteristic reliance of P. chrysosporium on a non-specific extracellular peroxidase system during the early stages of degradation (Fig. 3 C-E). These peroxidases continuously act on the “activated” chain ends and partially oxidized backbone regions of polystyrene following photooxidation, inducing long-chain scission and generating various small-molecule oxidation products through radical-mediated reactions. GC-MS results further show that PS subjected to synergistic aging releases substantial amounts of aromatic oxides and aliphatic degradation products during fungal metabolism, providing abundant substrates for subsequent incorporation into intracellular metabolic pathways. Cytochrome P450 (CYP450) serves as a core catalytic enzyme for diverse oxidation reactions by forming Fe (IV)-O high-valent iron-oxygen intermediates to activate and selectively oxidize inert C-H bonds 70 . Multiple P450-encoding genes with conserved domains were are extensively upregulated in P. chrysosporium (Table S5), further supporting the central role of the P450 family in PS activation, biodegradation, and integration into downstream metabolic pathways. Notably, cytochrome P450 monooxygenases represent the most functionally versatile subgroup within the P450 family. With broad substrate adaptability, diverse reaction capabilities, and atypical kinetic properties, they are considered among the most important oxidases in biocatalysis 71 . The fold changes observed in the P-PS and UV/H₂O₂-PS treatment groups were 1.53 and 1.84, respectively (Fig. 4 D). Based on the characteristics of metabolic products and the expression profiles of key enzyme genes, the downstream metabolism of PS can be summarized into three major pathways: (1) Aromatic oxidation products. 4-Methoxystyrene is first oxidized to cis-styrene ethylene glycol, which is subsequently converted into 4-hydroxy-2-oxovalerate. Under the action of 4-hydroxy-2-oxoglutarate aldolase (HOA), it is transformed into pyruvate, which is then converted into acetyl-CoA and enters the tricarboxylic acid cycle (TCA cycle). KEGG pathway analysis revealed that the expression level of the HOA-encoding gene was notably higher in the treatment group compared to the control group (Fig. 4 C), further supporting the activation of this metabolic pathway. In another aromatic oxidation pathway, 2-methyl-2-phenyl-Oxirane is initially converted into Styrene oxide, which is then catalyzed by styrene oxide isomerase (SOI) to produce phenylacetaldehyde. Phenylacetaldehyde is subsequently oxidized to homogentisic acid, which undergoes aromatic ring cleavage via homogentisic acid 1,2-dioxygenase (HGADO). The resulting 4-maleylacetoacetate is eventually metabolized to acetyl-CoA. Compared with the control group, the HGADO fold changes in the P-PS and UV/H₂O₂-PS treatment groups were 1.25 and 1.17, respectively (Fig. 4 E), indicating markedly enhanced aromatic ring cleavage following synergistic aging treatment. (2) Aliphatic oxidation products. Aliphatic compounds detected by GC-MS, such as cis-7-hexadecenoic acid, can be directly activated by acyl-CoA synthetase to form acyl-CoA. These intermediates enter the fatty acid β-oxidation pathway, undergoing progressive chain shortening to produce acetyl-CoA, which ultimately enters the TCA cycle to support energy metabolism and cellular biosynthesis 72 . (3) Oxygenated long-chain aromatic compounds such as Hexoxybenzene and Benzeneacetic acid, 2-tetradecyl ester, undergo hydroxylation, dealkylation, or oxidative cleavage catalyzed by cytochrome P450 monooxygenases, yielding shorter-chain aromatic intermediates. These products may be further transformed into aliphatic molecules by carboxylesterases (CES) and subsequently incorporated into aromatic-cleavage or β-oxidation pathways. Subsequent oxidation products 2-ethyl-1-butano, 2-isopropyl-5-methylhexan-1-ol, or Decane can be sequentially converted into their corresponding carboxylic acids by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), enabling further entry into the TCA cycle. Although GC-MS did not detect the corresponding carboxylic acids, their absence may be attributed to low production levels and rapid metabolic turnover, preventing accumulation to detectable concentrations. ADH catalyzes the interconversion of alcohols, aldehydes, and ketones, providing essential precursors for the irreversible oxidation mediated by ALDH. The fold changes in ADH expression in the P-PS and UV/H₂O₂-PS groups are 2.60 and 2.07, respectively (Fig. 4 F) 73 . ALDH further oxidizes a variety of aldehyde substrates to carboxylic acids, with fold changes of 1.04 and 1.33 in the two treatment groups (Table S5) 74 . Endocytosis is a major pathway for the internalization of plasma membrane proteins, lipids, and extracellular materials into the cytoplasm, playing a crucial role particularly in eukaryotic cells 75 . Transcriptomic analysis (Table S6) revealed that in both the P-PS and Aged-PS groups, genes associated with endocytosis, such as actin-like ATPase domain-containing protein and endocytosis protein end4were upregulated. In addition, several genes related to vesicle-mediated transport of plastic particles (NCS cytosine-purine permease and vesicle transport protein) also showed increased expression. These findings suggest that plastic particles may enter the hyphae of P. chrysosporium through endocytosis and are subsequently transported via intracellular vesicle trafficking 76 . However, the specific transmembrane processes involved remain unclear and will be further investigated in a separate study. Throughout the degradation process, the introduction of oxygen-containing functional groups (such as carbonyl, hydroxyl, and ether bonds) plays a crucial role. These groups decrease the electron density of certain carbon atoms in the substrate, thereby increasing their electrophilicity and making them more susceptible to oxidation or cleavage reactions, which in turn facilitates polymer degradation 77 . During enzymatic reactions, the presence of these oxidized functional groups enhances the binding and electronic interactions between the polymer and the enzyme’s active site, thus accelerating the degradation process 78 . Taking the carbonyl as an example (Fig. 4 D), the polarity of its covalent bond renders the carbon atom electrophilic and prone to nucleophilic attack. The enzyme, through the spatial conformation and electronic coordination of its active site, increases the sensitivity of the carbonyl carbon toward nucleophilic reagents (such as water molecules, cofactors, or enzyme residues), thereby inducing nucleophilic addition, breaking the π-bond of the C = O, and generating an oxyanion (O⁻) 79,80 . Through this mechanism, enzymatic reactions proceed more rapidly, although the precise triggering mechanism remains to be further elucidated. Transcriptomic analysis revealed upregulation of multiple pathways related to fatty acid degradation and the TCA cycle (Tables S7 and S8), indicating that enhanced availability of PS aging products enables microorganisms to more efficiently channel oxidized fragments into central metabolism. Differentially expressed genes and their enriched KEGG pathways are summarized in Fig. 4 C. Upregulation of 4-hydroxy-2-oxoglutarate aldolase (EC 4.1.3.16) and (R)-2-hydroxyglutarate-pyruvate transhydrogenase (EC 1.1.99.40) suggests that oxo-organic acids generated from aromatic ring cleavage are converted into TCA cycle precursors via the glycolate or oxaloacetate pathways 81 . Increased expression of alcohol dehydrogenase (EC 1.1.1.1) indicates that alcohol and aldehyde intermediates formed during PS oxidation undergo further dehydrogenation and oxidation, entering deeper metabolic routes. Concurrently, activation of acylglycerol lipase (EC 3.1.1.23) implies that longer-chain oxygenated fragments are enzymatically cleaved into shorter fatty acids or ester units, which subsequently enter β-oxidation and ultimately feed into the TCA cycle 82 . Collectively, these transcriptional changes depict a coordinated network in which PS oxidation products are progressively transformed into metabolites readily incorporated into central carbon metabolism. In summary, this study, together with existing literature, demonstrates that surface oxidation is a critical prerequisite for plastic biodegradation 83 . Physical or chemical pretreatments, such as UV irradiation, chemical oxidation, or thermal treatment, introduce oxygen-containing functional groups, including carbonyl, carboxyl, and hydroxyl groups, onto the polymer surface, thereby reducing hydrophobicity, activating polymer chains, and enhancing the accessibility of microbes and enzymes 44 . Using PS as a model, we further showed that these oxygen-containing groups promote electron transfer, accelerate free radical formation and backbone cleavage, thereby facilitating enzymatic degradation and microbial metabolism. The resulting oxidized fragments can be efficiently channeled into aromatic ring cleavage, fatty acid β-oxidation, and the TCA cycle, enabling the systematic conversion of high-molecular-weight polymers into central metabolites. This mechanism, in which surface oxidation enhances enzymatic degradation and subsequent metabolic integration, appears to be broadly applicable across different polymer systems, indicating that surface oxidation is not only a hallmark of polymer degradation but also an essential step for microbial utilization of high-molecular-weight materials 84 . Discussion This study constructs a systematical biodegradation method of PS by UV/H₂O₂-Aging. The results indicate that individual UV or H₂O₂ pretreatment had limited impact on plastic degradation, whereas combined UV/H₂O₂ aging significantly enhanced the surface hydrophilicity and biocompatibility of PS films. This treatment also generated micro-pores and cracks on the film surface, providing favorable sites for microbial biofilm formation and enzymatic action. Molecular docking simulations employing the pretreatment product 4,5-dimethoxy-2-hydroxyacetophenone as substrate, despite their limitations, provide valuable predictive insights into enzyme-catalyzed reactions. Building upon this foundation, they offer significant guidance regarding the enzymatic degradation process of substances. The pretreatment promoted fungal hyphal attachment and growth, thereby enhancing microbial degradation of the plastic. In addition, the introduction of oxygen-containing functional groups strengthens the interactions between the polymer and microbial enzymes via promotion of electron transfer, resulting in an enhancement of degradation process. The degradation of PS occurs through three main pathways: cleavage of the aromatic ring to generate metabolic intermediates, microbial assimilation of carboxyl-containing compounds, and further oxidation of these intermediates by CYP450. Overall, UV/H₂O₂ synergistic aging represents an effective strategy to enhance plastic biodegradation. By inducing surface modification and facilitating enzymatic reactions, it can significantly improve the degradation efficiency of PS films, providing both theoretical and practical insights for environmentally friendly plastic disposal. The following is the Supplementary material related to this article Video S1. Methods Experimental materials To prepare PS films, 3 g of PS plastic raw material was dissolved in 100 ml of xylene. Prior to experiments, the prepared PS films were sequentially rinsed with sterile water and 75% ethanol to remove impurities, followed by air-drying under aseptic conditions. After drying, the plastic films were cut into 30 mm × 30 mm pieces for subsequent use, serving as the original PS films 45 . P. chrysosporium (BKMF-1767, CCTCC No. AF-96007) was obtained from the China Center for Type Culture Collection (Wuhan, China) and preserved at 4°C in potato agar medium. All experiments were conducted using 3rd-generation cultures. The transfer method and the composition of the culture medium are provided in the Supplementary Information Text S1. UV/H₂O₂ pretreat of PS film The pretreatment experimental setup is illustrated in Figure S1 . The specific procedures were as follows: Dried pristine PS films were immersed in 30% H₂O₂ solution and irradiated using a 365 nm LED ultraviolet lamp with a power of 20 W. The distance between the UV lamp and the liquid surface was maintained at 15 cm. During the reaction process, the PS film was turned over every 12 hours to ensure uniform irradiation. Due to the consumption of H 2 O 2 , a certain volume of the reaction solution was extracted every 24 hours, filtered (0.22 µm Millipore filter), and detected the concentration of H 2 O 2 by titanium (Ⅳ) oxalate on a spectrophotometer at λ max = 400 nm 85 . A certain amount of H 2 O 2 was added in the suspension in order to maintain a stable oxidation rate of PS. To prevent temperature increase caused by ultraviolet irradiation and hydrogen peroxide reaction, the entire system was placed in a temperature-controlled chamber set at 25°C, with the equipment operating in a well-ventilated environment. After 7 days of pretreatment, the PS films were retrieved, soaked in deionized water for 2 hours with repeated rinsing, and finally dried in an oven at 40°C for 24 hours to obtain the pretreated PS films. Based on the aforementioned procedures, different treatment groups were established: Pristine PS served as the untreated control, H₂O₂ reaction under dark conditions, UV irradiation alone, and synergistic aging with combined UV/H₂O₂ treatment. The resulting PS films were designated as Pristine-PS (P-PS), H 2 O 2 -PS, UV-PS and UV/H 2 O 2 -PS, respectively. P. chrysosporium biofilm cultivation and biodegradation of PS film assays Low-carbon potato dextrose agar (LC-PDA) medium was prepared using five primary components: potato extract solution, glucose, KH₂PO₄, MgSO₄, and agar powder. The medium was dissolved in 1000 mL of potato extract, which was obtained by slicing potatoes, boiling them in water, and filtering the solution through gauze (detailed procedures are provided in Text S2). After the medium cooled and solidified, a small amount of P. chrysosporium was inoculated onto the plates using an inoculation loop. The cultures were incubated at 37°C in a constant-temperature incubator (SHP-250, SANFA, China) for 24 hours. Subsequently, the plates were removed, and PS films from different pretreatment groups were placed onto the surface of the white-rot fungal medium, ensuring direct contact between the fungal mycelia and the plastic films to facilitate co-cultivation. A control group with pure P. chrysosporium culture (without plastic films) was simultaneously prepared. PS films were collected on days 7, 14, 21, 28, and 35 of biodegradation. The degradation fragments were first immersed in 2% (w/v) sodium dodecyl sulfate (SDS) solution for 4 hours. Residual particles collected by vacuum pump filtration were resuspended in 20 mL of saturated saline solution, stirred for 15 min to ensure thorough mixing, and allowed to stand for 2 h. The supernatant mixture was then filtered through 0.25 µm vacuum-pumped glass fiber membrane, and microplastics with a particle size smaller than 0.25 µm were ignored. This collection process was repeated three times to minimize plastic loss. Finally, the plastic films were dried at 40°C for 24 h for mass determination. Plastic film degradation characterization Prior to analytical characterization, all plastic film samples underwent a cleaning protocol to remove surface impurities and residual microbial components. For post-pretreatment samples, immersion in deionized water for 2 h followed by repeated rinsing was performed. For post-biodegradation samples, the procedure involved: initial careful washing with distilled water, subsequent suspension in 25 mL distilled water containing 0.5 g SDS with agitation at 120 rpm for 2 h to eliminate biofilms, two additional washes with distilled water, and final recovery through vacuum filtration. Finally, all the samples were vacuum dried overnight in a vacuum drying oven. Surface hydrophobicity changes of plastic films before and after pretreatment, as well as after 35-day biodegradation, were quantitatively analyzed using a water contact angle goniometer (WCA). Morphological characteristics of the plastic films were examined using a thermal field emission scanning electron microscope (SEM) (Zeiss Gemini 500). Samples were sputter-coated with gold and observed under the TFE-SEM at an accelerating voltage of 2 kV 86 . Changes in surface chemical functional groups were detected by Fourier Transform Infrared Spectroscopy (FTIR) (Thermo Fisher IS50, USA). The changes in the weight average molecular weight (Mw) and number average molecular weight (Mn) of the PS plastics were determined via high-temperature gel permeation chromatography (GPC) (Agilent, PL220, USA). The PS plastic film was dissolved in tetrahydrofuran (High Performance Liquid Chromatography, Sigma), filtered through a 0.22 µm organic-phase microporous filter membrane, and placed in a 1 mL liquid-phase vial for measurement. 2.5. Physiological state and enzyme activity of P. chrysosporium Fungal colonization levels were evaluated by measuring the dry cell weight on plastic films, while cellular activity during growth was assessed to monitor physiological status. The activities of extracellular enzymes MnP, LiP and Lac were quantitatively determined 87, 88 . Detailed methodologies are provided in Text S2. Molecular docking simulations and analysis of results The molecular structure of 4,5-Dimethoxy-2-hydroxyacetophenone was obtained using GaussView 5.0 software, and the 3D structures of MnP, LiP, and Lac were downloaded from the Protein Data Bank (PDB; https://www.rcsb.org/ ), with protein codes 1LLP, 3M5Q, and 2HRG, respectively. MnP, LiP, and Lac are derived from white-rot fungi. Molecular docking was performed using AutoDock Vina to predict the optimal binding modes of 4,5-Dimethoxy-2-hydroxyacetophenone with LiP, MnP, and Lac. The docking score reflects the binding strength, with higher absolute values indicating stronger binding affinity 89 . Finally, the docking results were visualized using PyMOL for further analysis of the binding modes and affinities. Transcriptome and metabolome analysis After P. chrysosporium was cultured with plastic for 5 days, total RNA was isolated using the RNA Kit (15NT) (Agilent, DNF-471-1000), and a library was constructed by Personal Biotech Cp (Shanghai, China). Detailed experimental and analytical procedures are provided in Text S3. Characterization of degradation products by gas chromatography-mass spectrometry (GC-MS) 90 . Detailed methodologies are provided in Text S4. Data availability The data that support the findings of this study are available from the corresponding author upon request. Declarations Acknowledgements This work was funded by the National Natural Science Foundation of China (Grant NO. 42576166, 51809068 and 42107384); The Anhui Provincial Natural Science Foundation 2308085MD119. Author information These authors contributed equally: Zhi Guo, Yuanyuan Zha, Xingpan Guo. Authors and Affiliations School of Resources and Environmental Engineering, Hefei University of Technology, Hefei, China Zhi Guo, Yuanyuan Zha, Xinlei Ling, Lin Yao, Lishou Han, Fan Yang State Key Laboratory of Estuarine and Coastal Research, East China Normal University, 500 Dongchuan Road, Shanghai, China Xingpan Guo Anhui Ecological Civilization Research Institute, Hefei University of Technology, Hefei, China Zhi Guo, Yuanyuan Zha, Xinlei Ling, Lin Yao, Lishou Han, Fan Yang Contributions Z.G. and X.P. supervised and conceived the project. Y.Z. carried out the experiments, conducted the characterization, analyzed the data and wrote the paper. 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Sci. Technol. 40 , 4196–4199 (2006). Additional Declarations There is NO Competing Interest. Supplementary Files SupportingInfo.docx SupportingInformation.pdf Supporting Information VideoS1.mp4 Video S1 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8364324","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":616628627,"identity":"d38eac3a-079c-40b5-a136-ef0980ad9a03","order_by":0,"name":"Zhi Guo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYJACZgYDmwT2BhCTjXgtaQk8B5hJ0sJwmAQt8hHJhz8XFJzP45HIP8DwoewwA//sBvxaDG+kJRjPMLhdzCORzMA449xhBok7BwhomZFjkMxjcDtxP1ALM2/bYQYDiQTCWg7zGJxL7AFp+UuMFnmJHMNmHoMDEC2MxGgx4HmWzMxjkJzYw/PY4GDPuXQeiRuEbGkHhhjPH7vEHvbEhw9+lFnL8c8gZMsBJA6IzYNfPciWBoJKRsEoGAWjYMQDAFAuPwEPCjS5AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-9315-2788","institution":"School of Resources and Environmental Engineering, Hefei University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Zhi","middleName":"","lastName":"Guo","suffix":""},{"id":616628628,"identity":"2e17eae6-9789-442a-bbf2-9c735f8427c4","order_by":1,"name":"Yuanyuan Zha","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Zha","suffix":""},{"id":616628629,"identity":"42f09092-d5f9-438d-8206-0f871624ec3a","order_by":2,"name":"Xingpan Guo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xingpan","middleName":"","lastName":"Guo","suffix":""},{"id":616628630,"identity":"797ff707-3e6a-49a3-affb-40278dab8fb5","order_by":3,"name":"Xinlei Ling","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xinlei","middleName":"","lastName":"Ling","suffix":""},{"id":616628631,"identity":"dcb761f0-9361-46e1-b0a0-ac6daeab4f29","order_by":4,"name":"Lin Yao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Yao","suffix":""},{"id":616628632,"identity":"6ea4cea3-af4c-4bbd-bcca-6ee259c563ab","order_by":5,"name":"Lishou Han","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lishou","middleName":"","lastName":"Han","suffix":""},{"id":616628633,"identity":"ec091784-71a6-4142-9892-0e895f93e8e1","order_by":6,"name":"Fan Yang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Fan","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-12-15 09:27:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8364324/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8364324/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106078084,"identity":"c32f2b6c-4fdd-4e94-9eaa-850fb1c7bd4b","added_by":"auto","created_at":"2026-04-03 07:57:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":450024,"visible":true,"origin":"","legend":"\u003cp\u003ePretreatment and subsequent biodegradation processes. (A) Schematic illustration of the experimental procedure. (B) Colonization behavior of \u003cem\u003eP. chrysosporium\u003c/em\u003e on PS films subjected to different pretreatments, where visible mycelial colonies appeared on the film surfaces (highlighted by red circles). (C) Biomass produced by \u003cem\u003eP. chrysosporium\u003c/em\u003e colonization. Significant levels of correlation are indicated as follows: p \u0026lt; 0.05 *. (D) Activity of \u003cem\u003eP. chrysosporium\u003c/em\u003e. (E) Temporal changes in PS film mass during the biodegradation experiment. (F) Weight loss of PS films during the pretreatment and subsequent biodegradation processes.Different letters indicate significant differences among treatments (one-way ANOVA with Tukey’s multiple comparison test, p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8364324/v1/59439566958c038a544eb1df.png"},{"id":106078088,"identity":"cf46f498-202f-4a4e-b867-57b7d73f9d0e","added_by":"auto","created_at":"2026-04-03 07:57:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":792391,"visible":true,"origin":"","legend":"\u003cp\u003ePlastic film structure characterization. (A) Surface morphology of plastic films in different pretreatment groups and after 35 days of biodegradation. (B) Variation of contact angle on the surface of plastic film. (C) ATR-FTIR spectra of plastic film. (D, E,) Number average molecular weight (Mn, g/mol) and weight average molecular weight (Mw, g/mol) of PS plastic films after biodegradation in different pretreatment groups.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8364324/v1/2deaed760a7f4a03b638a074.png"},{"id":106078080,"identity":"bc9003a1-1c05-4981-8427-8c255d27424c","added_by":"auto","created_at":"2026-04-03 07:57:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":650688,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of cellular physiology and enzymatic degradation cycle. (A, B, C) Activity of the extracellular enzymes MnP, LiP and Lac secreted by \u003cem\u003eP. chrysosporium\u003c/em\u003e. Significant levels of correlation are indicated as follows: p \u0026lt; 0.05 *, p \u0026lt; 0.01 * *, p \u0026lt; 0.001 * * *. (D, E, F) Molecular docking results of 4,5-Dimethoxy-2-hydroxyacetophenone with MnP, LiP, and Lac (highest binding energy ranking). (G) Catalytic cycles of extracellular enzymes MnP, LiP, and Lac.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8364324/v1/561df3994cbd7b65b16aceb1.png"},{"id":106078090,"identity":"bc7e10ae-1959-4448-a136-21e4dfb7b51d","added_by":"auto","created_at":"2026-04-03 07:57:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":421501,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eP. chrysosporium \u003c/em\u003etranscription levels and metabolic model construction. (A) PS degradation pathway prediction. (B) Electron conjugation effect of oxygen-containing functional groups. (C) Differentially expressed gene-associated KEGG pathway. The red box indicates gene upregulation, with the numbers within the box representing different enzymes. \u003ca href=\"https://www.kegg.jp/entry/4.1.3.16\"\u003e4.1.3.16\u003c/a\u003e: 4-hydroxy-2-oxoglutarate aldolase, \u003ca href=\"https://www.kegg.jp/entry/1.1.99.40\"\u003e1.1.99.40\u003c/a\u003e: (R)-2-hydroxyglutarate-pyruvate transhydrogenase, \u003ca href=\"https://www.kegg.jp/entry/1.1.1.1\"\u003e1.1.1.1\u003c/a\u003e: alcohol dehydrogenase, 3.1.1.23: acylglycerol lipase. (B, C, D) \u003ca href=\"https://www.sciencedirect.com/topics/immunology-and-microbiology/transcriptome\" title=\"Learn more about Transcriptome from ScienceDirect's AI-generated Topic Pages\"\u003eTranscriptome\u003c/a\u003eexpression of enzymes related to intracellular metabolism.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8364324/v1/4967cb3af4d68672dd72d0d1.png"},{"id":106078170,"identity":"d4d30bc8-3e09-4d6d-ad61-1b125dbeded0","added_by":"auto","created_at":"2026-04-03 07:57:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3449496,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8364324/v1/241b2612-a82c-47f7-8a09-25d8e87c6b6a.pdf"},{"id":106078085,"identity":"96728164-0382-478e-aecf-2c23359e930f","added_by":"auto","created_at":"2026-04-03 07:57:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13921,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInfo.docx","url":"https://assets-eu.researchsquare.com/files/rs-8364324/v1/bea6a125ad632c3e4ed7d3f1.docx"},{"id":106078083,"identity":"bd6a2556-0756-49d9-83d9-b01ee48909e9","added_by":"auto","created_at":"2026-04-03 07:57:07","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1450294,"visible":true,"origin":"","legend":"Supporting Information","description":"","filename":"SupportingInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8364324/v1/90daf777f4d1ec4b95cdd8ae.pdf"},{"id":106078095,"identity":"a6b2cbe1-f0f5-4af3-8444-5b57472f7b55","added_by":"auto","created_at":"2026-04-03 07:57:12","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4395939,"visible":true,"origin":"","legend":"Video S1","description":"","filename":"VideoS1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8364324/v1/c279101a24686ab27108abf2.mp4"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Mechanistic Insights into UV/H₂O₂-Aged Polystyrene Enable an Optimized Protocol for Fungal Biodegradation","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlastic is a man-made long-chain and/or aromatic polymer material. Owing to its excellent properties, such as light weight, high plasticity and flexibility, corrosion resistance, and low cost, it is widely used in numerous applications, including construction, electronics industry, and packaging\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.The annual output of plastics soared to over 400\u0026nbsp;million metric tons in 2023\u003csup\u003e2\u003c/sup\u003e. As an agricultural application, mulch films can increase soil temperature, retain moisture, control weeds, and reduce crop diseases, thereby significantly improving the crop growth surroundings and agricultural cultivation patterns\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Therefore, the use of mulch films and facility-based farming has become increasingly widespread with the rapid development of agriculture\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, a large amount of discarded film is not properly treated in time resulting in widespread plastic residues in the soil environment, due to the lack of a well-established recycling system, effective scheduling, and scientific management guidance\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These residues gradually fragment into smaller particles (\u0026lt;\u0026thinsp;5 mm), known as microplastics and even nanoplastics, collectively referred to as micro/nanoplastics (MNPs), under prolonged exposure to sunlight and mechanical forces\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. MNPs can persist in the environment for hundreds or even thousands of years, leading to their gradual accumulation and widespread presence in the atmosphere, water bodies and terrestrial biosphere, and poses a serious threat to ecosystems\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. To date, there has no optimal resolving methods for environmental residual mulch film. Conventional plastic waste treatments, including landfill and incineration, are limited by low efficiency and secondary pollution\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Therefore, it is imperative to develop efficient and environmentally friendly methods to mitigate both the direct and indirect impacts of plastic pollution\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecent studies have identified four primary pathways for plastic degradation: biodegradation, photodegradation, chemical degradation, and thermal degradation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Among them, biodegradation typically proceeds slowly and exhibits limited efficiency. In cultivated soils rich in fungi, microorganisms, and invertebrates, the degradation rate of polystyrene (PS) remains below 1% after 90 days, with no significant increase observed thereafter\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Similarly, after an eight-week incubation, \u003cem\u003eCephalosporium\u003c/em\u003e species induced a weight loss of 2.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16% in PS, while \u003cem\u003eMucor\u003c/em\u003e species resulted in a 1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13% reduction\u003csup\u003e13\u003c/sup\u003e.Numerous investigations have demonstrated that appropriate physical or chemical pretreatment can substantially enhance the microbial degradation of plastics\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Such pretreatment promotes surface oxidation, decrease hydrophobicity, introduce oxygen-containing functional groups, and facilitate biofilm formation, thereby improving microbial attachment and activity\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. For instance, exposure of polyethylene (PE) to ultraviolet (UV) radiation and thermal treatment shortens polymer chains and generates hydroxyl, carboxyl, and carbonyl groups, which significantly accelerate its biodegradation by microorganisms\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Moreover, thermal pretreatment has proven particularly effective in promoting the enzymatic degradation of PS, whereas alkaline or acidic pretreatment generally enhances the biodegradability of bio-based plastic wastes\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Despite substantial progress, the fundamental mechanisms through which pretreatments accelerate biodegradation, particularly the pathways of bond cleavage and oxygen incorporation at the polymer surface, remain largely unresolved and warrant further investigation.\u003c/p\u003e \u003cp\u003eCurrently, exclusive reliance on biological degradation faces significant challenges\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The strong hydrophobicity and chemically stable structure of plastics hinder effective microbial colonization and biofilm formation on their surfaces, thereby limiting degradation efficiency\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. White-rot fungi exhibit strong oxidative decomposition capabilities through the secretion of extracellular ligninolytic enzymes such as manganese peroxidase (MnP), lignin peroxidase (LiP), and laccase (Lac)\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. These fungi can partially oxidize and depolymerize complex polymer structures owing to their non-specific oxidative systems, exhibiting considerable potential for plastic biodegradation\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, their attachment to plastic surfaces, enzyme stability, and long-term catalytic activity are often insufficient, resulting in slow and incomplete degradation\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.Recent studies suggest that coupling biological and photocatalytic processes for plastic degradation may effectively shorten degradation time and enhance efficiency, offering a promising strategy to overcome the limitations of purely biological methods\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. This provides an important direction for the present study, which aims to explore how pretreatment strategies can enhance the subsequent biodegradation efficiency of white-rot fungi.\u003c/p\u003e \u003cp\u003eTo further elucidate the pretreatment mechanisms of microplastics and their influence on subsequent biodegradation, this study proposes a novel strategy integrating physicochemical pretreatment with microbial degradation. The approach involves synergistic aging of plastics through combined UV irradiation and hydrogen peroxide treatment, followed by further fragmentation and mineralization by the typical white-rot fungus \u003cem\u003ePhanerochaete chrysosporium\u003c/em\u003e (\u003cem\u003eP. chrysposporium\u003c/em\u003e). The study aims that: (1) demonstrates the essential prerequisite of surface oxidation with the formation of oxygen-containing functional groups in plastic degradation; (2) illustrates the significance of oxygenated groups in enhancing microbial colonization, enzyme-mediated chain scission, and serving as electron donors that facilitate extracellular enzymatic cleavage of polymer chains; (3) integrates multi-omics analyses to identify intracellular metabolic pathways involved in PS degradation.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eVisualization process of pretreated collaborative fungi degrading plastics\u003c/h2\u003e \u003cp\u003eThis study used UV radiation and H₂O₂ as a synergistic pretreatment system to induce aging in pristine plastic films. Subsequently, the pretreated plastic films exhibit enhanced colonization by \u003cem\u003eP. chrysosporium\u003c/em\u003e, especially in the UV/H₂O₂ group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Upon co-cultivation with \u003cem\u003eP. chrysosporium\u003c/em\u003e, extensive filamentous networks and dense mycelial structures were formed on the pretreated film surfaces, which facilitated the physiological fragmentation of the films (Figure S2). The degree of cracking was more evident in the UV/H₂O₂-pretreated samples. By the end of the incubation period, the plastic films have disintegrated into irregular fragments (Figure S3). The colonization of biofilms on the PS film surface largely determines its potential biodegradability. Compared with pristine PS, aged PS films exhibit a higher tendency for biofilm formation\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, the attached biomass of \u003cem\u003eP. chrysosporium\u003c/em\u003e noticeably increased on pretreated films, with the UV/H₂O₂-PS group showing the strongest colonization ability. During the biodegradation process, the fungal cells maintained high viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), with activity levels of 86.3% (P-PS), 89.4% (H₂O₂-PS), 82.3% (UV-PS), and 70.4% (UV/H₂O₂-PS). Overall, UV/H₂O₂ pretreatment may effectively modify the surface characteristics of the plastics films, enhance biofilm formation, and consequently promote the biodegradation process\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCompared with the P-PS and pretreatment solely, the UV/H₂O₂-PS exhibits a significantly higher weight loss throughout the biodegradation process (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The mass of UV/H₂O₂-PS gradually decreased over 35 days, reaching a maximum weight loss of 25.75\u0026thinsp;\u0026plusmn;\u0026thinsp;5.08%, which was significantly higher than that of the P-PS (6.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89%), H₂O₂-PS (7.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31%), and UV-PS (7.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76%) groups. These findings clearly demonstrate that UV/H₂O₂ pretreatment substantially enhanced the biodegradability of PS films, making them more susceptible to colonization and degradation by \u003cem\u003eP. chrysosporium\u003c/em\u003e. The pronounced improvement in degradation efficiency is likely attributable to surface physicochemical modifications induced by UV/H₂O₂ pretreatment. Previous report has suggested that physical pretreatments, such as UV irradiation, ozonation, and plasma exposure, can disrupt the macromolecular aggregation of polymer chains, thereby increasing their accessibility to enzymatic and microbial attack\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In particular, UV/O₃ weathering has been reported to trigger physicochemical transformations in PS nanoparticles, including the generation of oxygen-containing functional groups and enhanced surface hydrophilicity\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. It is therefore plausible that the combined UV/H₂O₂ pretreatment induced similar modifications on the PS surface, facilitating microbial attachment and subsequent biodegradation. Collectively, these results suggest that such oxidative pretreatment may serve as a critical prerequisite for achieving efficient biological degradation of conventional plastics\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eP. chrysosporium\u003c/b\u003e \u003cb\u003edeeply oxidation of PS films\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSurface morphology characterization was performed to evaluate the effects of pretreatment on film aging, \u003cem\u003eP. chrysosporium\u003c/em\u003e colonization, and polymer degradation\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The pristine PS films and those films treated with UV or H₂O₂ alone exhibit relatively smooth surface textures without noticeable structural alterations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In contrast, the UV/H₂O₂-PS film displays distinct cracks and pits, indicating pronounced surface erosion and oxidation. These phenomena may be attributed to the high UV sensitivity of the benzene rings in PS molecules, which enables rapid photooxidation within a short time\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Meanwhile, the conjugated structures formed during aging further facilitate progressive degradation of the polymer backbone\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Once microcracks appear on the PS surface, H₂O₂ solution can infiltrate these sites, at which, reactive oxygen species (ROS) and carbon-centered radicals are generated under UV irradiation and in the presence of oxygen. These radicals sequentially cleave the PS molecular chains, accelerating surface oxidation and eventually resulting in the formation of pits. Biodegradation experiments further confirm strong colonization of \u003cem\u003eP. chrysosporium\u003c/em\u003e on the pretreated PS surfaces. SEM images reveal only minor wrinkles on the pristine PS films, whereas the pretreated films exhibit a roughened surface texture with visible fungal hyphae, indicating successful colonization. Notably, the UV/H₂O₂-PS show more pronounced groove-like wrinkles, larger pores, and spores embedded within the fractured layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Consistent with earlier findings, aged PS films exhibited a stronger tendency for biofilm formation compared with pristine PS films\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, which is likely attributed to increased surface roughness that facilitates microbial adhesion. In general, microorganisms adhere more easily to rough microplastic surfaces, thereby promoting subsequent biofilm formation and growth\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. These morphological and colonization results illustrate that UV/H₂O₂ synergistic aging markedly alters the surface properties of PS, eroding its originally surface structure and creating new sites for contaminant adsorption and radical attack, which in turn facilitates fungal attachment and biodegradation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo exploit the deeply molecular mechanism of PS film degradation by \u003cem\u003eP. chrysosporium\u003c/em\u003e, the hydrophilicity was employed. Increased hydrophilicity leads to a larger contact area and more adsorption sites, thereby enhancing electrostatic interactions with the PS film\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The hydrophilicity of plastic surfaces influences the adhesion of biofilms, with lower surface hydrophobicity being more conducive to microbial colonization\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, during the pretreatment stage, all applied pretreatment methods reduced the water contact angle of the plastic films. The contact angle of untreated P-PS was 91.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u0026deg;, which decreased to 88.66\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23\u0026deg; after H₂O₂ pretreatment, 88.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u0026deg; after UV pretreatment, and 85.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u0026deg; after UV/H₂O₂ co-pretreatment. This reduction may be attributed to the introduction of hydrophilic functional groups on the PS surface during pretreatment, thereby lowering its surface hydrophobicity\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Generally, evolution of polar carbonyl groups on MPs\u0026rsquo; main chain leads to increase of surface polarity and may increase the accessibility of water on the material surface\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. In the biodegradation stage, as more clearly observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the contact angles of all plastic films decreased markedly. After biodegradation, the contact angle of P-PS decreased to 78.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71\u0026deg;, H₂O₂-PS to 79.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u0026deg;, UV-PS to 75.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u0026deg;, while the UV/H₂O₂-PS showed the most pronounced reduction, reaching 66.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u0026deg;. Comparative data revealed that the contact angle of P-PS decreased by an average of 13.25\u0026deg; after direct biodegradation, whereas that of the UV/H₂O₂-PS followed by biodegradation decreased by an average of 25.8\u0026deg;. When \u003cem\u003eP. chrysosporium\u003c/em\u003e colonizes the plastic surface, its mycelia can penetrate the surface and spread into the matrix to absorb nutrients, enabling firm attachment to the plastic\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Hydrophobins, which are surfactant proteins produced by filamentous fungi, function as structural components involved in fungal growth and their interactions with the environment\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Acting as biosurfactants, fungal hydrophobins self-assemble into amphipathic protein membranes\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. These amphiphilic proteins involved in the formation of aerial structures such as spores or fruiting bodies. They form chemically robust layers which can only be dissolved in strong acids. These layers adhere to different surfaces, changing their wettability, and allow the binding of other proteins\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. These enhance the hydrophilicity of the plastic surface and promote the adhesion of the fungus to the polymer surface. Therefore, the colonization of \u003cem\u003eP. chrysosporium\u003c/em\u003e on plastic surfaces can reduce hydrophobicity and further promote microbial attachment\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eATR-FTIR spectra indicating significant changes in the surface functional groups of the PS plastic films before/after pretreatment and biodegradation, are well explained contact angle changes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, changes in absorption peaks were observed at 3025 cm⁻\u0026sup1; and 2922 cm⁻\u0026sup1; in the PS films before and after pretreatment, attributed to C-H stretching vibrations of the aromatic rings. A carbonyl (C\u0026thinsp;=\u0026thinsp;O) absorption peak was observed at 1602 cm⁻\u0026sup1;. The appearance of the carbonyl peak suggests the introduction of polar groups into the PS backbone, leading to a transition of the plastic from hydrophobic to hydrophilic. This change facilitates the attachment of \u003cem\u003eP. chrysosporium\u003c/em\u003e to the PS film. The increase in oxygen-containing functional groups may result from the cleavage of C-H bonds under ultraviolet light, followed by their reaction with oxygen to form peroxyl radicals (C-O\u0026bull;). These peroxyl radicals can abstract hydrogen atoms from the surrounding environment, forming hydroperoxide (COOH), which further decompose into other products such as carbonyl (C\u0026thinsp;=\u0026thinsp;O)\u003csup\u003e43\u003c/sup\u003e. Additionally, in the spectral region around 1100 cm⁻\u0026sup1;, corresponding to the stretching vibrations of oxygen-containing functional groups such as alcohols and ethers, changes in the carbon-oxygen (C-O) bond are observed. After co-cultivation with \u003cem\u003eP. chrysosporium\u003c/em\u003e, the C\u0026thinsp;=\u0026thinsp;O and the C-O absorption peak in the infrared spectra of the plastic films became more pronounced, indicating the effective usage of microorganisms to disintegrate pretreatment PS films. Pretreatment induces surface oxidation of plastics, generating oxygen-containing functional groups such as carbonyl, carboxyl, and hydroxyl. Microbial metabolism further reinforces this modification, enhancing surface polarity and hydrophilicity, destabilizing polymer bonds, and accelerating degradation. Based on these sense, oxygen-containing functional groups play a critical role in the transition of plastics from physicochemical aging to biological degradation\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. These results are also demonstrated by our previous research for \u003cem\u003eP. chrysosporium\u003c/em\u003e attacking O-containing groups, which cause plastic crack\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNumber average molecular weight (Mn, g/mol) and weight average molecular weight (Mw, g/mol) decrease of a polymer are important indicator of polymer degradation\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Changes in the average molecular weight of PS plastic films from different pretreatment groups and biodegradation groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE. It can be seen that the number average molecular weight of the P-PS sample is 161655 and the weight average molecular weight is 300642. After various pretreatments, both Mn and Mw decreased. The number average molecular weight of the directly biodegraded sample is 149757 and the weight average molecular weight is 297810, which were reduced by 7.36% and 0.94%, respectively, compared with the P-PS. In contrast, the number average molecular weight of UV/H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-PS after biodegradation is 77242, and the weight average molecular weight is 238639, which decreased by 52.22% and 20.62%, respectively, compared with the P-PS. This suggests the occurrence of endo-type depolymerization or random internal scission of chains in polymer macromolecules, which generated oligomeric products and mid-chain polymers\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Notably, the Mn and Mw of the biologically degraded samples are slightly higher than those of the pretreated samples. This can be ascribed to that \u003cem\u003eP. chrysosporium\u003c/em\u003e preferentially consume shorter polymer chains, resulting in a relative enrichment of longer chains or arise from oxidative reactions occurring during biodegradation\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Since the relative atomic mass of oxygen (16.00) is higher than that of carbon (12.01), the introduction of oxygen-containing functional groups would simultaneously lead to a relative increase in the average molecular weight of the polymer\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhysiological status and O-containing groups triggered enzymatic degradation reaction\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eExtracellular activities of oxidative enzymes MnP, LiP, and Lac in \u003cem\u003eP. chrysosporium\u003c/em\u003e exhibit significant variation across different pretreatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). The degradation system of white-rot fungi relies on the synergistic action of ligninolytic enzymes (LMEs), organic acids, mediators, and auxiliary enzymes, characterized by a non-specific oxidation mechanism driven by free radical generation\u003csup\u003e51\u003c/sup\u003e. Oxygen-containing functional groups (such as C\u0026thinsp;=\u0026thinsp;O、C-O) induce electronic density redistribution along the polymer chain through inductive and resonance effects, weakening the stability of adjacent C-C and C-H bonds\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. These \u0026ldquo;activated\u0026rdquo;chemical bonds are more susceptible to attack by oxidative intermediates (such as free radicals or peroxides) generated through enzyme catalysis, making them preferential sites for the cleavage of the polymer backbone\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Under the synergistic effect of ultraviolet radiation and hydrogen peroxide, PS forms multiple oxygen-containing functional groups, such as carbonyl and hydroxyl groups, which provide important chemical foundations for subsequent biodegradation. To further investigate the degradation mechanism of PS with extracellular enzymes secreted by \u003cem\u003eP. chrysosporium\u003c/em\u003e, we selected the product 4,5-Dimethoxy-2-hydroxyacetophenone as a substrate for molecular docking simulations. The simulation results show that these oxygen-containing functional groups can form stable hydrogen bonds with the enzyme active sites, and the minimum hydrogen bond energies with MnP, LiP, and Lac are \u0026minus;\u0026thinsp;5.9, -5.0, and \u0026minus;\u0026thinsp;5.6, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E). These results suggest that the enzymes can be able to bind these substrates, thereby carrying out subsequent degradation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThrough the synergistic action of multiple enzymes, the introduction of oxygen functional groups and the enzyme-catalyzed oxidative process form a positive feedback loop, substantially promoting the cleavage of the polymer backbone and resulting of biodegradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG)\u003csup\u003e54\u003c/sup\u003e. In the MnP system, the catalytic cycle is initiated by the reaction of H₂O₂ or organic peroxides with the heme center, first forming an Fe-peroxide complex\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. This intermediate undergoes O-O bond cleavage, transferring two electrons to heme and releasing one water molecule to form Compound I, which is a Fe\u003csup\u003e4+\u003c/sup\u003e-oxo-porphyrin-radical complex with a highly oxidized state\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Compound I possesses strong oxidizing power and can abstract one electron from an electron donor, thereby being reduced to compound II (Fe\u003csup\u003e4+\u003c/sup\u003e-oxo-porphyrin complex). This compound is subsequently further reduced back to Fe\u0026sup3;⁺, completing one catalytic cycle. During this process, Mn\u0026sup2;⁺ is oxidized to Mn\u0026sup3;⁺ and chelated with bidentate organic acids (oxalate, acetate) to form Mn\u0026sup3;⁺-ligand complexes\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. This complex acts as a diffusible redox mediator capable of migrating away from the enzyme molecule. It selectively oxidizes oxygen-containing functional groups or benzene ring side chains in localized regions on polystyrene (PS) surfaces via single-electron oxidation, abstracting hydrogen atoms to generate benzyl or phenoxy radicals. The generated radicals undergo resonance migration and rearrangement reactions, inducing C-C or C-H bond cleavage to form oxidation products such as carbonyl and carboxyl groups, thereby promoting polymer chain scission and degradation\u003csup\u003e58\u003c/sup\u003e. The presence of oxygen-containing functional groups enhances the interaction between the Mn\u0026sup3;⁺-ligand complex and the polymer surface, improving electron transfer efficiency and further accelerating the oxidative cracking process. Similarly, the LiP catalyzed cycle also initiates with the oxidation of Fe\u0026sup3;⁺ by H₂O₂, yielding compound I (Fe\u003csup\u003e4+\u003c/sup\u003e-oxo-porphyrin complex), an intermediate possessing an exceptionally high oxidation potential\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Compound I completes the cycle by sequentially abstracting two electrons from an aromatic substrate, forming compound II (Fe\u003csup\u003e4+\u003c/sup\u003e-oxo-porphyrin complex), and subsequently being reduced back to native Fe\u0026sup3;⁺. LiP directly extracts electrons from the aromatic ring of PS to generate a radical cation. Subsequently, the radical migrates via resonance between the benzene ring and side chain, triggering C-C bond cleavage to form oligomeric products or oxidized fragments\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Oxygen-containing functional groups lower the substrate's ionization potential, facilitating electron extraction and enhancing LiP' s oxidative efficiency. In the Lac system, electron transfer is initiated at the T₁ copper site. The copper centers of Lac oxidize substrates and transfer the acquired electrons to molecular oxygen\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Electrons from oxidized substrates first transfer to this site before sequentially moving to the trinuclear copper center (T₂/T₃)\u003csup\u003e62\u003c/sup\u003e An oxygen molecule binds to the T₃ copper cluster plane, accepting electrons from four substrates and being reduced to two water molecules. Phenoxy or benzylic radicals generated during catalysis can diffuse from the PS surface, initiating a series of non-enzymatic radical chain reactions. The introduction of oxygen-containing functional groups significantly reduces the oxidation potential of PS, promoting electron transfer from substrates to the copper centers of Lac\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. This accelerates radical generation and diffusion, thereby enhancing oxidative cleavage efficiency\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTranscriptome-level cellular degradation mechanisms and metabolic model construction\u003c/h3\u003e\n\u003cp\u003eThis study conducted GC-MS analysis on PS subjected to UV/H₂O₂ pretreatment followed by biodegradation to characterize metabolites formed during both the physicochemical pretreatment and microbial degradation processes (Tables S1 and S2). Based on the intermediates identified by GC-MS and the transcriptomic analysis of \u003cem\u003eP. chrysosporium\u003c/em\u003e, the proposed PS biodegradation pathway and mechanism are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. The PS backbone consists of a benzene ring and a carbon chain, with the benzene ring functioning as the primary chromophore. Upon UV irradiation, the benzene ring absorbs energy and undergoes electronic excitation, thereby decreasing the stability of the para-position C-H bond\u003csup\u003e65\u003c/sup\u003e. Under the synergistic action of UV and H₂O₂, hydroxyl radicals (\u0026bull;OH) produced from H₂O₂ photolysis further attack these activated sites, inducing hydroxylation on the aromatic ring or its para-substituent\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Subsequent dehydrogenation reactions may generate oxidized functional groups such as C\u0026thinsp;=\u0026thinsp;O\u003csup\u003e67\u003c/sup\u003e. As oxidation advances, the PS chains undergo consecutive photo-oxidation of side chains and backbone cleavage, producing highly reactive alkoxy and epoxy radical that drive continuous chain-scission reactions\u003csup\u003e68\u003c/sup\u003e. These intermediate radicals subsequently react with oxygen to form peroxides, which further decompose under UV exposure, leading to the detachment of aromatic structures from the backbone and the release of multiple low-molecular-weight aromatic organic compounds. In addition, hydroxyl and carboxyl groups formed during side-chain oxidation may undergo further esterification or condensation reactions with adjacent oxygen-containing groups, yielding oxidation derivatives such as 4,5-Dimethoxy-2-hydroxyacetophenone\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDuring the biodegradation phase, genes encoding extracellular enzymes such as MnP, LiP, and Lac were significantly upregulated (Table S4). This transcriptional pattern is consistent with the characteristic reliance of \u003cem\u003eP. chrysosporium\u003c/em\u003e on a non-specific extracellular peroxidase system during the early stages of degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-E). These peroxidases continuously act on the \u0026ldquo;activated\u0026rdquo; chain ends and partially oxidized backbone regions of polystyrene following photooxidation, inducing long-chain scission and generating various small-molecule oxidation products through radical-mediated reactions. GC-MS results further show that PS subjected to synergistic aging releases substantial amounts of aromatic oxides and aliphatic degradation products during fungal metabolism, providing abundant substrates for subsequent incorporation into intracellular metabolic pathways. Cytochrome P450 (CYP450) serves as a core catalytic enzyme for diverse oxidation reactions by forming Fe (IV)-O high-valent iron-oxygen intermediates to activate and selectively oxidize inert C-H bonds\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Multiple P450-encoding genes with conserved domains were are extensively upregulated in \u003cem\u003eP. chrysosporium\u003c/em\u003e (Table S5), further supporting the central role of the P450 family in PS activation, biodegradation, and integration into downstream metabolic pathways. Notably, cytochrome P450 monooxygenases represent the most functionally versatile subgroup within the P450 family. With broad substrate adaptability, diverse reaction capabilities, and atypical kinetic properties, they are considered among the most important oxidases in biocatalysis\u003csup\u003e71\u003c/sup\u003e. The fold changes observed in the P-PS and UV/H₂O₂-PS treatment groups were 1.53 and 1.84, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eBased on the characteristics of metabolic products and the expression profiles of key enzyme genes, the downstream metabolism of PS can be summarized into three major pathways: (1) Aromatic oxidation products. 4-Methoxystyrene is first oxidized to cis-styrene ethylene glycol, which is subsequently converted into 4-hydroxy-2-oxovalerate. Under the action of 4-hydroxy-2-oxoglutarate aldolase (HOA), it is transformed into pyruvate, which is then converted into acetyl-CoA and enters the tricarboxylic acid cycle (TCA cycle). KEGG pathway analysis revealed that the expression level of the HOA-encoding gene was notably higher in the treatment group compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), further supporting the activation of this metabolic pathway. In another aromatic oxidation pathway, 2-methyl-2-phenyl-Oxirane is initially converted into Styrene oxide, which is then catalyzed by styrene oxide isomerase (SOI) to produce phenylacetaldehyde. Phenylacetaldehyde is subsequently oxidized to homogentisic acid, which undergoes aromatic ring cleavage via homogentisic acid 1,2-dioxygenase (HGADO). The resulting 4-maleylacetoacetate is eventually metabolized to acetyl-CoA. Compared with the control group, the HGADO fold changes in the P-PS and UV/H₂O₂-PS treatment groups were 1.25 and 1.17, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), indicating markedly enhanced aromatic ring cleavage following synergistic aging treatment. (2) Aliphatic oxidation products. Aliphatic compounds detected by GC-MS, such as cis-7-hexadecenoic acid, can be directly activated by acyl-CoA synthetase to form acyl-CoA. These intermediates enter the fatty acid β-oxidation pathway, undergoing progressive chain shortening to produce acetyl-CoA, which ultimately enters the TCA cycle to support energy metabolism and cellular biosynthesis\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. (3) Oxygenated long-chain aromatic compounds such as Hexoxybenzene and Benzeneacetic acid, 2-tetradecyl ester, undergo hydroxylation, dealkylation, or oxidative cleavage catalyzed by cytochrome P450 monooxygenases, yielding shorter-chain aromatic intermediates. These products may be further transformed into aliphatic molecules by carboxylesterases (CES) and subsequently incorporated into aromatic-cleavage or β-oxidation pathways. Subsequent oxidation products 2-ethyl-1-butano, 2-isopropyl-5-methylhexan-1-ol, or Decane can be sequentially converted into their corresponding carboxylic acids by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), enabling further entry into the TCA cycle. Although GC-MS did not detect the corresponding carboxylic acids, their absence may be attributed to low production levels and rapid metabolic turnover, preventing accumulation to detectable concentrations. ADH catalyzes the interconversion of alcohols, aldehydes, and ketones, providing essential precursors for the irreversible oxidation mediated by ALDH. The fold changes in ADH expression in the P-PS and UV/H₂O₂-PS groups are 2.60 and 2.07, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF)\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. ALDH further oxidizes a variety of aldehyde substrates to carboxylic acids, with fold changes of 1.04 and 1.33 in the two treatment groups (Table S5)\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEndocytosis is a major pathway for the internalization of plasma membrane proteins, lipids, and extracellular materials into the cytoplasm, playing a crucial role particularly in eukaryotic cells\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. Transcriptomic analysis (Table S6) revealed that in both the P-PS and Aged-PS groups, genes associated with endocytosis, such as actin-like ATPase domain-containing protein and endocytosis protein end4were upregulated. In addition, several genes related to vesicle-mediated transport of plastic particles (NCS cytosine-purine permease and vesicle transport protein) also showed increased expression. These findings suggest that plastic particles may enter the hyphae of \u003cem\u003eP. chrysosporium\u003c/em\u003e through endocytosis and are subsequently transported via intracellular vesicle trafficking\u003csup\u003e76\u003c/sup\u003e. However, the specific transmembrane processes involved remain unclear and will be further investigated in a separate study.\u003c/p\u003e \u003cp\u003eThroughout the degradation process, the introduction of oxygen-containing functional groups (such as carbonyl, hydroxyl, and ether bonds) plays a crucial role. These groups decrease the electron density of certain carbon atoms in the substrate, thereby increasing their electrophilicity and making them more susceptible to oxidation or cleavage reactions, which in turn facilitates polymer degradation\u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. During enzymatic reactions, the presence of these oxidized functional groups enhances the binding and electronic interactions between the polymer and the enzyme\u0026rsquo;s active site, thus accelerating the degradation process\u003csup\u003e78\u003c/sup\u003e. Taking the carbonyl as an example (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), the polarity of its covalent bond renders the carbon atom electrophilic and prone to nucleophilic attack. The enzyme, through the spatial conformation and electronic coordination of its active site, increases the sensitivity of the carbonyl carbon toward nucleophilic reagents (such as water molecules, cofactors, or enzyme residues), thereby inducing nucleophilic addition, breaking the π-bond of the C\u0026thinsp;=\u0026thinsp;O, and generating an oxyanion (O⁻)\u003csup\u003e79,80\u003c/sup\u003e. Through this mechanism, enzymatic reactions proceed more rapidly, although the precise triggering mechanism remains to be further elucidated.\u003c/p\u003e \u003cp\u003eTranscriptomic analysis revealed upregulation of multiple pathways related to fatty acid degradation and the TCA cycle (Tables S7 and S8), indicating that enhanced availability of PS aging products enables microorganisms to more efficiently channel oxidized fragments into central metabolism. Differentially expressed genes and their enriched KEGG pathways are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC. Upregulation of 4-hydroxy-2-oxoglutarate aldolase (EC 4.1.3.16) and (R)-2-hydroxyglutarate-pyruvate transhydrogenase (EC 1.1.99.40) suggests that oxo-organic acids generated from aromatic ring cleavage are converted into TCA cycle precursors via the glycolate or oxaloacetate pathways\u003csup\u003e81\u003c/sup\u003e. Increased expression of alcohol dehydrogenase (EC 1.1.1.1) indicates that alcohol and aldehyde intermediates formed during PS oxidation undergo further dehydrogenation and oxidation, entering deeper metabolic routes. Concurrently, activation of acylglycerol lipase (EC 3.1.1.23) implies that longer-chain oxygenated fragments are enzymatically cleaved into shorter fatty acids or ester units, which subsequently enter β-oxidation and ultimately feed into the TCA cycle\u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. Collectively, these transcriptional changes depict a coordinated network in which PS oxidation products are progressively transformed into metabolites readily incorporated into central carbon metabolism.\u003c/p\u003e \u003cp\u003eIn summary, this study, together with existing literature, demonstrates that surface oxidation is a critical prerequisite for plastic biodegradation\u003csup\u003e83\u003c/sup\u003e. Physical or chemical pretreatments, such as UV irradiation, chemical oxidation, or thermal treatment, introduce oxygen-containing functional groups, including carbonyl, carboxyl, and hydroxyl groups, onto the polymer surface, thereby reducing hydrophobicity, activating polymer chains, and enhancing the accessibility of microbes and enzymes\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Using PS as a model, we further showed that these oxygen-containing groups promote electron transfer, accelerate free radical formation and backbone cleavage, thereby facilitating enzymatic degradation and microbial metabolism. The resulting oxidized fragments can be efficiently channeled into aromatic ring cleavage, fatty acid β-oxidation, and the TCA cycle, enabling the systematic conversion of high-molecular-weight polymers into central metabolites. This mechanism, in which surface oxidation enhances enzymatic degradation and subsequent metabolic integration, appears to be broadly applicable across different polymer systems, indicating that surface oxidation is not only a hallmark of polymer degradation but also an essential step for microbial utilization of high-molecular-weight materials\u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study constructs a systematical biodegradation method of PS by UV/H₂O₂-Aging. The results indicate that individual UV or H₂O₂ pretreatment had limited impact on plastic degradation, whereas combined UV/H₂O₂ aging significantly enhanced the surface hydrophilicity and biocompatibility of PS films. This treatment also generated micro-pores and cracks on the film surface, providing favorable sites for microbial biofilm formation and enzymatic action. Molecular docking simulations employing the pretreatment product 4,5-dimethoxy-2-hydroxyacetophenone as substrate, despite their limitations, provide valuable predictive insights into enzyme-catalyzed reactions. Building upon this foundation, they offer significant guidance regarding the enzymatic degradation process of substances. The pretreatment promoted fungal hyphal attachment and growth, thereby enhancing microbial degradation of the plastic. In addition, the introduction of oxygen-containing functional groups strengthens the interactions between the polymer and microbial enzymes via promotion of electron transfer, resulting in an enhancement of degradation process. The degradation of PS occurs through three main pathways: cleavage of the aromatic ring to generate metabolic intermediates, microbial assimilation of carboxyl-containing compounds, and further oxidation of these intermediates by CYP450. Overall, UV/H₂O₂ synergistic aging represents an effective strategy to enhance plastic biodegradation. By inducing surface modification and facilitating enzymatic reactions, it can significantly improve the degradation efficiency of PS films, providing both theoretical and practical insights for environmentally friendly plastic disposal.\u003c/p\u003e \u003cp\u003eThe following is the Supplementary material related to this article Video S1.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eExperimental materials\u003c/h2\u003e \u003cp\u003eTo prepare PS films, 3 g of PS plastic raw material was dissolved in 100 ml of xylene. Prior to experiments, the prepared PS films were sequentially rinsed with sterile water and 75% ethanol to remove impurities, followed by air-drying under aseptic conditions. After drying, the plastic films were cut into 30 mm \u0026times; 30 mm pieces for subsequent use, serving as the original PS films\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. chrysosporium\u003c/em\u003e (BKMF-1767, CCTCC No. AF-96007) was obtained from the China Center for Type Culture Collection (Wuhan, China) and preserved at 4\u0026deg;C in potato agar medium. All experiments were conducted using 3rd-generation cultures. The transfer method and the composition of the culture medium are provided in the Supplementary Information Text S1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eUV/H₂O₂ pretreat of PS film\u003c/h2\u003e \u003cp\u003eThe pretreatment experimental setup is illustrated in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The specific procedures were as follows: Dried pristine PS films were immersed in 30% H₂O₂ solution and irradiated using a 365 nm LED ultraviolet lamp with a power of 20 W. The distance between the UV lamp and the liquid surface was maintained at 15 cm. During the reaction process, the PS film was turned over every 12 hours to ensure uniform irradiation. Due to the consumption of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, a certain volume of the reaction solution was extracted every 24 hours, filtered (0.22 \u0026micro;m Millipore filter), and detected the concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e by titanium (Ⅳ) oxalate on a spectrophotometer at λ\u003csub\u003emax\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;400 nm\u003csup\u003e85\u003c/sup\u003e. A certain amount of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was added in the suspension in order to maintain a stable oxidation rate of PS. To prevent temperature increase caused by ultraviolet irradiation and hydrogen peroxide reaction, the entire system was placed in a temperature-controlled chamber set at 25\u0026deg;C, with the equipment operating in a well-ventilated environment. After 7 days of pretreatment, the PS films were retrieved, soaked in deionized water for 2 hours with repeated rinsing, and finally dried in an oven at 40\u0026deg;C for 24 hours to obtain the pretreated PS films.\u003c/p\u003e \u003cp\u003eBased on the aforementioned procedures, different treatment groups were established: Pristine PS served as the untreated control, H₂O₂ reaction under dark conditions, UV irradiation alone, and synergistic aging with combined UV/H₂O₂ treatment. The resulting PS films were designated as Pristine-PS (P-PS), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-PS, UV-PS and UV/H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-PS, respectively.\u003c/p\u003e \u003cp\u003e \u003cb\u003eP. chrysosporium\u003c/b\u003e \u003cb\u003ebiofilm cultivation and biodegradation of PS film assays\u003c/b\u003e\u003c/p\u003e \u003cp\u003eLow-carbon potato dextrose agar (LC-PDA) medium was prepared using five primary components: potato extract solution, glucose, KH₂PO₄, MgSO₄, and agar powder. The medium was dissolved in 1000 mL of potato extract, which was obtained by slicing potatoes, boiling them in water, and filtering the solution through gauze (detailed procedures are provided in Text S2). After the medium cooled and solidified, a small amount of \u003cem\u003eP. chrysosporium\u003c/em\u003e was inoculated onto the plates using an inoculation loop. The cultures were incubated at 37\u0026deg;C in a constant-temperature incubator (SHP-250, SANFA, China) for 24 hours. Subsequently, the plates were removed, and PS films from different pretreatment groups were placed onto the surface of the white-rot fungal medium, ensuring direct contact between the fungal mycelia and the plastic films to facilitate co-cultivation. A control group with pure \u003cem\u003eP. chrysosporium\u003c/em\u003e culture (without plastic films) was simultaneously prepared.\u003c/p\u003e \u003cp\u003ePS films were collected on days 7, 14, 21, 28, and 35 of biodegradation. The degradation fragments were first immersed in 2% (w/v) sodium dodecyl sulfate (SDS) solution for 4 hours. Residual particles collected by vacuum pump filtration were resuspended in 20 mL of saturated saline solution, stirred for 15 min to ensure thorough mixing, and allowed to stand for 2 h. The supernatant mixture was then filtered through 0.25 \u0026micro;m vacuum-pumped glass fiber membrane, and microplastics with a particle size smaller than 0.25 \u0026micro;m were ignored. This collection process was repeated three times to minimize plastic loss. Finally, the plastic films were dried at 40\u0026deg;C for 24 h for mass determination.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlastic film degradation characterization\u003c/h3\u003e\n\u003cp\u003ePrior to analytical characterization, all plastic film samples underwent a cleaning protocol to remove surface impurities and residual microbial components. For post-pretreatment samples, immersion in deionized water for 2 h followed by repeated rinsing was performed. For post-biodegradation samples, the procedure involved: initial careful washing with distilled water, subsequent suspension in 25 mL distilled water containing 0.5 g SDS with agitation at 120 rpm for 2 h to eliminate biofilms, two additional washes with distilled water, and final recovery through vacuum filtration. Finally, all the samples were vacuum dried overnight in a vacuum drying oven.\u003c/p\u003e \u003cp\u003eSurface hydrophobicity changes of plastic films before and after pretreatment, as well as after 35-day biodegradation, were quantitatively analyzed using a water contact angle goniometer (WCA). Morphological characteristics of the plastic films were examined using a thermal field emission scanning electron microscope (SEM) (Zeiss Gemini 500). Samples were sputter-coated with gold and observed under the TFE-SEM at an accelerating voltage of 2 kV\u003csup\u003e86\u003c/sup\u003e. Changes in surface chemical functional groups were detected by Fourier Transform Infrared Spectroscopy (FTIR) (Thermo Fisher IS50, USA).\u003c/p\u003e \u003cp\u003eThe changes in the weight average molecular weight (Mw) and number average molecular weight (Mn) of the PS plastics were determined via high-temperature gel permeation chromatography (GPC) (Agilent, PL220, USA). The PS plastic film was dissolved in tetrahydrofuran (High Performance Liquid Chromatography, Sigma), filtered through a 0.22 \u0026micro;m organic-phase microporous filter membrane, and placed in a 1 mL liquid-phase vial for measurement.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.5. Physiological state and enzyme activity of\u003c/b\u003e \u003cb\u003eP. chrysosporium\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFungal colonization levels were evaluated by measuring the dry cell weight on plastic films, while cellular activity during growth was assessed to monitor physiological status. The activities of extracellular enzymes MnP, LiP and Lac were quantitatively determined\u003csup\u003e87,\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e. Detailed methodologies are provided in Text S2.\u003c/p\u003e\n\u003ch3\u003eMolecular docking simulations and analysis of results\u003c/h3\u003e\n\u003cp\u003eThe molecular structure of 4,5-Dimethoxy-2-hydroxyacetophenone was obtained using GaussView 5.0 software, and the 3D structures of MnP, LiP, and Lac were downloaded from the Protein Data Bank (PDB; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with protein codes 1LLP, 3M5Q, and 2HRG, respectively. MnP, LiP, and Lac are derived from white-rot fungi. Molecular docking was performed using AutoDock Vina to predict the optimal binding modes of 4,5-Dimethoxy-2-hydroxyacetophenone with LiP, MnP, and Lac. The docking score reflects the binding strength, with higher absolute values indicating stronger binding affinity\u003csup\u003e\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e. Finally, the docking results were visualized using PyMOL for further analysis of the binding modes and affinities.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome and metabolome analysis\u003c/h2\u003e \u003cp\u003eAfter \u003cem\u003eP. chrysosporium\u003c/em\u003e was cultured with plastic for 5 days, total RNA was isolated using the RNA Kit (15NT) (Agilent, DNF-471-1000), and a library was constructed by Personal Biotech Cp (Shanghai, China). Detailed experimental and analytical procedures are provided in Text S3. Characterization of degradation products by gas chromatography-mass spectrometry (GC-MS)\u003csup\u003e90\u003c/sup\u003e. Detailed methodologies are provided in Text S4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis work was funded by\u0026nbsp;the\u0026nbsp;National Natural Science Foundation of China\u0026nbsp;(Grant NO.\u0026nbsp;42576166,\u0026nbsp;51809068\u0026nbsp;and\u0026nbsp;42107384);\u0026nbsp;The Anhui Provincial Natural Science Foundation 2308085MD119.\u003c/p\u003e\n\u003cp\u003eAuthor information\u003c/p\u003e\n\u003cp\u003eThese authors contributed equally: Zhi Guo, Yuanyuan Zha, Xingpan Guo.\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSchool of Resources and Environmental Engineering, Hefei University of Technology, Hefei, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhi Guo, Yuanyuan Zha, Xinlei Ling, Lin Yao, Lishou Han, Fan Yang\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eState Key Laboratory of Estuarine and Coastal Research, East China Normal University, 500 Dongchuan Road, Shanghai, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXingpan Guo\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnhui Ecological Civilization Research Institute, Hefei University of Technology, Hefei, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhi Guo, Yuanyuan Zha, Xinlei Ling, Lin Yao, Lishou Han, Fan Yang\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eContributions\u003c/p\u003e\n\u003cp\u003eZ.G. and X.P. supervised and conceived the project. Y.Z. carried out the experiments, conducted the characterization, analyzed the data and wrote the paper. X.L.\u0026nbsp;carried out molecular docking simulations. L.Y., L.H. and F.Y. reviewed, and revised the manuscript.\u003c/p\u003e\n\u003cp\u003eCorresponding authors\u003c/p\u003e\n\u003cp\u003eCorrespondence to\u0026nbsp;Zhi Guo Xingpan Guo\u003c/p\u003e\n\u003cp\u003eEthics declarations\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZimmermann, W. Polyester-degrading enzymes in a circular economy of plastics. \u003cem\u003eNat\u003c/em\u003e\u003cem\u003e. Rev\u003c/em\u003e\u003cem\u003e. Bioeng\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 681\u0026ndash;696 (2025).\u003c/li\u003e\n\u003cli\u003eXu, Y. et al. 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Technol.\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 4196\u0026ndash;4199 (2006).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8364324/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8364324/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlastic pollution is a global environmental challenge, yet plastic biodegradation remains inefficient and poorly understood. In this study, a degradation strategy for polystyrene (PS) films was developed by combining ultraviolet (UV) irradiation and hydrogen peroxide (H₂O₂) pretreatment with subsequent biodegradation by \u003cem\u003ePhanerochaete chrysosporium\u003c/em\u003e. UV/H₂O₂ pretreatment proved optimal, resulting in a mass loss of up to 25.75% and inducing the formation of oxygen-containing functional groups, including carbonyl, hydroxyl, and carboxyl groups. These groups act as electron donors, facilitating extracellular enzymatic chain-cleavage reactions, while also promoting fungal colonization and enhancing the activities of manganese peroxidase, lignin peroxidase, and laccase. The pretreated PS was degraded through three main pathways prior to entering the tricarboxylic acid cycle: direct assimilation of carboxylated compounds, enzymatic aromatic ring cleavage, and cytochrome P450-mediated oxidation. Overall, UV/H₂O₂ pretreatment significantly improves surface oxidation, microbial activity, and enzymatic reactivity, offering an effective strategy to accelerate plastic biodegradation.\u003c/p\u003e","manuscriptTitle":"Mechanistic Insights into UV/H₂O₂-Aged Polystyrene Enable an Optimized Protocol for Fungal Biodegradation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-03 07:55:33","doi":"10.21203/rs.3.rs-8364324/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"75823749-6d42-44a3-a929-65543074204d","owner":[],"postedDate":"April 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":65618705,"name":"Earth and environmental sciences/Environmental sciences"},{"id":65618706,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-03T07:55:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-03 07:55:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8364324","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8364324","identity":"rs-8364324","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}