Chitosan Nanoparticles as Interfacial Adjuvants to Enhance the Biodegradation of Polyethylene Terephthalate by Ideonella sakaiensis

Chitosan Nanoparticles as Interfacial Adjuvants to Enhance the Biodegradation of Polyethylene Terephthalate by Ideonella sakaiensis | 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 Research Article Chitosan Nanoparticles as Interfacial Adjuvants to Enhance the Biodegradation of Polyethylene Terephthalate by Ideonella sakaiensis Zhichao Shi, Tingbiao Wu, Lihong Wu, Xiaomin Chi, Yingjuan Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9055686/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 The escalating global crisis of polyethylene terephthalate (PET) pollution necessitates the development of efficient biological recycling strategies. While the bacterium Ideonella sakaiensis can utilize PET as a sole carbon source, its practical application as a biocatalyst is hampered by poor interfacial adhesion to hydrophobic plastic surfaces and limited degradation efficiency under ambient conditions. In this study, we developed chitosan nanoparticles (CS-NPs) via ionic crosslinking to serve as a novel electrostatic bridging adjuvant. The synthesized CS-NPs exhibited a uniform particle size of 360.1 ± 10.8 nm and a high positive zeta potential of + 40.1 ± 2.2 mV. Supplementation with an optimal dosage of CS-NPs (3 mL per 30 mL medium) significantly promoted the adhesion of Ideonella sakaiensis to diverse PET substrates. Under optimized environmental conditions (28°C, pH 7.0), CS-NPs were introduced as adhesion adjuvants, achieving a 91.2% degradation efficiency of PET fluorescent microspheres over 14 days compared to 58.9% without CS-NPs. Furthermore, application of the CS-NPs adjuvant to macroscopic PET films resulted in dense surface colonization and severe structural disruption, characterized by deep physical pitting and cracking. High-performance liquid chromatography-mass spectrometry (LC-MS) confirmed the robust hydrolytic depolymerization of PET into its constituent monomers: terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET), and bis(2-hydroxyethyl) terephthalate (BHET). While challenges remain regarding degradation of CS-NPs environmental fate and microbial competition, this work provides the CS-NPs adjuvant as promising tools for the bioremediation of PET pollution. Polyethylene terephthalate (PET) Biodegradation Ideonella sakaiensis Chitosan nanoparticles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key points • The development of an interfacial adjuvant chitosan nanoparticles. • Addition of chitosan nanoparticles enhanced the degradation efficiency of PET. • Chitosan nanoparticles achieve accelerated degradation through electrostatic attraction. Introduction Polyethylene terephthalate (PET) is one of the most widely applied synthetic polymers globally, utilized extensively in packaging, textiles, electronics, and automotive industries due to its exceptional mechanical durability, chemical resistance, and processability (Ji 2013 ; Nisticò 2020 ). However, the inherent chemical stability of PET renders it highly recalcitrant to natural degradation, with environmental half-lives spanning decades (Dhaka et al. 2022 ; Muringayil Joseph et al. 2024 ). The continuous accumulation of discarded PET and its subsequent fragmentation into microplastics renders their removal exceedingly difficult, posing severe, long-term threats to marine ecosystems, food chains, and human health (Dhaka et al. 2022 ; Webb et al. 2012 ). Current remediation strategies primarily rely on mechanical recycling and chemical degradation; the former is limited by low recycling efficiency and downcycling, while the latter requires harsh conditions (high temperature and pressure) that often cause secondary pollution (Abedsoltan 2023 ; Al-Sabagh et al. 2016 ; Suhaimi et al. 2022 ). Consequently, the development of mild, efficient, and sustainable biodegradation technologies has emerged as a central research focus (Koshti et al. 2018 ; Kushwaha et al. 2023 ). Ideonella sakaiensis 201-F6, a bacterium uniquely capable of utilizing PET as its sole carbon and energy source, is recognized as a model organism for plastic biodegradation (Yoshida et al. 2016 ). The bacterium secretes two specific hydrolases, PETase and MHETase, which synergistically hydrolyze PET into biologically assimilable monomers: terephthalic acid (TPA) and ethylene glycol (EG) (Yoshida et al. 2016 ). Despite this breakthrough, the deployment of native Ideonella sakaiensis for industrial-scale bioremediation remains constrained by critical bottlenecks. Specifically, the strain exhibits weak initial adhesion and colonization on highly hydrophobic PET surfaces, and its degradation kinetics are severely restricted by environmental factors and substrate morphologies (Palm et al. 2019 ; Wallace et al. 2020 ; Yoshida et al. 2021 ). Previous studies suggest that microbial adhesion to the plastic surface is the critical first step in biodegradation (Sun et al. 2020 ). Enhancing this interfacial interaction using biocompatible adjuvants represents a promising strategy to amplify biocatalytic turnover (Yang et al. 2025 ). Chitosan (CS), a naturally occurring and biodegradable polysaccharide, can be formulated into highly stable chitosan nanoparticles (CS-NPs) via ionic crosslinking (Des Bouillons-Gamboa et al. 2024 ; Sun et al. 2021 ). CS-NPs possess excellent biocompatibility, dispersibility, and strong electrostatic adsorptive capabilities (Jiang et al. 2024 ; Ullah et al. 2025 ). Due to their high density of positive surface charges, CS-NPs can theoretically function as a biomimetic electrostatic bridge, simultaneously binding to negatively charged microbial cell envelopes and the hydrophobic PET surface (Benettayeb et al. 2023 ; Poznanski et al. 2023 ). This dual function acting both as a microbial carrier and a plastic surface modifier has demonstrated potential in broader environmental remediation contexts, yet its specific application as an adjuvant for Ideonella sakaiensis -mediated PET degradation remains unexplored. Inspired by our recent research on the use of CS-NPs as an adhesive adjuvant to further enhance the degradation of PU substrates by Cladosporium oxysporum SCSIO 81042 (Zeng et al. 2026 ), we proposed whether CS-NPs could also enhance the biodegradation of microplastics by bacteria. In this study, we synthesized CS-NPs via an ionic gelation method and systematically evaluated their ability to promote PET depolymerization by Ideonella sakaiensis . Utilizing diverse substrates including PET fluorescent microspheres, amorphous PET films, and nano-PET powders, we determined the optimal adjuvant dosage of CS-NPs and environmental conditions for maximum hydrolytic efficiency. Furthermore, we elucidated the underlying mechanism of CS-NPs enhanced biodegradation through optical microscopy and LC-MS. This work provided a promising interfacial adjuvant CS-NPs to enhance the biodegradation of polyethylene terephthalate by Ideonella sakaiensis for the future bioremediation of microplastic pollution. Materials and Methods Materials Nano-scale PET powder was sourced from Xinmiao New Materials Co., Ltd. (Dongguan, China), and amorphous PET films were purchased from Goodfellow GmbH (Shanghai, China). Green PET fluorescent nanoparticles were obtained from Beijing Zhongke Keyou Nano Technology Co., Ltd. (Beijing, China). All other reagents, unless specified otherwise, were purchased from Sigma-Aldrich (Missouri, USA). R2A medium was obtained from HuanKai Microbial. Chitosan (CS, Mw = 100 kDa) and standard reference materials for TPA and bis(2-hydroxyethyl) terephthalate (BHET) were purchased from Meilun Biotechnology Co., Ltd. (Dalian, China). Preparation and Characterization of CS-NPs CS-NPs were prepared utilizing an ionic gelation method. Briefly, 0.2 g of CS (Mw = 100 kDa) was dissolved in 100 mL of deionized water containing 1% (v/v) glacial acetic acid under continuous magnetic stirring for 12 hours until complete dissolution (2 mg/mL CS solution). The pH of the solution was adjusted to 4.5–5.5. Subsequently, a crosslinking solution containing 8.4 mg/mL of sodium tripolyphosphate (TPP) was added dropwise into the CS solution at a TPP-to-CS mass ratio of 1:5. The mixture was stirred for 30 minutes to facilitate robust ionic crosslinking (Des Bouillons-Gamboa et al. 2024 ). The synthesized CS-NPs suspension was characterized using a Zetasizer Nano ZSE (Malvern, UK) to determine the hydrodynamic size distribution, polydispersity index (PDI), and zeta potential. Morphological analysis was conducted via cryo-transmission electron microscopy (Cryo-TEM, Thermo Fisher Scientific Tundra, USA) at an acceleration voltage of 100 kV. Degradation Assays using PET Fluorescent Microspheres Standard R2A medium was sterilized and aliquoted into 100 mL Erlenmeyer flasks (30 mL per flask) (Charnock 2021 ). Single colonies of Ideonella sakaiensis were pre-cultured in R2A medium to an optical density (OD₆₀₀) of 0.5. Green PET fluorescent microspheres (1 µm diameter, 10 mg/L, ex/em 488/518 nm) were used as visual substrate probes. To evaluate the adjuvant effect, 30 µL of the microsphere stock was added to 30 mL of R2A medium. The experimental groups included: blank control, bacteria only, PET microspheres only, bacteria + PET microspheres, and the CS-NPs-adjuvanted group (3 mL CS-NPs [2 mg/mL] + bacteria + PET microspheres). To assess environmental conditions, temperature (28°C and 37°C) and pH (6.0, 7.0, and 8.0) gradients were tested (cultured in combined illumination and shaking incubator, ZQZY-BGS8, Shanghai Zhichu Instrument Co., Ltd, China). Samples were continuously agitated in an orbital shaker. Degradation was quantified daily by measuring the fluorescence intensity (488/518 nm) of 0.5 mL aliquots, supplemented by UV 365 nm excitation imaging. All experiments were conducted in triplicate. Biodegradation of Macroscopic PET Films Pre-cultured Ideonella sakaiensis (OD₆₀₀ = 0.5) was inoculated into 60 mL of R2A medium, centrifuged, and resuspended in 60 mL of Minimal Salt Medium (MSM). Amorphous PET films (1 cm × 1 cm) were sterilized using 75% ethanol and 30 minutes of UV irradiation, then introduced into 50 mL of sterile R2A medium. The experimental matrix comprised 4 groups (PET film, CS-NPs + PET film, Ide + PET film, Ide + CS-NPs + PET film) to accurately isolate the adjuvant effect. The CS-NP solution (1.82 mg/mL) was added at a volume of 3 mL per reaction. Incubations were maintained at 28°C and 180–200 rpm for 14 to 21 days. Post-incubation, the films were meticulously washed with ultrapure water and subjected to sonication to remove residual biofilm before scanning electron microscopy (SEM, Gemini 1 360, Germany) structural analysis. All experiments were conducted in triplicate. Depolymerization of PET Powders and Metabolite Identification Assays utilizing nano-PET powders and CS-NPs were conducted in 30 mL of R2A medium inoculated with pre-cultured Ideonella sakaiensis . UV-sterilized PET powder (100 mg) was introduced alongside 3 mL of CS-NPs adjuvant (1.82 mg/mL). The flasks were incubated at 28°C and 200 rpm for 14 days. Following the reaction, the total matrix was collected, washed, and centrifuged at 8000 × g for 10 minutes. The supernatant was filtered through a 0.22 µm membrane. Metabolic intermediates derived from PET were analyzed using a Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, San Jose, USA) coupled with a Vanquish ultrahigh-performance liquid chromatography system. The liquid chromatography system did not serve a separation function, it served to introduce the sample into the mass spectrometer. The mobile phase consisted of 0.1% formic acid in water (A) and methanol (B), delivered at a ratio of 20% A to 80% B for 2 minutes. The flow rate was maintained at 0.2 mL/min, with an injection volume of 2 µL. All experiments were conducted in triplicate. All results from independent experiments were statistically analyzed using one-way analysis of variance (ANOVA): *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control. And data were expressed as mean ± SD. Results and Discussion Preparation and Characterization of the CS-NPs Adjuvant Chitosan nanoparticles were successfully engineered via ionic gelation, relying on the robust crosslinking between the polycationic CS chains and multivalent anionic TPP at a pH of 5.0 (Des Bouillons-Gamboa et al. 2024 ). The reaction yielded a highly stable, milky-white nano-suspension within 30 minutes. Cryo-TEM analysis revealed that the CS-NPs possessed a uniform, network-like morphology with smooth surfaces and a distinct lack of visible aggregation ( Fig. 1 ) . Dynamic Light Scattering (DLS) confirmed a unimodal hydrodynamic diameter of 360.1 ± 10.8 nm alongside a low PDI of 0.370 ± 0.01, indicative of a highly homogenous particle distribution. Crucially, the surface zeta potential was recorded at + 40.1 ± 2.2 mV. This strong positive charge density ensures robust electrostatic stability in aqueous environments, establishing the physical prerequisite for the nanoparticles to act as effective bridging agents between bacterial cells and plastic substrates. Adjuvant-Enhanced Degradation of PET Fluorescent Microspheres by Ideonella sakaiensis PET fluorescent microspheres were employed as a visual substrate probe to precisely evaluate the degradation efficiency of Ideonella sakaiensis and CS-NPs. Due to their intrinsic fluorescence, the gradual depolymerization of these PET substrates can be directly quantified. Scanning electron microscopy (SEM) analysis confirmed the regular spherical morphology of the microspheres ( Fig. 2 A ) . Further physicochemical characterization revealed an average particle size of 1044.3 nm, a polydispersity index (PDI) of 0.11 ± 0.01 (indicating a highly uniform size distribution), and a mildly negative surface zeta potential of -0.645 mV ( Table S1 ). To establish the baseline degradation performance of Ideonella sakaiensis , depolymerization kinetics were evaluated in a 50% R2A medium across various temperatures (28°C and 37°C) and pH values (6.0, 7.0, and 8.0) ( Fig. S1 ). The results revealed that the optimal environmental parameters for PET microsphere degradation were 28°C and pH 7.0 ( Fig. 3 ) . Furthermore, the degradation efficiency was found to be highly dependent on substrate concentration; as the concentration of the PET microspheres increased, the degradation rate exhibited a corresponding decrease. Under optimized baseline conditions (28°C, pH 7.0, and low substrate concentration), native Ideonella sakaiensis achieved a degradation efficiency of approximately 58.9% over a 14-day incubation period, demonstrating robust intrinsic degradation capacity. Building upon these optimal conditions, the promotional effect of varying volumes (3, 6, and 9 mL) of the chitosan nanoparticle (CS-NPs) adjuvant on PET depolymerization was systematically investigated ( Fig. 2 B-D ) . Using an adjuvant-free system (" Ide + PET fluorescent microspheres") as the control, the degradation rates across all experimental groups continuously increased as the culture time extended. Notably, the experimental group supplemented with 3 mL of CS-NPs demonstrated the most pronounced synergistic effect. After 14 days of co-incubation, the PET degradation rate in the 3 mL CS-NP group was significantly higher than that of both the untreated control and the higher-dosage CS-NPs groups. The attenuated promotional effect observed in the 6 mL and 9 mL dosage groups indicates a critical concentration threshold, suggesting that excessive adjuvant volumes may lead to nanoparticle agglomeration, thereby hindering optimal interfacial contact between the bacteria and the plastic substrate. Structural Disruption of Macroscopic PET Films To validate the system's efficacy against standard commercial plastics, the degradation of amorphous PET films by Ideonella sakaiensis and CS-NPs was examined. Following 14 days of incubation, robust mycelial networks and dense bacterial colonization were visibly apparent on the adjuvant-treated films. Subsequent SEM analysis (conducted after rigorous biofilm removal) confirmed severe structural degradation. Unlike the smooth topology of the untreated controls, the PET films treated with the combined Ideonella /CS-NPs system exhibited pronounced physical pitting, extensive surface erosion, and deep micro-cracking ( Fig. 4 ) . These morphological disruptions confirm that the CS-NPs adjuvant facilitates robust cellular adhesion, allowing the biocatalyst to physically penetrate and aggressively depolymerize the amorphous PET films. Identification of Degradation Metabolites and Elucidation of the Depolymerization Pathway In order to verify whether CS-NPs would have any effect on the degradation of PET monomers by Ideonella sakaiensis , mass spectrometry (MS) analysis was conducted on the reaction supernatant to identify the intermediate metabolites of PET degradation. As illustrated in Fig. 5 A-C, three distinct metabolites were detected in the experimental group, all of which were entirely absent in the uninoculated control. Metabolites A (m/z 165.01853, [M–H]⁻), B (m/z 209.04514, [M–H]⁻), and C (m/z 255.08593, [M + H]⁺) were definitively identified as terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET), and bis(2-hydroxyethyl) terephthalate (BHET), respectively. This is consistent with the results reported in previous literature regarding the degradation of PET by Ideonella sakaiensis (Yoshida et al. 2016 ), indicating that CS-NPs not only enhances the efficiency but also does not interfere with the degradation process of PET. To confirm the origin of the detected monomers, we performed LC-MS analysis on control groups. No characteristic peaks of terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET), or bis(2-hydroxyethyl) terephthalate (BHET) were observed in the PET-only control ( Fig. S2 ) or the bacteria-only control ( Fig. S3 ), verifying that these monomers were exclusively generated from the enzymatic hydrolysis of PET, rather than from spontaneous degradation or bacterial metabolism. The accumulation of these specific products confirms the successful hydrolytic cleavage of the ester bonds within the PET polymer chains. These metabolic products align perfectly with the established two-step synergistic depolymerization mechanism of Ideonella sakaiensis . The biodegradation pathway initiates with the robust adhesion of the bacterium to the plastic surface, followed by the extracellular secretion of the primary hydrolase, PETase. This enzyme specifically recognizes and binds to the ester linkages within the PET macromolecules. Utilizing its highly conserved catalytic triad (Ser160-His237-Asp206) at the active site, PETase executes a nucleophilic attack on the ester bonds, efficiently cleaving the long-chain PET polymer into the primary intermediate, MHET (Burgin et al. 2024 ; Magalhães et al. 2022 ; Yoshida et al. 2021 ). Mechanism of Interfacial Bridging and Enhanced Adhesion To determine whether CS-NPs improve degradation efficiency by promoting the adhesion of Ideonella sakaiensis to the plastic surface, an assay was conducted using PET fluorescent microspheres as the sole carbon source in R2A medium over a 21-day incubation period. Optical microscopy was utilized to elucidate the physical mechanism driving the enhanced degradation (Fig. 6 ). After 7 days of co-incubation, TEM imaging was used to visualize the interfacial interaction ( Fig. S4 ). In the presence of CS-NPs, rod-shaped Ideonella sakaiensis cells were tightly adsorbed onto the surface of PET microspheres, whereas no such adhesion was observed in the absence of bacteria. This indicates that CS-NPs act as an interfacial mediator to enhance bacterial adhesion. Meanwhile, the CS-NPs-treated group displayed a massively increased accumulation of PET microspheres physically tethered directly to the bacterial cell surfaces, in stark contrast to the adjuvant-free control group ( Fig. 6 D and E) . The underlying mechanism for this observed synergy is governed by electrostatic attraction, Zeta potential measurements further elucidated the electrostatic basis of this mediation ( Table. S1 ). CS-NPs exhibited a strong positive charge (+ 40.1 ± 2.20 mV), while both Ideonella sakaiensis (-10.1 ± 1.19 mV) and PET microspheres (-0.645 ± 0.01 mV) were negatively charged, providing a driving force for the electrostatic attraction (Zeng et al. 2026 ). By forming electrostatic attraction, the adjuvant physically tethers the biocatalyst to the interface. These results demonstrate that CS-NPs enhance the degradation of PET microplastics by promoting robust adhesion between Ideonella sakaiensis and the substrate, effectively overcoming the thermodynamic barriers of hydrophobic exclusion. Conclusion This investigation successfully established chitosan nanoparticles as a potent, biomimetic interfacial adjuvant capable of enhancing the biodegradation of PET plastics. Synthesized via mild ionic gelation, the strongly cationic CS-NPs (360.1 nm, + 40.1 mV) acted as an electrostatic bridge, facilitating robust adhesion between Ideonella sakaiensis and highly recalcitrant PET substrates. At an optimal dosage (3 mL per 30 mL reaction), the CS-NPs maximized the depolymerization of fluorescent PET microspheres (91.2% degradation at 28°C, pH 7.0 compared to 58.9% without CS-NPs) and induced profound structural pitting in macroscopic PET films. The accumulation of terminal TPA and MHET monomers confirmed authentic hydrolytic cleavage. While high concentrations of the adjuvant induced physical cellular agglomeration, the optimized system successfully achieved the goal of enhancing efficacy. Although our research has demonstrated the enhancement of CS-NPs on the degradation of microplastics by bacteria, the extent of this enhancement is still rather limited. Therefore, future studies should explore the optimization of CS-NPs physicochemical properties including precise size tuning and targeted surface modifications to further maximize their interfacial tethering capacity. Abbreviations PET Polyethylene terephthalate SEM Scanning electron microscopy CS-NPs Chitosan nanoparticles TEM Transmission electron microscopy CS Chitosan Cryo-TEM Cryo-transmission electron microscopy TPP Sodium tripolyphosphate DLS Dynamic light scattering I. sakaiensis Ideonella sakaiensis PDI Polydispersity index Ide Ideonella sakaiensis R2A R2A medium TPA Terephthalic acid MSM Minimal salt medium MHET Mono(2-hydroxyethyl) terephthalate SD Standard deviation BHET Bis(2-hydroxyethyl) terephthalate ANOVA One-way analysis of variance PETase PET hydrolase LC-MS Liquid chromatography-mass spectrometry MHETase MHET hydrolase Declarations Supplementary Information The online version contains supplementary material available at https://doi.org/xxxxx. Acknowledgements We thank Prof. Qinglian Li from South China Sea Institute of Oceanology, Chinese Academy of Sciences for providing the bacterial strains and technical assistance in the cultivation of the strains. We thank Mr. Kai Yang and Ms. Shan Liu from Bioimaging Platform at Shenzhen Bay Laboratory for their technical assistance and valuable advice in electron microscopy. Author contribution Z.S.: Conceptualization, Data curation, Methodology, Investigation, Formal analysis, Writing – review & editing. T.W.: Methodology, Investigation, Formal analysis, Visualization, Writing – original draft. L.W.: Methodology, Investigation, Formal analysis, Visualization. X.C.: Formal analysis, Investigation. Y.W.: Formal analysis, Investigation. T.F.: Funding acquisition, Writing – review & editing, Supervision. Y.J.: Project administration, Resources, Supervision. Funding This work was supported by the National Key Research and Development Program (2023YFC3903300). Data availability The original contributions presented in this study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author(s). 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Science 351(6278):1196–1199. 10.1126/science.aad6359 Yoshida S, Hiraga K, Taniguchi I, Oda K (2021) Ideonella sakaiensis, PETase, and MHETase: From identification of microbial PET degradation to enzyme characterization. Methods Enzymol 648:187–205. 10.1016/bs.mie.2020.12.007 Zeng Q, Shi Z, Deng S, Wu T, Wu L, Guo Q, Yin J, Fan T, Tian X, Li Q (2026) Biodegradation of polyurethane by marine-derived Cladosporium oxysporum SCSIO 81042 under seawater conditions and its enhancement by chitosan nanoparticles as adjuvant. Environ Res 296:123981. 10.1016/j.envres.2026.123981 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials.docx 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. 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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-9055686","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":605759172,"identity":"67cd779d-4084-4b3c-8dcb-35942c3461e2","order_by":0,"name":"Zhichao Shi","email":"","orcid":"","institution":"Shenzhen Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Zhichao","middleName":"","lastName":"Shi","suffix":""},{"id":605759175,"identity":"dbed3f78-027f-4399-8879-d64cabc84d85","order_by":1,"name":"Tingbiao Wu","email":"","orcid":"","institution":"Shenzhen Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Tingbiao","middleName":"","lastName":"Wu","suffix":""},{"id":605759177,"identity":"6143abf4-1c00-441c-9ff7-dafed2165f52","order_by":2,"name":"Lihong Wu","email":"","orcid":"","institution":"Shenzhen Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Lihong","middleName":"","lastName":"Wu","suffix":""},{"id":605759179,"identity":"6b3a83bd-a9d7-4869-8392-bfa05b33273b","order_by":3,"name":"Xiaomin Chi","email":"","orcid":"","institution":"National Innovation Center for Molecular Drug, GrandBio Molecular Drug Innovations Center Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Xiaomin","middleName":"","lastName":"Chi","suffix":""},{"id":605759182,"identity":"3dd07660-df70-490f-9563-3e142f1c3565","order_by":4,"name":"Yingjuan Wang","email":"","orcid":"","institution":"National Innovation Center for Molecular Drug, GrandBio Molecular Drug Innovations Center Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Yingjuan","middleName":"","lastName":"Wang","suffix":""},{"id":605759187,"identity":"8567ac6d-d12e-40b7-8adf-dd09651081a6","order_by":5,"name":"Tingting Fan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYBAC9gYQWcHAYMBOrBaeAwyMDQfOALUwk6TlYBtJWtjPHn/8cV6dvTkzA+OHHwx2eYS18OQlNhzcdpjZspmBWbKHIbmYoBZ7hhxDoJYDbAaHGRikGRgOJDYQtIX/DVDLnDoeoBbm38RpkQDZ0sAsAdTCRqQtEm8MZ5w5dtjAspmxzbLHIJkYh+UYfKioAYYYe/PhGz8q7AhrQQKMQMUGJKgfBaNgFIyCUYAbAADC3Dgm0jknJwAAAABJRU5ErkJggg==","orcid":"","institution":"Shenzhen Bay Laboratory","correspondingAuthor":true,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Fan","suffix":""},{"id":605759189,"identity":"dfe8130c-9b8d-4bbe-87ab-932b624e5ff7","order_by":6,"name":"Yuyang Jiang","email":"","orcid":"","institution":"Shenzhen Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Yuyang","middleName":"","lastName":"Jiang","suffix":""}],"badges":[],"createdAt":"2026-03-07 05:53:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9055686/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9055686/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104876772,"identity":"2c4677c8-c17b-4fd4-86de-c7106348cbd8","added_by":"auto","created_at":"2026-03-18 08:43:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":262995,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePreparation and characterization of CS-NPs.\u003c/strong\u003e Schematic diagram of CS-NPs synthesis. Morphological features of CS-NPs observed using TEM. Scale bar: 100 nm.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9055686/v1/ba88c389c2f70d070cd8339e.png"},{"id":104876680,"identity":"7e86e89e-45f1-4823-b8f1-91ab06e37891","added_by":"auto","created_at":"2026-03-18 08:43:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":243751,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDegradation of PET fluorescent microspheres by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIdeonella sakaiensis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (A) The size of PET fluorescent microspheres; (B-D) Degradation of PET fluorescent microspheres with different concentrations CS-NPs by \u003cem\u003eIdeonella sakaiensis.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9055686/v1/1d92da3269883b44473855b9.png"},{"id":104876527,"identity":"6f423f2b-88b1-4bb3-9b85-f44a47ebc142","added_by":"auto","created_at":"2026-03-18 08:42:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":259876,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of temperature, pH, and substrate concentration on the degradation efficiency of PET fluorescent microspheres by \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9055686/v1/e30e28d5a756cfaa2323fb38.png"},{"id":104876554,"identity":"2250a4bd-490e-41ec-9d57-642ae79fc855","added_by":"auto","created_at":"2026-03-18 08:42:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":177691,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDegradation of PET film by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIdeonella sakaiensis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and CS-NPs\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003e SEM analysis of the surface of PET film, Scale bar: 100 nm.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9055686/v1/1c727bf502dd3cc295908e51.png"},{"id":104876628,"identity":"ed02f658-c109-4468-a695-f3408f750032","added_by":"auto","created_at":"2026-03-18 08:43:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":140664,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolite analysis (A-C) and proposed depolymerization pathway (D) of PET with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIdeonella sakaiensis \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand CS-NPs. \u003c/strong\u003eThe measured mass-to-charge ratios of all molecular ions exhibited deviations within 5 ppm from the theoretical values of the proposed metabolites, which is within the acceptable analytical error range.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9055686/v1/c748bdccab0b9a1673437445.png"},{"id":104876627,"identity":"160db151-1300-455b-8705-8da8f2eca7d0","added_by":"auto","created_at":"2026-03-18 08:43:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4413315,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe CS-NPs adjuvant enhances the adhesion to and degradation of PET fluorescent microspheres by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIdeonella sakaiensis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e(A-D) Optical microscopy images of \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e, CS-NPs, and PET fluorescent microspheres. (E) Adsorption efficacy during the co-culture of \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e with PET fluorescent microspheres and CS-NPs using TEM. Scale bar: 1 μm. (F) Predicted mechanistic model of the CS-NP adjuvant promoting the adhesion and degradation of PET fluorescent microspheres by \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9055686/v1/79e3d24b673b6bc1957ad3dd.png"},{"id":104876897,"identity":"0d4450b6-71d5-48c1-a6d7-a488633b83ae","added_by":"auto","created_at":"2026-03-18 08:44:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6386915,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9055686/v1/f35d993a-ab48-429e-bb5c-3bd5de6eaa79.pdf"},{"id":104876502,"identity":"77c954de-c288-472b-a9c4-316d95dad14e","added_by":"auto","created_at":"2026-03-18 08:42:41","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":448155,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-9055686/v1/15a425554a3a8e5d4c303dc2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chitosan Nanoparticles as Interfacial Adjuvants to Enhance the Biodegradation of Polyethylene Terephthalate by Ideonella sakaiensis","fulltext":[{"header":"Key points","content":"\u003cp\u003e\u0026bull; The development of an interfacial adjuvant chitosan nanoparticles.\u003c/p\u003e\u003cp\u003e\u0026bull; Addition of chitosan nanoparticles enhanced the degradation efficiency of PET.\u003c/p\u003e\u003cp\u003e\u0026bull; Chitosan nanoparticles achieve accelerated degradation through electrostatic attraction.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003ePolyethylene terephthalate (PET) is one of the most widely applied synthetic polymers globally, utilized extensively in packaging, textiles, electronics, and automotive industries due to its exceptional mechanical durability, chemical resistance, and processability (Ji \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e; Nisticò \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, the inherent chemical stability of PET renders it highly recalcitrant to natural degradation, with environmental half-lives spanning decades (Dhaka et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Muringayil Joseph et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). The continuous accumulation of discarded PET and its subsequent fragmentation into microplastics renders their removal exceedingly difficult, posing severe, long-term threats to marine ecosystems, food chains, and human health (Dhaka et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Webb et al. \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). Current remediation strategies primarily rely on mechanical recycling and chemical degradation; the former is limited by low recycling efficiency and downcycling, while the latter requires harsh conditions (high temperature and pressure) that often cause secondary pollution (Abedsoltan \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e; Al-Sabagh et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Suhaimi et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). Consequently, the development of mild, efficient, and sustainable biodegradation technologies has emerged as a central research focus (Koshti et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kushwaha et al. \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e 201-F6, a bacterium uniquely capable of utilizing PET as its sole carbon and energy source, is recognized as a model organism for plastic biodegradation (Yoshida et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). The bacterium secretes two specific hydrolases, PETase and MHETase, which synergistically hydrolyze PET into biologically assimilable monomers: terephthalic acid (TPA) and ethylene glycol (EG) (Yoshida et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Despite this breakthrough, the deployment of native \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e for industrial-scale bioremediation remains constrained by critical bottlenecks. Specifically, the strain exhibits weak initial adhesion and colonization on highly hydrophobic PET surfaces, and its degradation kinetics are severely restricted by environmental factors and substrate morphologies (Palm et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wallace et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yoshida et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Previous studies suggest that microbial adhesion to the plastic surface is the critical first step in biodegradation (Sun et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Enhancing this interfacial interaction using biocompatible adjuvants represents a promising strategy to amplify biocatalytic turnover (Yang et al. \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eChitosan (CS), a naturally occurring and biodegradable polysaccharide, can be formulated into highly stable chitosan nanoparticles (CS-NPs) \u003cem\u003evia\u003c/em\u003e ionic crosslinking (Des Bouillons-Gamboa et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e; Sun et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). CS-NPs possess excellent biocompatibility, dispersibility, and strong electrostatic adsorptive capabilities (Jiang et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ullah et al. \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e). Due to their high density of positive surface charges, CS-NPs can theoretically function as a biomimetic electrostatic bridge, simultaneously binding to negatively charged microbial cell envelopes and the hydrophobic PET surface (Benettayeb et al. \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e; Poznanski et al. \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). This dual function acting both as a microbial carrier and a plastic surface modifier has demonstrated potential in broader environmental remediation contexts, yet its specific application as an adjuvant for \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e -mediated PET degradation remains unexplored. Inspired by our recent research on the use of CS-NPs as an adhesive adjuvant to further enhance the degradation of PU substrates by \u003cem\u003eCladosporium oxysporum\u003c/em\u003e SCSIO 81042 (Zeng et al. \u003cspan class=\"CitationRef\"\u003e2026\u003c/span\u003e), we proposed whether CS-NPs could also enhance the biodegradation of microplastics by bacteria.\u003c/p\u003e \u003cp\u003eIn this study, we synthesized CS-NPs \u003cem\u003evia\u003c/em\u003e an ionic gelation method and systematically evaluated their ability to promote PET depolymerization by \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e. Utilizing diverse substrates including PET fluorescent microspheres, amorphous PET films, and nano-PET powders, we determined the optimal adjuvant dosage of CS-NPs and environmental conditions for maximum hydrolytic efficiency. Furthermore, we elucidated the underlying mechanism of CS-NPs enhanced biodegradation through optical microscopy and LC-MS. This work provided a promising interfacial adjuvant CS-NPs to enhance the biodegradation of polyethylene terephthalate by \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e for the future bioremediation of microplastic pollution.\u003c/p\u003e\n\n"},{"header":"Materials and Methods","content":"\u003ch2\u003eMaterials\u003c/h2\u003e\u003cp\u003eNano-scale PET powder was sourced from Xinmiao New Materials Co., Ltd. (Dongguan, China), and amorphous PET films were purchased from Goodfellow GmbH (Shanghai, China). Green PET fluorescent nanoparticles were obtained from Beijing Zhongke Keyou Nano Technology Co., Ltd. (Beijing, China). All other reagents, unless specified otherwise, were purchased from Sigma-Aldrich (Missouri, USA). R2A medium was obtained from HuanKai Microbial. Chitosan (CS, Mw = 100 kDa) and standard reference materials for TPA and bis(2-hydroxyethyl) terephthalate (BHET) were purchased from Meilun Biotechnology Co., Ltd. (Dalian, China).\u003c/p\u003e\n\u003ch3\u003ePreparation and Characterization of CS-NPs\u003c/h3\u003e\n\u003cp\u003eCS-NPs were prepared utilizing an ionic gelation method. Briefly, 0.2 g of CS (Mw\u0026thinsp;=\u0026thinsp;100 kDa) was dissolved in 100 mL of deionized water containing 1% (v/v) glacial acetic acid under continuous magnetic stirring for 12 hours until complete dissolution (2 mg/mL CS solution). The pH of the solution was adjusted to 4.5\u0026ndash;5.5. Subsequently, a crosslinking solution containing 8.4 mg/mL of sodium tripolyphosphate (TPP) was added dropwise into the CS solution at a TPP-to-CS mass ratio of 1:5. The mixture was stirred for 30 minutes to facilitate robust ionic crosslinking (Des Bouillons-Gamboa et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe synthesized CS-NPs suspension was characterized using a Zetasizer Nano ZSE (Malvern, UK) to determine the hydrodynamic size distribution, polydispersity index (PDI), and zeta potential. Morphological analysis was conducted \u003cem\u003evia\u003c/em\u003e cryo-transmission electron microscopy (Cryo-TEM, Thermo Fisher Scientific Tundra, USA) at an acceleration voltage of 100 kV.\u003c/p\u003e\n\u003ch3\u003eDegradation Assays using PET Fluorescent Microspheres\u003c/h3\u003e\n\u003cp\u003eStandard R2A medium was sterilized and aliquoted into 100 mL Erlenmeyer flasks (30 mL per flask) (Charnock \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Single colonies of \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e were pre-cultured in R2A medium to an optical density (OD₆₀₀) of 0.5. Green PET fluorescent microspheres (1 \u0026micro;m diameter, 10 mg/L, ex/em 488/518 nm) were used as visual substrate probes. To evaluate the adjuvant effect, 30 \u0026micro;L of the microsphere stock was added to 30 mL of R2A medium. The experimental groups included: blank control, bacteria only, PET microspheres only, bacteria\u0026thinsp;+\u0026thinsp;PET microspheres, and the CS-NPs-adjuvanted group (3 mL CS-NPs [2 mg/mL] + bacteria\u0026thinsp;+\u0026thinsp;PET microspheres). To assess environmental conditions, temperature (28\u0026deg;C and 37\u0026deg;C) and pH (6.0, 7.0, and 8.0) gradients were tested (cultured in combined illumination and shaking incubator, ZQZY-BGS8, Shanghai Zhichu Instrument Co., Ltd, China). Samples were continuously agitated in an orbital shaker. Degradation was quantified daily by measuring the fluorescence intensity (488/518 nm) of 0.5 mL aliquots, supplemented by UV 365 nm excitation imaging. All experiments were conducted in triplicate.\u003c/p\u003e\n\u003ch3\u003eBiodegradation of Macroscopic PET Films\u003c/h3\u003e\n\u003cp\u003ePre-cultured \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e (OD₆₀₀ = 0.5) was inoculated into 60 mL of R2A medium, centrifuged, and resuspended in 60 mL of Minimal Salt Medium (MSM). Amorphous PET films (1 cm \u0026times; 1 cm) were sterilized using 75% ethanol and 30 minutes of UV irradiation, then introduced into 50 mL of sterile R2A medium. The experimental matrix comprised 4 groups (PET film, CS-NPs\u0026thinsp;+\u0026thinsp;PET film, \u003cem\u003eIde\u003c/em\u003e\u0026thinsp;+\u0026thinsp;PET film, \u003cem\u003eIde\u003c/em\u003e\u0026thinsp;+\u0026thinsp;CS-NPs\u0026thinsp;+\u0026thinsp;PET film) to accurately isolate the adjuvant effect. The CS-NP solution (1.82 mg/mL) was added at a volume of 3 mL per reaction. Incubations were maintained at 28\u0026deg;C and 180\u0026ndash;200 rpm for 14 to 21 days. Post-incubation, the films were meticulously washed with ultrapure water and subjected to sonication to remove residual biofilm before scanning electron microscopy (SEM, Gemini 1 360, Germany) structural analysis. All experiments were conducted in triplicate.\u003c/p\u003e\n\u003ch3\u003eDepolymerization of PET Powders and Metabolite Identification\u003c/h3\u003e\n\u003cp\u003eAssays utilizing nano-PET powders and CS-NPs were conducted in 30 mL of R2A medium inoculated with pre-cultured \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e. UV-sterilized PET powder (100 mg) was introduced alongside 3 mL of CS-NPs adjuvant (1.82 mg/mL). The flasks were incubated at 28\u0026deg;C and 200 rpm for 14 days. Following the reaction, the total matrix was collected, washed, and centrifuged at 8000 \u0026times; g for 10 minutes. The supernatant was filtered through a 0.22 \u0026micro;m membrane. Metabolic intermediates derived from PET were analyzed using a Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, San Jose, USA) coupled with a Vanquish ultrahigh-performance liquid chromatography system. The liquid chromatography system did not serve a separation function, it served to introduce the sample into the mass spectrometer. The mobile phase consisted of 0.1% formic acid in water (A) and methanol (B), delivered at a ratio of 20% A to 80% B for 2 minutes. The flow rate was maintained at 0.2 mL/min, with an injection volume of 2 \u0026micro;L. All experiments were conducted in triplicate.\u003c/p\u003e \u003cp\u003eAll results from independent experiments were statistically analyzed using one-way analysis of variance (ANOVA): *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, compared with the control. And data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePreparation and Characterization of the CS-NPs Adjuvant\u003c/h2\u003e \u003cp\u003eChitosan nanoparticles were successfully engineered \u003cem\u003evia\u003c/em\u003e ionic gelation, relying on the robust crosslinking between the polycationic CS chains and multivalent anionic TPP at a pH of 5.0 (Des Bouillons-Gamboa et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The reaction yielded a highly stable, milky-white nano-suspension within 30 minutes. Cryo-TEM analysis revealed that the CS-NPs possessed a uniform, network-like morphology with smooth surfaces and a distinct lack of visible aggregation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Dynamic Light Scattering (DLS) confirmed a unimodal hydrodynamic diameter of 360.1\u0026thinsp;\u0026plusmn;\u0026thinsp;10.8 nm alongside a low PDI of 0.370\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01, indicative of a highly homogenous particle distribution. Crucially, the surface zeta potential was recorded at +\u0026thinsp;40.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 mV. This strong positive charge density ensures robust electrostatic stability in aqueous environments, establishing the physical prerequisite for the nanoparticles to act as effective bridging agents between bacterial cells and plastic substrates.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAdjuvant-Enhanced Degradation of PET Fluorescent Microspheres by\u003c/b\u003e \u003cb\u003eIdeonella sakaiensis\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePET fluorescent microspheres were employed as a visual substrate probe to precisely evaluate the degradation efficiency of \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e and CS-NPs. Due to their intrinsic fluorescence, the gradual depolymerization of these PET substrates can be directly quantified. Scanning electron microscopy (SEM) analysis confirmed the regular spherical morphology of the microspheres \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Further physicochemical characterization revealed an average particle size of 1044.3 nm, a polydispersity index (PDI) of 0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (indicating a highly uniform size distribution), and a mildly negative surface zeta potential of -0.645 mV (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTo establish the baseline degradation performance of \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e, depolymerization kinetics were evaluated in a 50% R2A medium across various temperatures (28\u0026deg;C and 37\u0026deg;C) and pH values (6.0, 7.0, and 8.0) (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). The results revealed that the optimal environmental parameters for PET microsphere degradation were 28\u0026deg;C and pH 7.0 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Furthermore, the degradation efficiency was found to be highly dependent on substrate concentration; as the concentration of the PET microspheres increased, the degradation rate exhibited a corresponding decrease. Under optimized baseline conditions (28\u0026deg;C, pH 7.0, and low substrate concentration), native \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e achieved a degradation efficiency of approximately 58.9% over a 14-day incubation period, demonstrating robust intrinsic degradation capacity.\u003c/p\u003e \u003cp\u003eBuilding upon these optimal conditions, the promotional effect of varying volumes (3, 6, and 9 mL) of the chitosan nanoparticle (CS-NPs) adjuvant on PET depolymerization was systematically investigated \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D\u003cb\u003e)\u003c/b\u003e. Using an adjuvant-free system (\"\u003cem\u003eIde\u003c/em\u003e\u0026thinsp;+\u0026thinsp;PET fluorescent microspheres\") as the control, the degradation rates across all experimental groups continuously increased as the culture time extended. Notably, the experimental group supplemented with 3 mL of CS-NPs demonstrated the most pronounced synergistic effect. After 14 days of co-incubation, the PET degradation rate in the 3 mL CS-NP group was significantly higher than that of both the untreated control and the higher-dosage CS-NPs groups. The attenuated promotional effect observed in the 6 mL and 9 mL dosage groups indicates a critical concentration threshold, suggesting that excessive adjuvant volumes may lead to nanoparticle agglomeration, thereby hindering optimal interfacial contact between the bacteria and the plastic substrate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStructural Disruption of Macroscopic PET Films\u003c/h3\u003e\n\u003cp\u003eTo validate the system's efficacy against standard commercial plastics, the degradation of amorphous PET films by \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e and CS-NPs was examined. Following 14 days of incubation, robust mycelial networks and dense bacterial colonization were visibly apparent on the adjuvant-treated films. Subsequent SEM analysis (conducted after rigorous biofilm removal) confirmed severe structural degradation. Unlike the smooth topology of the untreated controls, the PET films treated with the combined \u003cem\u003eIdeonella\u003c/em\u003e/CS-NPs system exhibited pronounced physical pitting, extensive surface erosion, and deep micro-cracking \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. These morphological disruptions confirm that the CS-NPs adjuvant facilitates robust cellular adhesion, allowing the biocatalyst to physically penetrate and aggressively depolymerize the amorphous PET films.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of Degradation Metabolites and Elucidation of the Depolymerization Pathway\u003c/h2\u003e \u003cp\u003eIn order to verify whether CS-NPs would have any effect on the degradation of PET monomers by \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e, mass spectrometry (MS) analysis was conducted on the reaction supernatant to identify the intermediate metabolites of PET degradation. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C, three distinct metabolites were detected in the experimental group, all of which were entirely absent in the uninoculated control. Metabolites \u003cb\u003eA\u003c/b\u003e (m/z 165.01853, [M\u0026ndash;H]⁻), \u003cb\u003eB\u003c/b\u003e (m/z 209.04514, [M\u0026ndash;H]⁻), and \u003cb\u003eC\u003c/b\u003e (m/z 255.08593, [M\u0026thinsp;+\u0026thinsp;H]⁺) were definitively identified as terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET), and bis(2-hydroxyethyl) terephthalate (BHET), respectively. This is consistent with the results reported in previous literature regarding the degradation of PET by \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e (Yoshida et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), indicating that CS-NPs not only enhances the efficiency but also does not interfere with the degradation process of PET. To confirm the origin of the detected monomers, we performed LC-MS analysis on control groups. No characteristic peaks of terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET), or bis(2-hydroxyethyl) terephthalate (BHET) were observed in the PET-only control (\u003cb\u003eFig. S2\u003c/b\u003e) or the bacteria-only control (\u003cb\u003eFig. S3\u003c/b\u003e), verifying that these monomers were exclusively generated from the enzymatic hydrolysis of PET, rather than from spontaneous degradation or bacterial metabolism. The accumulation of these specific products confirms the successful hydrolytic cleavage of the ester bonds within the PET polymer chains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese metabolic products align perfectly with the established two-step synergistic depolymerization mechanism of \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e. The biodegradation pathway initiates with the robust adhesion of the bacterium to the plastic surface, followed by the extracellular secretion of the primary hydrolase, PETase. This enzyme specifically recognizes and binds to the ester linkages within the PET macromolecules. Utilizing its highly conserved catalytic triad (Ser160-His237-Asp206) at the active site, PETase executes a nucleophilic attack on the ester bonds, efficiently cleaving the long-chain PET polymer into the primary intermediate, MHET (Burgin et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Magalh\u0026atilde;es et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yoshida et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMechanism of Interfacial Bridging and Enhanced Adhesion\u003c/h2\u003e \u003cp\u003eTo determine whether CS-NPs improve degradation efficiency by promoting the adhesion of \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e to the plastic surface, an assay was conducted using PET fluorescent microspheres as the sole carbon source in R2A medium over a 21-day incubation period. Optical microscopy was utilized to elucidate the physical mechanism driving the enhanced degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). After 7 days of co-incubation, TEM imaging was used to visualize the interfacial interaction (\u003cb\u003eFig. S4\u003c/b\u003e). In the presence of CS-NPs, rod-shaped \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e cells were tightly adsorbed onto the surface of PET microspheres, whereas no such adhesion was observed in the absence of bacteria. This indicates that CS-NPs act as an interfacial mediator to enhance bacterial adhesion. Meanwhile, the CS-NPs-treated group displayed a massively increased accumulation of PET microspheres physically tethered directly to the bacterial cell surfaces, in stark contrast to the adjuvant-free control group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD \u003cb\u003eand E)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe underlying mechanism for this observed synergy is governed by electrostatic attraction, Zeta potential measurements further elucidated the electrostatic basis of this mediation (\u003cb\u003eTable. S1\u003c/b\u003e). CS-NPs exhibited a strong positive charge (+\u0026thinsp;40.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.20 mV), while both \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e (-10.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19 mV) and PET microspheres (-0.645\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mV) were negatively charged, providing a driving force for the electrostatic attraction (Zeng et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). By forming electrostatic attraction, the adjuvant physically tethers the biocatalyst to the interface. These results demonstrate that CS-NPs enhance the degradation of PET microplastics by promoting robust adhesion between \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e and the substrate, effectively overcoming the thermodynamic barriers of hydrophobic exclusion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis investigation successfully established chitosan nanoparticles as a potent, biomimetic interfacial adjuvant capable of enhancing the biodegradation of PET plastics. Synthesized \u003cem\u003evia\u003c/em\u003e mild ionic gelation, the strongly cationic CS-NPs (360.1 nm, +\u0026thinsp;40.1 mV) acted as an electrostatic bridge, facilitating robust adhesion between \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e and highly recalcitrant PET substrates. At an optimal dosage (3 mL per 30 mL reaction), the CS-NPs maximized the depolymerization of fluorescent PET microspheres (91.2% degradation at 28\u0026deg;C, pH 7.0 compared to 58.9% without CS-NPs) and induced profound structural pitting in macroscopic PET films. The accumulation of terminal TPA and MHET monomers confirmed authentic hydrolytic cleavage. While high concentrations of the adjuvant induced physical cellular agglomeration, the optimized system successfully achieved the goal of enhancing efficacy. Although our research has demonstrated the enhancement of CS-NPs on the degradation of microplastics by bacteria, the extent of this enhancement is still rather limited. Therefore, future studies should explore the optimization of CS-NPs physicochemical properties including precise size tuning and targeted surface modifications to further maximize their interfacial tethering capacity.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.9873%;\"\u003e\n \u003cp\u003ePET\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 31.1031%;\"\u003e\n \u003cp\u003ePolyethylene terephthalate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6365%;\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2731%;\"\u003e\n \u003cp\u003eScanning electron microscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.9873%;\"\u003e\n \u003cp\u003eCS-NPs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 31.1031%;\"\u003e\n \u003cp\u003eChitosan nanoparticles\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6365%;\"\u003e\n \u003cp\u003eTEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2731%;\"\u003e\n \u003cp\u003eTransmission electron microscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.9873%;\"\u003e\n \u003cp\u003eCS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 31.1031%;\"\u003e\n \u003cp\u003eChitosan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6365%;\"\u003e\n \u003cp\u003eCryo-TEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2731%;\"\u003e\n \u003cp\u003eCryo-transmission electron microscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.9873%;\"\u003e\n \u003cp\u003eTPP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 31.1031%;\"\u003e\n \u003cp\u003eSodium tripolyphosphate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6365%;\"\u003e\n \u003cp\u003eDLS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2731%;\"\u003e\n \u003cp\u003eDynamic light scattering\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.9873%;\"\u003e\n \u003cp\u003e\u003cem\u003eI. sakaiensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 31.1031%;\"\u003e\n \u003cp\u003e\u003cem\u003eIdeonella sakaiensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6365%;\"\u003e\n \u003cp\u003ePDI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2731%;\"\u003e\n \u003cp\u003ePolydispersity index\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.9873%;\"\u003e\n \u003cp\u003eIde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 31.1031%;\"\u003e\n \u003cp\u003e\u003cem\u003eIdeonella sakaiensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6365%;\"\u003e\n \u003cp\u003eR2A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2731%;\"\u003e\n \u003cp\u003eR2A medium\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.9873%;\"\u003e\n \u003cp\u003eTPA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 31.1031%;\"\u003e\n \u003cp\u003eTerephthalic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6365%;\"\u003e\n \u003cp\u003eMSM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2731%;\"\u003e\n \u003cp\u003eMinimal salt medium\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.9873%;\"\u003e\n \u003cp\u003eMHET\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 31.1031%;\"\u003e\n \u003cp\u003eMono(2-hydroxyethyl) terephthalate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6365%;\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2731%;\"\u003e\n \u003cp\u003eStandard deviation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.9873%;\"\u003e\n \u003cp\u003eBHET\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 31.1031%;\"\u003e\n \u003cp\u003eBis(2-hydroxyethyl) terephthalate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6365%;\"\u003e\n \u003cp\u003eANOVA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2731%;\"\u003e\n \u003cp\u003eOne-way analysis of variance\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.9873%;\"\u003e\n \u003cp\u003ePETase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 31.1031%;\"\u003e\n \u003cp\u003ePET hydrolase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6365%;\"\u003e\n \u003cp\u003eLC-MS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2731%;\"\u003e\n \u003cp\u003eLiquid chromatography-mass spectrometry\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 18.9873%;\"\u003e\n \u003cp\u003eMHETase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 31.1031%;\"\u003e\n \u003cp\u003eMHET hydrolase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6365%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33.2731%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available at https://doi.org/xxxxx.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Qinglian Li from South China Sea Institute of Oceanology, Chinese Academy of Sciences for providing the bacterial strains and technical assistance in the cultivation of the strains. We thank Mr. Kai Yang and Ms. Shan Liu from Bioimaging Platform at Shenzhen Bay Laboratory for their technical assistance and valuable advice in electron microscopy.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZ.S.:\u003c/strong\u003e Conceptualization, Data curation, Methodology, Investigation, Formal analysis, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eT.W.:\u003c/strong\u003e Methodology, Investigation, Formal analysis, Visualization, Writing \u0026ndash; original draft. \u003cstrong\u003eL.W.:\u003c/strong\u003e Methodology, Investigation, Formal analysis, Visualization. \u003cstrong\u003eX.C.:\u0026nbsp;\u003c/strong\u003eFormal analysis, Investigation. \u003cstrong\u003eY.W.:\u003c/strong\u003e Formal analysis, Investigation. \u003cstrong\u003eT.F.:\u003c/strong\u003e Funding acquisition, Writing \u0026ndash; review \u0026amp; editing, Supervision. \u003cstrong\u003eY.J.:\u003c/strong\u003e Project administration, Resources, Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Research and Development Program (2023YFC3903300).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in this study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author(s).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbedsoltan H (2023) A focused review on recycling and hydrolysis techniques of polyethylene terephthalate. 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Environ Res 296:123981. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.envres.2026.123981\u003c/span\u003e\u003cspan address=\"10.1016/j.envres.2026.123981\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\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":"Polyethylene terephthalate (PET), Biodegradation, Ideonella sakaiensis, Chitosan nanoparticles","lastPublishedDoi":"10.21203/rs.3.rs-9055686/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9055686/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe escalating global crisis of polyethylene terephthalate (PET) pollution necessitates the development of efficient biological recycling strategies. While the bacterium \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e can utilize PET as a sole carbon source, its practical application as a biocatalyst is hampered by poor interfacial adhesion to hydrophobic plastic surfaces and limited degradation efficiency under ambient conditions. In this study, we developed chitosan nanoparticles (CS-NPs) \u003cem\u003evia\u003c/em\u003e ionic crosslinking to serve as a novel electrostatic bridging adjuvant. The synthesized CS-NPs exhibited a uniform particle size of 360.1\u0026thinsp;\u0026plusmn;\u0026thinsp;10.8 nm and a high positive zeta potential of +\u0026thinsp;40.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 mV. Supplementation with an optimal dosage of CS-NPs (3 mL per 30 mL medium) significantly promoted the adhesion of \u003cem\u003eIdeonella sakaiensis\u003c/em\u003e to diverse PET substrates. Under optimized environmental conditions (28\u0026deg;C, pH 7.0), CS-NPs were introduced as adhesion adjuvants, achieving a 91.2% degradation efficiency of PET fluorescent microspheres over 14 days compared to 58.9% without CS-NPs. Furthermore, application of the CS-NPs adjuvant to macroscopic PET films resulted in dense surface colonization and severe structural disruption, characterized by deep physical pitting and cracking. High-performance liquid chromatography-mass spectrometry (LC-MS) confirmed the robust hydrolytic depolymerization of PET into its constituent monomers: terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET), and bis(2-hydroxyethyl) terephthalate (BHET). While challenges remain regarding degradation of CS-NPs environmental fate and microbial competition, this work provides the CS-NPs adjuvant as promising tools for the bioremediation of PET pollution.\u003c/p\u003e","manuscriptTitle":"Chitosan Nanoparticles as Interfacial Adjuvants to Enhance the Biodegradation of Polyethylene Terephthalate by Ideonella sakaiensis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-18 08:39:42","doi":"10.21203/rs.3.rs-9055686/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":"289421e3-8529-494a-a962-0cf8e77a6ced","owner":[],"postedDate":"March 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-16T14:08:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-18 08:39:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9055686","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9055686","identity":"rs-9055686","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}