Discovery of a polyvinyl alcohol-degrading strain of the ascomycete Fusarium oxysporum and optimizing of its degradation performance of PVA

Discovery of a polyvinyl alcohol-degrading strain of the ascomycete Fusarium oxysporum and optimizing of its degradation performance of PVA | 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 Discovery of a polyvinyl alcohol-degrading strain of the ascomycete Fusarium oxysporum and optimizing of its degradation performance of PVA Xin Zhang, Juyi Song, Chang Liu, Hui Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3834003/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 Massive accumulation of plastics into environment has caused enormous pressure on the ecosystem. Efficient and environmentally friendly plastics degradation technologies have evolved into a global ecological challenge. Microbial degradation, as an eco-friendly plastic treatment technology, is confronted with a problem of low efficiency in its current application. Hence, it is crucial to discovery plastic biodegradable microorganisms and find the optimal conditions for their action. The aim of our study is to isolate plastic-biodegrading fungi and explore optimum conditions for their action. A strain isolate of Fusarium oxysporum was obtained from a degraded plastic handle through screening, separation, and purification and designated PDBF01 (CGMCC No.40272). In a screening assay of plastic polymers, PDBF01 only exhibited the degradability to polyvinyl alcohol (PVA), with no activity toward polyvinyl chloride, polypropylene, or polylactic acid. PVA degradation efficiency of PDBF01 was significantly affected by inoculum concentration, temperature, and degradation time. PDBF01 produced significant degradation of PVA under 28°C and 25% inoculum concentration. Moreover, the highest degradation rate reached 51.26% after 21 days. PVA degradation rate of PDBF01 was further increased to 58.83% by the addition of electrolytes (K + , Mg 2+ , Fe 2+ , and Ca 2+ ). Our results suggested PDBF01 can be used as a potential and efficient PVA-degrading strain in practical applications. Biological sciences/Biotechnology Biological sciences/Biotechnology/Environmental biotechnology Fusarium oxysporum polyvinyl alcohol biological identification degradation properties Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction There is a growing, world-wide interest in addressing the issue of plastic polymer pollution and developing strategies to mitigate its negative environmental effects [ 1 ]. Currently, microbial degradation is the major approach towards treatment of plastic waste since it is ecologically-compatible and resource-friendly [ 2 , 3 ]. Therefore, many attempts have been made to target biodegradation of specific plastics by cultivating polymer-selective microorganisms [ 4 – 7 ]. Polyvinyl alcohol (PVA), a water-soluble macromolecular compound, has been widely applied in the textile and agriculture industries due to excellent film strength, durability, and low toxicity [ 8 , 9 ]. However, the applications of PVA are restricted by degradation methods that are critical for ecosystem health. It is necessary, therefore, to find an efficient and environmentally-friendly PVA degradation method to ensure its better application in actual production. Many attempts have been made to explore degrading PVA with low energy consumption and high efficiency [ 10 , 11 , 12 ]. The ascomycete Fusarium oxysporum is a notable facultative parasitic fungus that can infect plants and survive in soil. It presents narrow host specificity [ 13 ]. To date, research on F. oxysporum has been mainly focused on biological control of Fusarium disease by exploring the mode of action of its non-pathogenic strains [ 14 , 15 ]. However, the fungus has also been successfully used to degrade some materials, such as swine hair and corn stalks [ 16 , 17 ]. In this study, we report the isolation and identification of a F. oxysporum strain (PDBF01) with selective degradation abilities for PVA plastic. Moreover, we assessed the optimal environmental conditions to facilitate efficient PVA degradation. We foresee the potential for widespread use of the PDBF01 strain in future bioremediation efforts. 2. Results 2.1. Identification of strains PDBF01 colony morphology is shown in Fig. 1 A. The macro morphology of PDBF01 presented a white center with reddish brown periphery. Colony diameters ranged between 65–68 mm. No exudate or soluble pigment was observed. Hypha were wrapped, transparent, and smooth with a diameter of 1.5–3.5 µm (Fig. 1 B). Bottle stem singly occurred in aerial hypha with a 10–35 µm size. The microspore was slightly curved, fusiform, or rod-shaped with sizes ranging from 4.5–11 × 1.5–2.8 µm. The megaspore was a curved sickle shape with 1–3 diaphragms and ranged in size from 15–25 × 2.8–3.8 µm. Phylogenetic analysis identified PDBF01 as Fusarium oxysporum (Fig. 2 ). Data is deposited in China National Microbiology Data Center (NMDC) with accession numbers NMDCN0002P0P ( https://nmdc.cn/resource/genomics/sequence/detail/NMDCN002P0P ). 2.2. Assessmen of plastic degradation by PDBF01 PDBF01 was capable of growing on media with PVA as the sole carbon source. In contrast, PDBF01 did not grow on media containing PLA, PP, or PVC as the sole carbon source, consistent with polymer specificity for biodegradation. After culturing PDBF01 (160mg dry weight) in 100mL pure PVA solution for 7 days, it was found that the dry weight of PDBF01 increased by 76 mg. The results showed that PDBF01 grew on PVA as a nutrient, further indicating PDBF01 has a degradation effect on PVA. 2.3. Effect of temperature on PVA degradation rate Temperature is an important element affecting microbial metabolism and can directly influence degradative properties of microorganisms by affecting activity of secreted degrading enzymes [ 18 , 19 ]. PDBF01 exhibited the highest degradation rate of PVA at 28°C (Fig. 3 A). The degradation rate presented a decreasing trend as the temperature continues to rise. Higher temperatures resulted in reduced degradation rates, attributable to adverse effects on enzymatic activity. Similar phenomena were observed in a previous study [ 20 ]. PDBF01 grew among the experimental temperature (24–40°C), but the growth rate decreased after exceeding 28 ° C (Fig. 3 B), which was conformed to the law of temperature affecting the growth of fungi. Excessive temperature can lead to protein denaturation and death of fungal cells, while low temperature can inhibit fungal metabolic activity and reproductive ability. 2.4. Effect of inoculum concentration of strain PDBF01 on PVA degradation rate Inoculum concentration can affect growth rate by directly affecting thermodynamic properties and patterns of gene expression, thus affecting the properties of strains [ 21 ]. Degradation rate of PDBF01 increased rapidly with increase of inoculum concentration up to 25% yet was stable at higher inoculum concentrations. Meanwhile PDBF01 gained 207.66 mg weight at a 25% inoculum concentration compared to the initial gauge (Fig. 4 ). 2.5. Degradation time on PVA degradation rate The microbial degradation of plastics is extremely slow and the optimal degradation time of different plastics is variable and strain-dependent [ 22 , 23 ]. PDBF01 degradation rate of PVA increased with the increase of degradation time to a stable maximum at 21 d post-inoculation (Fig. 5 ). 2.6. Composition of medium on PVA degradation rate Medium composition can affect culture growth and product formation of microorganisms, further affecting the properties of microbial strains. Different substances added to medium can satisfy specific growth or property requirements [ 24 , 25 ]. For example, Jingjing et al. [ 26 ] previously reported that addition of potassium ions could promote growth of Lactobacillus plantarum and improve the freeze-drying resistance and storage stability of the strain. In our study, the degradation rate of PVA by PDBF01 increased to 58.83% under the same degradation conditions after the addition of electrolytes, which may relate to the influence of these ions on metabolism and physiological function regulation of the strain and/or the regulation of enzyme activity. 3. Discussion PVA is widely used in multiple industries due to its excellent physical and chemical properties. However, excessive accumulation of PVA in the environment can affect or even destroy the balance of the ecosystem due to the difficulty in recyclingof PVA [ 27 , 28 ]. With the increasing severity of PVA pollution, increasingly strict environmental awareness have prompted researchers to seek effective methods to eliminate PVA waste and conducted extensive research on accelerating the degradation of PVA [ 29 ]. The significant effectiveness of microbial degradation of PVA and its environmentally friendly advantages have promoted it becoming a popular research topic [ 30 ]. Currently, various microbial strains capable of degrading PVA have been discovered, such as Pseudomonas sp., Bacillus sp., Aspergillus sp ., etc. [ 31 , 32 , 33 ], but there are few efficient strains. Fusarium oxysporum is a widely occurring bacterium in nature, which has been proven to have great potential for microbial degradation [ 34 ]. Kim et al., [ 35 ] previously found F. oxysporum could effectively degrade an endocrine disrupting chemical, DEHP. In this study, we successfully obtained and identified through sequencing analysis an ascomycete strain from a degraded plastic handle as Fusarium oxysporum . The strain was designated PDBF01 (CGMCC No.40272). There are differences in temperature demands for the growth of various microbial strains [ 36 ]. Meanwhile, it is generally necessary to choose the most suitable temperature range for their growth as the temperature for the degradation reaction to ensure high activity and vigorous metabolism of the strain, thus achieving efficient plastic degradation. Excessive temperature induce protein denaturation and accumulations of toxic substances such as reactive oxygen species, which leading to DNA damage and other direct impacts on the growth of the strains [ 37 , 38 ]. Higher temperature may lead a decrease or loss of enzyme activity. The loss of key metabolic enzyme activity affects the degradation performance of the strain [ 39 ]. Previous research has found that fungi may enter a dormant state to adapt to low-temperature environment, and the growth rate of the strain slows down or stagnates [ 40 ]. Previous researches found the growth of fungi will significantly decrease under low-temperatures [ 41 ], which may related to the changes of metabolic pathways inside the fungi or inhibiting the activity of certain key enzymes. Therefore, selecting the appropriate temperature is crucial for the growth and potential application of the strain. In our study, the optimal temperature for PVA degradation by Fusarium oxysporum PDBF01 was 28 ℃, and the degradation efficiency of PDBF01 varied with temperature. Wilkes, R. A., & Aristilde, L. [ 42 ] reported that the degradation rate of PVA by Pseudomonas spp . was also affected by temperature, showing the same trend as our results. Normally, the degradation rate of plastic by microorganisms increases with the inoculum concentration of microbial strains [ 43 ]. However, exceeding the upper limit concentration can actually cause a decrease in growth rate. These changes may be related to rate-limiting factors such as excessive consumption of nutrients in medium, accumulation of metabolites (forming metabolic feedback and inhibition), and altered metabolic pathway functions [ 44 , 45 , 46 ],thereby affecting the degradation efficiency of plastics. Similar phenomena were observed in previous research. Wolski et al. [ 47 ] observed similar changes in pentachlorophenol degradation by Pseudomonas aeruginosa . Hence, in our research, we validated different inoculation concentrations and selected the appropriate strain PDBF01 inoculation concentration (25%) to obtain better degradation effect on PVA. The degradation time of microbial degradation of plastics significantly impact the degradation rate. The degradation rate of plastic degraded by microorganisms will gradually increase with the extension of degradation time, which is related to adaption time and reproduction of strains in the process of degrading plastics to achieve efficient degradation [ 48 , 49 ]. The adaptation period, reproduction period and degradation period of microorganisms may be influenced by various elements. In our study, it was found that after 28 days of degradation, further prolongation of degradation time did not show significant impacts on the growth of strain PDBF01 and its degradation rate of PVA. It may mainly be due to the reduction of substrate after PVA degradation, limiting the access to carbon sources and energy, weakening the growth momentum of the strain [ 50 , 51 ].The growth of strain PDBF01 has reached a saturation state in such specific environment. The changes may be related to an altered culture environment that is no longer suitable for the growth of the strain, as described previously [ 52 ]. Puiggené et al. [ 53 ] has also reported similar results and ascribed this effect as relating to the growth characteristics of the strain itself. A sharp decline in viable count occurred due to the abnormal differentiation and release of metabolite. Gao, R., Liu, R., & Sun, C. [ 54 ] also observed the same phenomenon and was committed to reducing the time of microbial degradation of plastics. 4. Materials and methods 4.1. Materials and reagents The PBDF01 F. oxysporum strain was isolated from a degraded plastic handle. Polyvinyl alcohol (PVA1788; polymerization degree of 1700, alcoholysis degree of 88%, purity of 99%) was purchased from Qiansheng Biotechnology Co. Ltd. (Hefei, China). Potato dextrose agar medium (PDA) and potato dextrose broth medium (PDB) were purchased from Aisiwei Biochemical Co., Ltd. (Hangzhou, China). Boric acid, iodine, potassium Iodide and other chemicals were analytical grade. 4.2. Screening and identification of strains 4.2.1. Screening and purification of strains The strains were screened and purified by previously-established methods with slight modification [ 55 ]. Environmental isolates (three replicate samples) were cultured for 30 d in a shaking incubator (28°C, 150 r/min oscillation) in 50 mL PDB medium. Mixed strain cultures displaying adequate growth potential were streaked on PDA medium and cultured at 30°C in the constant temperature incubator for 10 d. The three main colonies grown were isolated and inoculated in PDB medium for culture expansion. Cultures were reinoculated to PDA medium through repeated growth cycles to isolate and purify the strains until a single dominant colony per plate was produced. 4.2.2. Identification of strains Preliminary identification of the strain isolate was performed by microscopy for assessment of colony and microscopic morphology. We further characterized the isolate strain through molecular identification, DNA extraction, PCR amplification, electrophoresis detection, ITS rDNA sequencing, EF-1α sequence analysis and homology comparison according to the FMIC-QO01-003 fungal polyphase identification and detection method and QO-03-02 operating procedures for Molecular Biological Identification of Microbial Strains [ 56 ]. Specifically, the strains obtained were streak cultured on PDA medium for 7 days to observe the size, color, edge type and surface characteristics of the colonies. A single colony was selected for microscopic observation to examine its morphology, hyphal morphology, spore morphology and other characteristics. After preparing a single strain into a fungal suspension, it was mechanically crushed. Then SDS was added for cell lysis. DNA was extracted and precipitated using PCI. Design primers for PCR amplification of the ITS region of the strain by using the universal primers ITS1 and ITS4 reported in previous reports. The PCR amplification were performed under the following procedur: pre denaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 45 seconds, annealing at 55 ° C for 45 seconds, extension at 72 ° C for 1 minute, and 35 cycles. The specific bands of PCR products were detected by 1.5% agarose gel electrophoresis and DNAMARKER was set as a molecular weight indicator. PCR products for Sanger sequencing was recovered by gel recovery kit. Perform Blastn analysis on the sequencing results in the NCBI database and select the species with the highest similarity for phylogenetic analysis. Neighbor joining phylogenetic trees in ITS rDNA sequence and EF-1α sequence of the strain for bootstrap analysis was constructed using MEGA5.0 software. 4.3. Screening of degradable plastics Assessment of plastic polymer degradation by the PDBF01 isolate was performed by previous methods, with modification [ 57 ]. The isolated strain was cultured in PDA medium for 3d before transferring the mycelium from the young margin of the colony to PDB liquid medium for 7d. Then, PDBF01 cultures (1 mL) were diluted to an OD600 solution value between 0.8 and 1. Wash the precipitated strain with clean water after centrifuging the strain solution under 5000rpm for 10 minutes, and perform vacuum drying at -40 ℃ before weighing the dry weight. Polyvinyl chloride (PVC), polypropylene (PP), PVA, or polylactic acid (PLA; 0.2 g ea.) was added as the sole carbon source to sterilized, carbon-free medium, inoculated with diluted PDBF01 (16mg of dry weight), and cultured for 30 days at 28°C. Identification of polymer degradation was performed by observation of colony growth pattern and weight changes in dry weight of the strain PDBF01. 4.4. Optimization of degradation conditions of PVA PVA content was assessed as reported with slight modifications. Briefly, the PVA standard solution (20 mg/L) was scanned at full wavelength ranging from 400 to 700 nm, and the maximum absorbance was found at 690nm. The absorbance of the solution at 690 mm wavelength was determined by spectrophotometry after PVA reacting with iodine in boric acid medium. PVA was quantified by standard curve. 4.4.1. Degradation temperature 20mL of PDBF01 liquid solutions (160 mg dry weight of PDBF01; 20% solution, v/v; 3 replicates ea.) cultured under 2.3 conditions was inoculated to 100mL PVA solution (1g/L) and incubated in a shaking incubator (35 d, 150 r/min oscillation) at 24°C, 28°C, 32°C, 36°C, or 40°C. Content of PVA and changes in dry weight of PDBF01 was assessed at 35 d post-inoculation. Solutions only with PVA were used as controls. 4.4.2. Inoculum concentration of strain PDBF01 (i.e. strain with dry weight of 40mg, 120mg, 200mg, 280mg, 360mg) at concentrations of 5%, 15%, 25%, 35%, or 45% (v/v; 3 replicates ea.) cultured under 2.3 conditions was inoculated to 100mL PVA solution (1g/L) and cultured in a shaking incubator (28°C, 150 r/min oscillation) for 35 d. Content of PVA and changes in dry weight of PDBF01 was assessed at 35 d post-inoculation. Solutions only with PVA were used as controls. 4.4.3. Degradation time 25mL of PDBF01 liquid solutions (200 mg dry weight of PDBF01; 20% solution, v/v; 3 replicates ea.) cultured under 2.3 conditions was inoculated to 100mL PVA solution (1g/L) and incubated in a shaking incubator (28°C, 150 r/min oscillation). Content of PVA and changes in dry weight of PDBF01 was assessed at 7, 14, 21, 28, and 35 d post-inoculation. Solutions only with PVA were used as controls. 4.4.4. Composition of medium on PVA degradation rate 25mL of PDBF01 liquid solutions (200 mg dry weight of PDBF01; 25% solution, v/v; 3 replicates ea.) cultured under 2.3 conditions was inoculated to 100mL PVA solution (1g/L). Then 10 mg K + , Mg 2+ , Fe 2+ , and Ca 2+ (10%, w/v) was added into culture solutions and incubated in a shaking incubator (28°C, 150 r/min oscillation). Content of PVA and changes in dry weight of PDBF01 was assessed at 35 d post-inoculation. Solutions only with PVA were used as controls. 4.5. Statistical analysis Bonferroni-corrected (α = 0.05) multiple comparisons were performed by one-way ANOVA followed by a Duncan means separation test using the SPSS 13.0 software package. Data are represented as means (± SD). Declarations Author Contributions Conceptualization, methodology, software, validation, formal analysis, , investigation, resources, Z.X., L.C. and C.H.; Fund acquisition, data curation, S.J.Y. and C.H.; Writing—original draft preparation, writing—review and editing, supervision, Z.X., L.C. S.J.Y. and C.H. All authors have read and agreed to the published version of the manuscript. Funding This work was financed by the Jiangsu Agriculture Science and Technology Innovation Fund (JASTIF) (CX(21)3113). Youth Science and Technology Fund (YJ(2021)009) of Jiangsu Yanjiang Institute of Agricultural Science. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The sequence data for the strain used in this study is deposited in China National Microbiology Data Center (NMDC) with accession numbers NMDCN0002P0P (https://nmdc.cn/resource/genomics/sequence/detail/NMDCN002P0P). Acknowledgments We would like to acknowledge Jiangsu Yanjiang Institution of Agricultural Science for assistance in this work. We thank LetPub (www.letpub.com) for linguistic assistance and pre-submission expert review. Conflicts of Interest The authors declare no conflict of interest. References Law, K. L., & Narayan, R. (2022). Reducing environmental plastic pollution by designing polymer materials for managed end-of-life. Nature Reviews Materials, 7(2), 104-116. Kale, S. K., Deshmukh, A. G., Dudhare, M. S., & Patil, V. B. (2015). Microbial degradation of plastic: a review. Journal of Biochemical Technology, 6(2), 952-961. Jaiswal, S., Sharma, B., & Shukla, P. (2020). Integrated approaches in microbial degradation of plastics. 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Water SA, 32(1), 93-97. Yuan, J., Ma, J., Sun, Y., Zhou, T., Zhao, Y., & Yu, F. (2020). Microbial degradation and other environmental aspects of microplastics/plastics. Science of the Total Environment, 715, 136968. Chamas, A., Moon, H., Zheng, J., Qiu, Y., Tabassum, T., Jang, J. H., ... & Suh, S. (2020). Degradation rates of plastics in the environment. ACS Sustainable Chemistry & Engineering, 8(9), 3494-3511. Zhang, X., Wu, W., Zhang, Y., Wang, J., Liu, Q., Geng, C., & Lu, J. (2007). Screening of efficient hydrocarbon-degrading strains and study on influence factors of degradation of refinery oily sludge. Industrial & engineering chemistry research, 46(26), 8910-8917. Pimda, W., & Bunnag, S. (2015). Biodegradation of waste motor oil by Nostoc hatei strain TISTR 8405 in water containing heavy metals and nutrients as co-contaminants. Journal of Industrial and Engineering Chemistry, 28, 117-123. Vrabl, P., Schinagl, C. W., Artmann, D. J., Heiss, B., & Burgstaller, W. (2019). Fungal growth in batch culture–what we could benefit if we start looking closer. Frontiers in microbiology, 10, 2391. Puiggené, Ò., Espinosa, M. J. C., Schlosser, D., Thies, S., Jehmlich, N., Kappelmeyer, U.,. .. & Eberlein, C. (2022). Extracellular degradation of a polyurethane oligomer involving outer membrane vesicles and further insights on the degradation of 2, 4-diaminotoluene in Pseudomonas capeferrum TDA1. Scientific Reports, 12(1), 1-12. Gao, R., Liu, R., & Sun, C. (2022). A marine fungus Alternaria alternata FB1 efficiently degrades polyethylene. Journal of Hazardous Materials, 431, 128617. Hao, X., Zhang, X., Duan, B., Huo, S., Lin, W., Xia, X., & Liu, K. (2018). Screening and genome sequencing of deltamethrin-degrading bacterium ZJ6. Current microbiology, 75, 1468-1476. Rojas, O. C., Bonifaz, A., Campos, C., Treviño-Rangel, R. D. J., González-Álvarez, R., & González, G. M. (2018). Molecular identification, antifungal susceptibility, and geographic origin of clinical strains of Sporothrix schenckii complex in Mexico. Journal of Fungi, 4(3), 86. Nadeem, H., Alia, K. B., Muneer, F., Rasul, I., Siddique, M. H., Azeem, F., & Zubair, M. (2021). Isolation and identification of low-density polyethylene degrading novel bacterial strains. Archives of Microbiology, 203(9), 5417-5423. Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.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. 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. 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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-3834003","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":265665559,"identity":"6375b54e-7c98-4632-a847-873c3f7256be","order_by":0,"name":"Xin Zhang","email":"","orcid":"","institution":"Jiangsu Yanjiang Institute of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Zhang","suffix":""},{"id":265665560,"identity":"87690d23-26af-4e78-951b-34ed07b2bef7","order_by":1,"name":"Juyi Song","email":"","orcid":"","institution":"Jiangsu Yanjiang Institute of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Juyi","middleName":"","lastName":"Song","suffix":""},{"id":265665561,"identity":"6da2e7f7-d919-40ee-9d03-4dd5dbf74d0d","order_by":2,"name":"Chang Liu","email":"","orcid":"","institution":"Jiangsu Yanjiang Institute of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Chang","middleName":"","lastName":"Liu","suffix":""},{"id":265665562,"identity":"15b6fc70-9916-46c2-aa55-5b6c94e29bb8","order_by":3,"name":"Hui Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYDACCQhZb9/AwPiAgeEA8VoSDBgYmA1I0cIA0sImQZQW/tnNxx7zVFjkmbOfPVb5o+JOYgP74aMb8Fpy51i6Mc8ZiWLLnry02zxnniU28KSl3cCnxUAix0w6t02CseEGj9ltxrbDiQ0SPGYEtOR/k879B9FS+JM4LTls0rkNEokbgFoYeInRInEjzUz6zzEJY8meHGNpnjOHjdsI+YV/RvIzyRk1dXL87GcMP/6oOCzbz374GF4tmICNNOWjYBSMglEwCrABAD4LSZfZvZf6AAAAAElFTkSuQmCC","orcid":"","institution":"Jiangsu Yanjiang Institute of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Hui","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-01-04 08:15:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3834003/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3834003/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49333400,"identity":"8e892868-bdf3-48e1-bc79-cef88a816ef2","added_by":"auto","created_at":"2024-01-08 19:48:08","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":99796,"visible":true,"origin":"","legend":"\u003cp\u003eMacroscopic and microscopic colony morphology of PDBF01.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3834003/v1/802924506a8708fe19714a68.jpg"},{"id":49333399,"identity":"1067dcd1-a5a4-425d-b017-1a9ada01a55c","added_by":"auto","created_at":"2024-01-08 19:48:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":178247,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of PDBF01.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3834003/v1/ad0985048c9cb2f6a7c55afc.jpg"},{"id":49333169,"identity":"635f26d0-1528-4bd4-a729-a049d3240ca2","added_by":"auto","created_at":"2024-01-08 19:40:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":61224,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of temperature on PVA degradation rate of PDBF01.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3834003/v1/8bc2c64ada15535949720470.jpg"},{"id":49333165,"identity":"35e94d68-febf-46da-89a8-f3f3a8f3c52f","added_by":"auto","created_at":"2024-01-08 19:40:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":64212,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of inoculum concentration on PVA degradation rate of PDBF01.\u003c/p\u003e\n\u003cp\u003e*Values are given as mean ± SD (n = 3). Different letters in the same column indicate significantly different (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3834003/v1/a5e65ca0a417b9f972489d32.jpg"},{"id":49333496,"identity":"76b404af-2f2b-4e9d-8d18-429b5ab503d6","added_by":"auto","created_at":"2024-01-08 19:56:08","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":69679,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of degradation time on PVA degradation rate of PDBF01.\u003c/p\u003e\n\u003cp\u003e*Values are given as mean ± SD (n = 3). Different letters in the same column indicate significantly different (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3834003/v1/e0225b7961eeb38bee3b59cd.jpg"},{"id":55264780,"identity":"80f5f6f6-4b53-4eea-ae02-4c5161b194dd","added_by":"auto","created_at":"2024-04-25 01:49:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":825562,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3834003/v1/f1200784-942f-4a1a-b6e4-03e35a6ed2f4.pdf"},{"id":49333170,"identity":"9b890b49-990c-4abb-93f2-d3d7dd988445","added_by":"auto","created_at":"2024-01-08 19:40:08","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2105097,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-3834003/v1/757443e60d8538e924ef43c7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Discovery of a polyvinyl alcohol-degrading strain of the ascomycete Fusarium oxysporum and optimizing of its degradation performance of PVA","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThere is a growing, world-wide interest in addressing the issue of plastic polymer pollution and developing strategies to mitigate its negative environmental effects [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Currently, microbial degradation is the major approach towards treatment of plastic waste since it is ecologically-compatible and resource-friendly [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, many attempts have been made to target biodegradation of specific plastics by cultivating polymer-selective microorganisms [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePolyvinyl alcohol (PVA), a water-soluble macromolecular compound, has been widely applied in the textile and agriculture industries due to excellent film strength, durability, and low toxicity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, the applications of PVA are restricted by degradation methods that are critical for ecosystem health. It is necessary, therefore, to find an efficient and environmentally-friendly PVA degradation method to ensure its better application in actual production. Many attempts have been made to explore degrading PVA with low energy consumption and high efficiency [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe ascomycete \u003cem\u003eFusarium oxysporum\u003c/em\u003e is a notable facultative parasitic fungus that can infect plants and survive in soil. It presents narrow host specificity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To date, research on F. oxysporum has been mainly focused on biological control of Fusarium disease by exploring the mode of action of its non-pathogenic strains [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, the fungus has also been successfully used to degrade some materials, such as swine hair and corn stalks [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we report the isolation and identification of a \u003cem\u003eF. oxysporum\u003c/em\u003e strain (PDBF01) with selective degradation abilities for PVA plastic. Moreover, we assessed the optimal environmental conditions to facilitate efficient PVA degradation. We foresee the potential for widespread use of the PDBF01 strain in future bioremediation efforts.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Identification of strains\u003c/h2\u003e\n \u003cp\u003ePDBF01 colony morphology is shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA. The macro morphology of PDBF01 presented a white center with reddish brown periphery. Colony diameters ranged between 65\u0026ndash;68 mm. No exudate or soluble pigment was observed. Hypha were wrapped, transparent, and smooth with a diameter of 1.5\u0026ndash;3.5 \u0026micro;m (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Bottle stem singly occurred in aerial hypha with a 10\u0026ndash;35 \u0026micro;m size. The microspore was slightly curved, fusiform, or rod-shaped with sizes ranging from 4.5\u0026ndash;11 \u0026times; 1.5\u0026ndash;2.8 \u0026micro;m. The megaspore was a curved sickle shape with 1\u0026ndash;3 diaphragms and ranged in size from 15\u0026ndash;25 \u0026times; 2.8\u0026ndash;3.8 \u0026micro;m. Phylogenetic analysis identified PDBF01 as \u003cem\u003eFusarium oxysporum\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Data is deposited in China National Microbiology Data Center (NMDC) with accession numbers NMDCN0002P0P (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://nmdc.cn/resource/genomics/sequence/detail/NMDCN002P0P\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Assessmen of plastic degradation by PDBF01\u003c/h2\u003e\n \u003cp\u003ePDBF01 was capable of growing on media with PVA as the sole carbon source. In contrast, PDBF01 did not grow on media containing PLA, PP, or PVC as the sole carbon source, consistent with polymer specificity for biodegradation. After culturing PDBF01 (160mg dry weight) in 100mL pure PVA solution for 7 days, it was found that the dry weight of PDBF01 increased by 76 mg. The results showed that PDBF01 grew on PVA as a nutrient, further indicating PDBF01 has a degradation effect on PVA.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Effect of temperature on PVA degradation rate\u003c/h2\u003e\n \u003cp\u003eTemperature is an important element affecting microbial metabolism and can directly influence degradative properties of microorganisms by affecting activity of secreted degrading enzymes [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. PDBF01 exhibited the highest degradation rate of PVA at 28\u0026deg;C (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The degradation rate presented a decreasing trend as the temperature continues to rise. Higher temperatures resulted in reduced degradation rates, attributable to adverse effects on enzymatic activity. Similar phenomena were observed in a previous study [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. PDBF01 grew among the experimental temperature (24\u0026ndash;40\u0026deg;C), but the growth rate decreased after exceeding 28 \u0026deg; C (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB), which was conformed to the law of temperature affecting the growth of fungi. Excessive temperature can lead to protein denaturation and death of fungal cells, while low temperature can inhibit fungal metabolic activity and reproductive ability.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Effect of inoculum concentration of strain PDBF01 on PVA degradation rate\u003c/h2\u003e\n \u003cp\u003eInoculum concentration can affect growth rate by directly affecting thermodynamic properties and patterns of gene expression, thus affecting the properties of strains [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. Degradation rate of PDBF01 increased rapidly with increase of inoculum concentration up to 25% yet was stable at higher inoculum concentrations. Meanwhile PDBF01 gained 207.66 mg weight at a 25% inoculum concentration compared to the initial gauge (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. Degradation time on PVA degradation rate\u003c/h2\u003e\n \u003cp\u003eThe microbial degradation of plastics is extremely slow and the optimal degradation time of different plastics is variable and strain-dependent [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. PDBF01 degradation rate of PVA increased with the increase of degradation time to a stable maximum at 21 d post-inoculation (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6. Composition of medium on PVA degradation rate\u003c/h2\u003e\n \u003cp\u003eMedium composition can affect culture growth and product formation of microorganisms, further affecting the properties of microbial strains. Different substances added to medium can satisfy specific growth or property requirements [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. For example, Jingjing et al. [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e] previously reported that addition of potassium ions could promote growth of Lactobacillus plantarum and improve the freeze-drying resistance and storage stability of the strain. In our study, the degradation rate of PVA by PDBF01 increased to 58.83% under the same degradation conditions after the addition of electrolytes, which may relate to the influence of these ions on metabolism and physiological function regulation of the strain and/or the regulation of enzyme activity.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003ePVA is widely used in multiple industries due to its excellent physical and chemical properties. However, excessive accumulation of PVA in the environment can affect or even destroy the balance of the ecosystem due to the difficulty in recyclingof PVA [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. With the increasing severity of PVA pollution, increasingly strict environmental awareness have prompted researchers to seek effective methods to eliminate PVA waste and conducted extensive research on accelerating the degradation of PVA [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The significant effectiveness of microbial degradation of PVA and its environmentally friendly advantages have promoted it becoming a popular research topic [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Currently, various microbial strains capable of degrading PVA have been discovered, such as \u003cem\u003ePseudomonas sp., Bacillus sp., Aspergillus sp\u003c/em\u003e., etc. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], but there are few efficient strains. \u003cem\u003eFusarium oxysporum\u003c/em\u003e is a widely occurring bacterium in nature, which has been proven to have great potential for microbial degradation [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Kim et al., [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] previously found \u003cem\u003eF. oxysporum\u003c/em\u003e could effectively degrade an endocrine disrupting chemical, DEHP. In this study, we successfully obtained and identified through sequencing analysis an ascomycete strain from a degraded plastic handle as \u003cem\u003eFusarium oxysporum\u003c/em\u003e. The strain was designated PDBF01 (CGMCC No.40272).\u003c/p\u003e \u003cp\u003eThere are differences in temperature demands for the growth of various microbial strains [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Meanwhile, it is generally necessary to choose the most suitable temperature range for their growth as the temperature for the degradation reaction to ensure high activity and vigorous metabolism of the strain, thus achieving efficient plastic degradation. Excessive temperature induce protein denaturation and accumulations of toxic substances such as reactive oxygen species, which leading to DNA damage and other direct impacts on the growth of the strains [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Higher temperature may lead a decrease or loss of enzyme activity. The loss of key metabolic enzyme activity affects the degradation performance of the strain [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Previous research has found that fungi may enter a dormant state to adapt to low-temperature environment, and the growth rate of the strain slows down or stagnates [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Previous researches found the growth of fungi will significantly decrease under low-temperatures [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], which may related to the changes of metabolic pathways inside the fungi or inhibiting the activity of certain key enzymes. Therefore, selecting the appropriate temperature is crucial for the growth and potential application of the strain. In our study, the optimal temperature for PVA degradation by \u003cem\u003eFusarium oxysporum\u003c/em\u003e PDBF01 was 28 ℃, and the degradation efficiency of PDBF01 varied with temperature. Wilkes, R. A., \u0026amp; Aristilde, L. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] reported that the degradation rate of PVA by \u003cem\u003ePseudomonas spp\u003c/em\u003e. was also affected by temperature, showing the same trend as our results.\u003c/p\u003e \u003cp\u003eNormally, the degradation rate of plastic by microorganisms increases with the inoculum concentration of microbial strains [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. However, exceeding the upper limit concentration can actually cause a decrease in growth rate. These changes may be related to rate-limiting factors such as excessive consumption of nutrients in medium, accumulation of metabolites (forming metabolic feedback and inhibition), and altered metabolic pathway functions [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e],thereby affecting the degradation efficiency of plastics. Similar phenomena were observed in previous research. Wolski et al. [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] observed similar changes in pentachlorophenol degradation by \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. Hence, in our research, we validated different inoculation concentrations and selected the appropriate strain PDBF01 inoculation concentration (25%) to obtain better degradation effect on PVA.\u003c/p\u003e \u003cp\u003eThe degradation time of microbial degradation of plastics significantly impact the degradation rate. The degradation rate of plastic degraded by microorganisms will gradually increase with the extension of degradation time, which is related to adaption time and reproduction of strains in the process of degrading plastics to achieve efficient degradation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The adaptation period, reproduction period and degradation period of microorganisms may be influenced by various elements. In our study, it was found that after 28 days of degradation, further prolongation of degradation time did not show significant impacts on the growth of strain PDBF01 and its degradation rate of PVA. It may mainly be due to the reduction of substrate after PVA degradation, limiting the access to carbon sources and energy, weakening the growth momentum of the strain [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].The growth of strain PDBF01 has reached a saturation state in such specific environment. The changes may be related to an altered culture environment that is no longer suitable for the growth of the strain, as described previously [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Puiggen\u0026eacute; et al. [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] has also reported similar results and ascribed this effect as relating to the growth characteristics of the strain itself. A sharp decline in viable count occurred due to the abnormal differentiation and release of metabolite. Gao, R., Liu, R., \u0026amp; Sun, C. [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] also observed the same phenomenon and was committed to reducing the time of microbial degradation of plastics.\u003c/p\u003e"},{"header":"4. Materials and methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Materials and reagents\u003c/h2\u003e \u003cp\u003eThe PBDF01 F. oxysporum strain was isolated from a degraded plastic handle. Polyvinyl alcohol (PVA1788; polymerization degree of 1700, alcoholysis degree of 88%, purity of 99%) was purchased from Qiansheng Biotechnology Co. Ltd. (Hefei, China). Potato dextrose agar medium (PDA) and potato dextrose broth medium (PDB) were purchased from Aisiwei Biochemical Co., Ltd. (Hangzhou, China). Boric acid, iodine, potassium Iodide and other chemicals were analytical grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Screening and identification of strains\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e4.2.1. Screening and purification of strains\u003c/h2\u003e \u003cp\u003eThe strains were screened and purified by previously-established methods with slight modification [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Environmental isolates (three replicate samples) were cultured for 30 d in a shaking incubator (28\u0026deg;C, 150 r/min oscillation) in 50 mL PDB medium. Mixed strain cultures displaying adequate growth potential were streaked on PDA medium and cultured at 30\u0026deg;C in the constant temperature incubator for 10 d. The three main colonies grown were isolated and inoculated in PDB medium for culture expansion. Cultures were reinoculated to PDA medium through repeated growth cycles to isolate and purify the strains until a single dominant colony per plate was produced.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e4.2.2. Identification of strains\u003c/h2\u003e \u003cp\u003ePreliminary identification of the strain isolate was performed by microscopy for assessment of colony and microscopic morphology. We further characterized the isolate strain through molecular identification, DNA extraction, PCR amplification, electrophoresis detection, ITS rDNA sequencing, EF-1α sequence analysis and homology comparison according to the FMIC-QO01-003 fungal polyphase identification and detection method and QO-03-02 operating procedures for Molecular Biological Identification of Microbial Strains [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSpecifically, the strains obtained were streak cultured on PDA medium for 7 days to observe the size, color, edge type and surface characteristics of the colonies. A single colony was selected for microscopic observation to examine its morphology, hyphal morphology, spore morphology and other characteristics. After preparing a single strain into a fungal suspension, it was mechanically crushed. Then SDS was added for cell lysis. DNA was extracted and precipitated using PCI. Design primers for PCR amplification of the ITS region of the strain by using the universal primers ITS1 and ITS4 reported in previous reports. The PCR amplification were performed under the following procedur: pre denaturation at 94 \u0026deg; C for 5 minutes, denaturation at 94 \u0026deg; C for 45 seconds, annealing at 55 \u0026deg; C for 45 seconds, extension at 72 \u0026deg; C for 1 minute, and 35 cycles. The specific bands of PCR products were detected by 1.5% agarose gel electrophoresis and DNAMARKER was set as a molecular weight indicator. PCR products for Sanger sequencing was recovered by gel recovery kit. Perform Blastn analysis on the sequencing results in the NCBI database and select the species with the highest similarity for phylogenetic analysis. Neighbor joining phylogenetic trees in ITS rDNA sequence and EF-1α sequence of the strain for bootstrap analysis was constructed using MEGA5.0 software.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Screening of degradable plastics\u003c/h2\u003e \u003cp\u003eAssessment of plastic polymer degradation by the PDBF01 isolate was performed by previous methods, with modification [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The isolated strain was cultured in PDA medium for 3d before transferring the mycelium from the young margin of the colony to PDB liquid medium for 7d. Then, PDBF01 cultures (1 mL) were diluted to an OD600 solution value between 0.8 and 1. Wash the precipitated strain with clean water after centrifuging the strain solution under 5000rpm for 10 minutes, and perform vacuum drying at -40 ℃ before weighing the dry weight. Polyvinyl chloride (PVC), polypropylene (PP), PVA, or polylactic acid (PLA; 0.2 g ea.) was added as the sole carbon source to sterilized, carbon-free medium, inoculated with diluted PDBF01 (16mg of dry weight), and cultured for 30 days at 28\u0026deg;C. Identification of polymer degradation was performed by observation of colony growth pattern and weight changes in dry weight of the strain PDBF01.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Optimization of degradation conditions of PVA\u003c/h2\u003e \u003cp\u003ePVA content was assessed as reported with slight modifications. Briefly, the PVA standard solution (20 mg/L) was scanned at full wavelength ranging from 400 to 700 nm, and the maximum absorbance was found at 690nm. The absorbance of the solution at 690 mm wavelength was determined by spectrophotometry after PVA reacting with iodine in boric acid medium. PVA was quantified by standard curve.\u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e4.4.1. Degradation temperature\u003c/h2\u003e \u003cp\u003e20mL of PDBF01 liquid solutions (160 mg dry weight of PDBF01; 20% solution, v/v; 3 replicates ea.) cultured under 2.3 conditions was inoculated to 100mL PVA solution (1g/L) and incubated in a shaking incubator (35 d, 150 r/min oscillation) at 24\u0026deg;C, 28\u0026deg;C, 32\u0026deg;C, 36\u0026deg;C, or 40\u0026deg;C. Content of PVA and changes in dry weight of PDBF01 was assessed at 35 d post-inoculation. Solutions only with PVA were used as controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e4.4.2. Inoculum concentration of strain\u003c/h2\u003e \u003cp\u003ePDBF01 (i.e. strain with dry weight of 40mg, 120mg, 200mg, 280mg, 360mg) at concentrations of 5%, 15%, 25%, 35%, or 45% (v/v; 3 replicates ea.) cultured under 2.3 conditions was inoculated to 100mL PVA solution (1g/L) and cultured in a shaking incubator (28\u0026deg;C, 150 r/min oscillation) for 35 d. Content of PVA and changes in dry weight of PDBF01 was assessed at 35 d post-inoculation. Solutions only with PVA were used as controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e4.4.3. Degradation time\u003c/h2\u003e \u003cp\u003e25mL of PDBF01 liquid solutions (200 mg dry weight of PDBF01; 20% solution, v/v; 3 replicates ea.) cultured under 2.3 conditions was inoculated to 100mL PVA solution (1g/L) and incubated in a shaking incubator (28\u0026deg;C, 150 r/min oscillation). Content of PVA and changes in dry weight of PDBF01 was assessed at 7, 14, 21, 28, and 35 d post-inoculation. Solutions only with PVA were used as controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e4.4.4. Composition of medium on PVA degradation rate\u003c/h2\u003e \u003cp\u003e25mL of PDBF01 liquid solutions (200 mg dry weight of PDBF01; 25% solution, v/v; 3 replicates ea.) cultured under 2.3 conditions was inoculated to 100mL PVA solution (1g/L). Then 10 mg K\u003csup\u003e+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, and Ca\u003csup\u003e2+\u003c/sup\u003e (10%, w/v) was added into culture solutions and incubated in a shaking incubator (28\u0026deg;C, 150 r/min oscillation). Content of PVA and changes in dry weight of PDBF01 was assessed at 35 d post-inoculation. Solutions only with PVA were used as controls.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.5. Statistical analysis\u003c/h2\u003e \u003cp\u003eBonferroni-corrected (α\u0026thinsp;=\u0026thinsp;0.05) multiple comparisons were performed by one-way ANOVA followed by a Duncan means separation test using the SPSS 13.0 software package. Data are represented as means (\u0026plusmn;\u0026thinsp;SD).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, methodology, software, validation, formal analysis, , investigation, resources, Z.X., L.C. and C.H.; Fund acquisition, data curation, S.J.Y. and C.H.; Writing\u0026mdash;original draft preparation, writing\u0026mdash;review and editing, supervision, Z.X., L.C. S.J.Y. and C.H. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financed by the Jiangsu Agriculture Science and Technology Innovation Fund (JASTIF) (CX(21)3113). Youth Science and Technology Fund (YJ(2021)009) of Jiangsu Yanjiang Institute of Agricultural Science.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequence data for the strain used in this study is deposited in China National Microbiology Data Center (NMDC) with accession numbers NMDCN0002P0P (https://nmdc.cn/resource/genomics/sequence/detail/NMDCN002P0P).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge Jiangsu Yanjiang Institution of Agricultural Science for assistance in this work. We thank LetPub (www.letpub.com) for linguistic assistance and pre-submission expert review.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLaw, K. L., \u0026amp; Narayan, R. (2022). Reducing environmental plastic pollution by designing polymer materials for managed end-of-life. Nature Reviews Materials, 7(2), 104-116.\u003c/li\u003e\n\u003cli\u003eKale, S. K., Deshmukh, A. G., Dudhare, M. S., \u0026amp; Patil, V. B. (2015). Microbial degradation of plastic: a review. Journal of Biochemical Technology, 6(2), 952-961.\u003c/li\u003e\n\u003cli\u003eJaiswal, S., Sharma, B., \u0026amp; Shukla, P. (2020). Integrated approaches in microbial degradation of plastics. Environmental Technology \u0026amp; Innovation, 17, 100567.\u003c/li\u003e\n\u003cli\u003eBooth, G. H., Cooper, A. W., \u0026amp; Robb, J. 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Archives of Microbiology, 203(9), 5417-5423.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Fusarium oxysporum, polyvinyl alcohol, biological identification, degradation properties","lastPublishedDoi":"10.21203/rs.3.rs-3834003/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3834003/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMassive accumulation of plastics into environment has caused enormous pressure on the ecosystem. Efficient and environmentally friendly plastics degradation technologies have evolved into a global ecological challenge. Microbial degradation, as an eco-friendly plastic treatment technology, is confronted with a problem of low efficiency in its current application. Hence, it is crucial to discovery plastic biodegradable microorganisms and find the optimal conditions for their action. The aim of our study is to isolate plastic-biodegrading fungi and explore optimum conditions for their action. A strain isolate of \u003cem\u003eFusarium oxysporum\u003c/em\u003e was obtained from a degraded plastic handle through screening, separation, and purification and designated PDBF01 (CGMCC No.40272). In a screening assay of plastic polymers, PDBF01 only exhibited the degradability to polyvinyl alcohol (PVA), with no activity toward polyvinyl chloride, polypropylene, or polylactic acid. PVA degradation efficiency of PDBF01 was significantly affected by inoculum concentration, temperature, and degradation time. PDBF01 produced significant degradation of PVA under 28\u0026deg;C and 25% inoculum concentration. Moreover, the highest degradation rate reached 51.26% after 21 days. PVA degradation rate of PDBF01 was further increased to 58.83% by the addition of electrolytes (K\u003csup\u003e+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, and Ca\u003csup\u003e2+\u003c/sup\u003e). Our results suggested PDBF01 can be used as a potential and efficient PVA-degrading strain in practical applications.\u003c/p\u003e","manuscriptTitle":"Discovery of a polyvinyl alcohol-degrading strain of the ascomycete Fusarium oxysporum and optimizing of its degradation performance of PVA","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-08 19:40:03","doi":"10.21203/rs.3.rs-3834003/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":"a42ee31a-5c3d-4f60-b1c3-211820f4bb47","owner":[],"postedDate":"January 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":27991990,"name":"Biological sciences/Biotechnology"},{"id":27991991,"name":"Biological sciences/Biotechnology/Environmental biotechnology"}],"tags":[],"updatedAt":"2024-04-22T03:24:42+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-08 19:40:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3834003","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3834003","identity":"rs-3834003","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}