Feasible Analytical Protocol of Residual polymers in Culture Medium after Biodegradation Testing

Feasible Analytical Protocol of Residual polymers in Culture Medium after Biodegradation Testing | 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 Feasible Analytical Protocol of Residual polymers in Culture Medium after Biodegradation Testing Yuta Sawanaka, Junko Torii, Yuya Tachibana, Ken-ichi Kasuya This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6191631/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 development of biodegradable polymers and their certification require an analytical method that is both reliable and practical. Although biochemical oxygen demand (BOD) testing remains a robust method for confirming polymer metabolism, it does not provide precise information about the residual compounds during and after biodegradation. Moreover, direct analysis these residues is challenging, particularly when seawater interferes with analysis. In this study, we propose an extraction/chemical-structure-analysis/molecular-mass-analysis protocol as an enhanced analytical approach for investigating the culture medium post-BOD biodegradation testing in seawater. We conducted BOD biodegradation test of poly(3-hydroxybutyrate- co -3-hydroxyvalerate), poly( ε -caprolactone), and poly(butylene succinate) in seawater. Following testing, 1 H NMR analysis of the extracts identified the chemical structures of the residual polymers and enabled the assessment of NMR degradability, which aligned well with the BOD biodegradability trend. Additionally, molecular-mass-analysis revealed changes in the molecular mass, supporting evaluation of the chain scission of polymer. This study advances analytical methods in the field of biodegradable polymers. Physical sciences/Chemistry/Green chemistry/Sustainability Physical sciences/Materials science/Techniques and instrumentation/Characterization and analytical techniques Figures Figure 1 Figure 2 Figure 3 Introduction Biodegradable polymers have attracted much attention as a potential solution to the environmental pollution caused by plastic waste 1 , 2 . In particular, plastic pollution in the ocean is a serious problem because the ocean is the final definition of plastics. To develop biodegradable polymers and certify their biodegradability, it is indispensable to establish a feasible and reliable evaluation method 3 . Although the weight loss method 4 does not provide direct information on biodegradability, it is widely used for monitoring polymer disintegration because of its simplicity. In contrast, methods 5 – 7 such as measuring carbon dioxide evolution (CO 2 evolution) 8 , 9 and monitoring O 2 consumption through biochemical oxygen demand (BOD) measurements 10 , 11 have been developed to evaluate polymer biodegradability and have been applied in studies of biodegradable polymers. Since polymer degradation involves hydrolysis followed by microbial metabolism of the hydrolysates 6 , 12 , CO 2 evolution and O 2 consumption serve as suitable indicators. As a result, the International Organization for Standardization (ISO), American Society for Testing and Materials (ASTM), and Organization for Economic Co-operation and Development (OECD) have standardized various testing protocols. 5 – 7 These include CO 2 evolution measurements, as outlined in ISO 9439, 13 and OECD 301 B 14 , as well as BOD measurements detailed in ISO 18830 15 and ISO23977 16 . To gain a comprehensive understanding of the biodegradation process, researchers often combined additional methods, such as total organic carbon (TOC) analysis 17 , 18 , infrared (IR) spectroscopy 19 , and mass spectrometry (MS) 20 . These techniques offer deeper insights into the residual polymers, hydrolysates, and biomass. TOC analysis, which measures the carbon content in a sample, is a standard method for detecting organic substances in the medium after biodegradation testing based on O 2 consumption and CO 2 evolution. However, this method does not provide information on the chemical structures, molecular mass, and composition ratios of the residual polymer, partially degraded compounds, and microbially derived biomass. To obtain these details, some researchers employ IR spectroscopy, liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) as post-biodegradation evaluation methods. 21 , 22 In addition to the difficulty of analyzing residual polymers during ongoing biodegradation, organic substances from microorganisms can interfere with these analyses and further complicate data interpretation. Furthermore, the inorganic and organic substances in seawater add complexity because seawater contains 35 g L − 1 inorganic compounds, 0.9 mg L − 1 organic compounds. 23 In this study, we propose a refined analytical approach to examine the culture medium post-BOD biodegradation testing in seawater. First, we confirmed the quantitative recovery of the polymer from the culture medium in seawater using an extraction protocol. Next, we performed BOD biodegradation testing on the biodegradable polymers 24 , 25 i.e., poly(3-hydroxybutyrate- co -3-hydroxyvalerate) (PHBV) 26 , poly( ε -caprolactone) (PCL) 26 , and poly(butylene succinate) (PBSu) 27 , in seawater. The organic substances were extracted from the culture medium for further analyses. SEC analysis revealed that these extracted organic substances contained the residual polymers with or without hydrolysis. Additionally, a correlation was identified between the degradability estimates derived from 1 H NMR analyses and BOD biodegradability. Results and Discussion Extraction efficiency. To confirm the quantifiability of the extraction protocol from the culture medium of BOD biodegradation testing, a mixture of BOD culture medium using seawater and PHBV, PCL, or PBSu, which can dissolve in chloroform, was prepared. Because the terminal carboxylic groups were in the carboxylate form in the pH 7.8 medium, the medium was acidified to pH 4 to protonate the carboxylate group back to the carboxylic acid form. The organic substances were extracted with chloroform. The extraction efficiency of each polymer was determined from 1 H NMR spectra, using nitrobenzene as an internal standard, yielding the average extraction efficiencies of PHBV, PCL, and PBSu were found to be 101 ± 4%, 104 ± 3%, and 99 ± 4%, respectively. The SEC curves of the extracted residue overlapped with those of the corresponding polymers before extraction, as shown in Figure S1 , confirming the quantitative recovery without any loss in molecular mass. Therefore, this protocol ensures reliable polymer quantification in BOD biodegradation studied. BOD biodegradation testing. Next, we used the extraction protocol to evaluate residual polymers in BOD biodegradation tests with seawater. PHBV and PCL are recognized as biodegradable polymers in seawater. On the other hand, PBSu is employed as a hardly degradable polymer in seawater, while it well degraded in soil and compost. 28 To recover the residual polymers during the biodegradation in progress, the BOD biodegradation testing of each culture media of PHBV, PCL, and PBSu was stopped at 6 days (Fig. 1 a, Figure S2, and Table 1 ). The BOD biodegradability of PHBV, PCL, and PBSu after 6 days was 62%, 18%, and 4%, respectively. Additionally, the other culture media were incubated for 30 days (Fig. 1 b, Figure S3, and Table 1 ). The biodegradability after 30 days was 84%, 37%, and 6%, respectively. The BOD curve of PHBV reached a plateau after 30 days, indicating near-complete biodegradation. The shortfall from 100% is typically attributed to biomass formation by the microorganisms. 29 Table 1 BOD biodegradability and NMR degradability of polymers. Polymer Incubation period / day Run BOD biodegradability / % Average BOD degradability / % NMR degradability a / % Average NMR degradability / % PHBV 6 1 56 63 ± 9 80 80 ± 13 2 – b 67 3 69 93 30 1 85 84 ± 1 > 99 > 99 ± n.d. 2 83 > 99 3 84 > 99 PCL 6 1 25 18 ± 10 34 25 ± 13 2 23 31 3 6 9 30 1 38 37 ± 6 46 46 ± 9 2 42 55 3 30 37 PBSu 6 1 < 1 4 ± 6 < 1 2 ± 3 2 11 < 1 3 < 1 5 30 1 1 6 ± 8 11 7 ± 4 2 15 3 3 3 5 a Estimated from the 1 H NMR spectra with nitrobenzene as an internal standard. b BOD biodegradability was negative owing to the significant failure. To obtain chemical information from the culture medium after BOD biodegradation testing, we analyzed the extract from the culture medium by 1 H NMR and SEC measurements (Fig. 2 , Fig. 3 , and Figure S4 − 14). In the 1 H NMR spectra of the extract from the blank media, the peaks around 0.8–1.2, and 4.3 ppm were observed (Figure S4). The SEC chart also reveals peaks in the lower molecular region (Figure S5). These peaks were derived from the organic substances present in the seawater and were observed in the spectra of the extract with the polymers. We used the peak of proton a adjacent to the oxygen (− O−C H R − or − O−C H 2 −) moiety in the polymer to quantitatively evaluate each residual polymer. The changes in the peak of each proton a of the extracts from the media at 0, 6, and 30 days are depicted in Fig. 2 . In PHBV, the peak of proton a in decreased with ongoing biodegradation and was mostly absent after 30 days (Fig. 2 a). In PCL, the peak of proton a also declined (Fig. 2 b). In PBSu, the peak of proton a showed only a slight decrease after 30 days (Fig. 2 c). NMR degradability. The decrease in the peak intensity reflects polymer loss during BOD biodegradation testing. To compare the decrease in peak intensity in 1 H NMR spectra and BOD biodegradability, we introduce “NMR degradability” in Eq. (3), which was derived from the reduction of the polymer in the 1 H NMR spectra. Although the culture medium contains numerous organic substances, hydrolysates, e.g. hydroxycarboxylic acid, diol, and dicarboxylic acid, are rapidly metabolized or remain unextractable in chloroform. Some metabolites could be extracted, but their peaks were identified in the blank extract and thus did not interfere with the analysis. Consequently, NMR degradability indicates the degradation of the polymer. The BOD biodegradability and NMR degradability values were listed in Table 1 . The NMR degradabilities of PHBV and PCL were marginally higher than their BOD biodegradabilities, suggesting that some of the polymers transformed into other organic substances via microbial assimilation rather than being fully mineralized to a carbon dioxide. Notably, the BOD biodegradability of PHBV after 6 days in Run 2 was negative, while its NMR degradability remained comparable to that of other runs. It is uncertain whether this anomaly reflects a true measurement or an error caused by external factors or instrumentation. Indeed, the BOD equipment employed in this study often produces such large errors. 29 The NMR degradability thus helps validate the results. In contrast, the BOD biodegradabilities and NMR degradabilities of PBSu were nearly identical, indicating extremely low biodegradability in seawater and complete recovery of residual PBSu. SEC analysis of extracts. Molecular mass is crucial for assessing the biodegradation process, because polymer biodegradation initially involves fragmentation into low-molecular mass compounds assimilable by microorganisms. To examine the molecular mass of the residual polymer, SEC analysis of the extracts was performed (Fig. 3 ). In the blank extracts, the peak appeared at 18 − 20 min, corresponding to the organic substances present in seawater. (Figure S5). 23 For the extracts of PHBV media at 0 day, the peaks of the extracts were observed at 11 − 18 min, and the average peak molecular weight was 155 kg·mol − 1 (Fig. 3 a). After 6 days and 30 days, the SEC curves nearly overlapped with those of the blank extract (Fig. 3 a), suggesting a low recovery due to extensive PHBV degradation in the culture media. For PCL on 0 day, the peaks appeared at 13 − 18 min, with the average peak molecular weight of 50 kg·mol − 1 (Fig. 3 b). At 30 days, the average peak molecular weight shifted to 39 kg·mol − 1 and the peak broadened with the incubation period (Fig. 3 b), indicating PCL hydrolysis during the BOD biodegradation testing. For PBSu on 0 day, peaks were observed at 15 − 20 min and the average peak molecular weight was 12 kg·mol − 1 (Fig. 3 c). At 30 days, the average peak molecular weight shifted to 9.6 kg·mol − 1 , broadening over time, indicating slight PBSu hydrolysis despite its negligible BOD biodegradability within 30 days (Fig. 3 c). This implies that PBSu underwent further degradation over an extended period. Conclusion Until now, the direct analysis of residual polymers from incubation media has been challenging due to the lack of established analytical protocols and the interfering effects of seawater. We demonstrated that our chloroform-based extraction method serves as a feasible analytical approach for post-BOD biodegradation testing. The extraction of PHBV, PCL, and PBSu from BOD biodegradation media was quantitative. Subsequently, 1 H NMR analysis of the extract not only identified the chemical structures of residual polymers but also allowed NMR degradability to be evaluated, which correlated with BOD biodegradability. Additionally, SEC analysis revealed changes in the molecular mass, clarifying polymer hydrolysis. Hence, the extraction–NMR–SEC protocol proposed in this study enables effective analysis of residual polymers after BOD biodegradation. Furthermore, it is both accessible and reliable and requires no specialized equipment or technicians. Methods Reagents and materials. Poly(hydroxybutyrate- co -hydroxyvalerate) (PHBV) was purchased from HighChem Co., Ltd. Polycaprolactone (PCL) was kindly supplied from Daicel Co., Ltd. Succinic acid (SA), KH 2 PO 4 , and NH 4 Cl were purchased from FUJIFILM Wako Pure Chemical Co. (Osaka, Japan). Hydrochloric acid (HCl), chloroform (CHCl 3 ), chloroform- d (CDCl 3 ), and Na 2 SO 4 was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Methanol and 1,4-butanediol (BD) were purchased from Kishida Chemical Co., Ltd. (Osaka, Japan). N -allylthiourea and titanium tetraisopropoxide (Ti(O i Pr) 4 ) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All chemicals used were reagent grade. PHBV, PCL and PBSu were purified by reprecipitation before using with chloroform and methanol. Other chemicals were used without further purifications. Recovery efficiency of polymer from BOD solution with seawater. The polymer i.e., PHBV (8 mg), PCL (6 mg), and PBSu (8 mg), was added to the BOD culture medium (200 mL) with seawater based on ASTMD6691-17 and the mixture was stirred for 1 h. After acidification with 1 M hydrochloric acid (2.0 mL), the culture medium was extracted three times with chloroform (50 mL, 30 mL, and 30 mL). The organic layers were combined and dried over with anhydrous sodium sulfate (Na 2 SO 4 ). The filtrate was evaporated in vacuo to obtain the residue. After adding nitrobenzene (0.49 mmol) as an internal standard for 1 H NMR analysis, all the residues were dissolved in chloroform- d 1 (2.0 mL). Recovery efficiency (%) was estimated using the following equation: Recovery efficiency (%) = x f / x i × 100 (1) where ( x i ) is the initial weight of polymer, ( x f ) is the residual weight of polymer estimated from the integral ratio between the protons of o -position of nitro benzene and the proton(s) adjacent to the oxygen (− O−C H R − or − O−C H 2 −) moiety in polymer. BOD biodegradation testing. The BOD biodegradability of the samples by the aerobic microorganisms from seawater was determined by measuring oxygen consumption with a BOD instrumentation (OxiTop-C measuring head with a 300-mL BOD reactor, WTW GmbH, Weilheim, Germany) referring to ASTM D6691-17. Seawater collected from quay of Yokosuka Headquarters in Japan agency marine-earth science and technology (JAMSTEC), which was filtered through a filter paper (ADVANTEC No. 2), was used as the base medium for a BOD biodegradation testing. Then, the filtrate was allowed to stand for 1 day before the testing. The base medium (1.0 L) was filtrated using a membrane filter (MEMBRANE FILTER mixed cellulose ester, φ = 0.45 µm, ADVANTEC) and the filter was added to the base medium (10 mL) to prepare the condensed seawater. The BOD medium contained 199.0 mL of the base medium and KH 2 PO 4 , 0.1 g·L − 1 , NH 4 Cl, 0.5 g·L − 1 , N -allylthiourea 5.0 mg·L − 1 , 1.0 mL of the concentrated seawater as an additional inoculum. A sample (ca. 4 mg) was placed in the BOD reactor, and 200 mL of the BOD medium was added to the BOD reactor. Two bottles without a sample were also prepared as a blank medium. The OxiTop-C measuring head was attached to the head of a BOD reactor, and the reactor was incubated at 30°C. The BOD biodegradability of BD, SA, and PBSu was defined as follows: BOD biodegradability (%) = (BOD sample (mg) – BOD blank (mg)) / ThOD (mg) × 100 (2) Here, BOD sample and BOD blank are the experimentally observed values of oxygen demand of BD, SA, and PBSu media and the blank media, respectively. ThOD is a theoretically calculated value of oxygen demand of a sample, which was obtained by assuming that the sample completely degraded into CO 2 and H 2 O. Synthesis of PBSu. A 100 mL flask was equipped with a nitrogen gas inlet and outlet. BD (92 mmol), SA (20 mmol), and titanium tetraisopropoxide (77 µmol) was added to the flask under a dry nitrogen gas flow. The reaction mixture was stirred at 200 ℃ under a dry nitrogen gas flow to remove the water and BD produced during the esterification reaction. After 1 h, titanium tetraisopropoxide (77 µmol) was added and the mixture was stirred at 200 ℃ under reduced pressure (50 Pa) for 1 h. PBSu was corrected as a white solid. PBSu was dissolved in a chloroform (50 mL) and then the solution was poured into a methanol (700 mL). PBSu (11.5 g) was obtained as a white solid. Before BOD biodegradation testing, the PBSu was reprecipitated with a chloroform and methanol, which was used for the experiments. Declarations Author contributions statement Y. S and Y. T. designed the study. Y. S. wrote original draft and Y. S., Y. T. and K. K. wrote the paper. Y. S. and J. T. conducted all experiments. Y. T. and K. K. are the corresponding authors. All the authors participated in the analysis and discussion of the results. Declaration of Competing Interest The 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. Data and materials availability All data needed to evaluate the conclusions in the paper are present in the paper and the Supplementally Information. Any additional data related to this paper may be requested from Yuya Tachibana ( [email protected] ) or Ken-ichi Kasuya ( [email protected] ). Author Contribution Y. S and Y. T. designed the study. Y. S. wrote original draft and Y. S., Y. T. and K. K. wrote the paper. Y. S. and J. T. conducted all experiments. Y. T. and K. K. are the corresponding authors. All the authors participated in the analysis and discussion of the results. Acknowledgement Yuya Tachibana is grateful for financial support from JSPS KAKENHI Grant-in-Aid for Scientific Research (C) Number 20K12233. Ken-ichi Kasuya is grateful for financial support from the New Energy and Industrial Technology Development Organization (NEDO), project code JPNP14004. NMR measurements were performed on a NM-ECS400 NMR spectrometer and JNM-ECA600 NMR spectrometer (JEOL Ltd., Tokyo, Japan) at the Center for Instrumental Analysis of Gunma University. The seawater was collected from the shore of the Japan Agency for Marine -Earth Science and Technology (JAMSTEC) facing the Tokyo Bay of Japan. Data Availability All data needed to evaluate the conclusions in the paper are present in the paper and the Supplementally Information. Any additional data related to this paper may be requested from Yuya Tachibana ( [email protected] ) or Ken-ichi Kasuya ( [email protected] ). References Narancic, T. et al. Biodegradable Plastic Blends Create New Possibilities for End-of-Life Management of Plastics but They Are Not a Panacea for Plastic Pollution. Environ. Sci. Technol. 52 , 10441–10452 (2018). Fagnani, D. E. et al. 100th Anniversary of Macromolecular Science Viewpoint: Redefining Sustainable Polymers. ACS Macro Lett. 10 , 41–53 (2021). Kim, M. S. et al. A Review of Biodegradable Plastics: Chemistry, Applications, Properties, and Future Research Needs. Chem. Rev. 123 , 9915–9939 (2023). Sheth, M., Kumar, R. A., Davé, V., Gross, R. 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Controlling the Marine Biodegradation Profile and Mechanical Properties of Poly(ε-caprolactone) with Hydrophobic Water-Responsive Linkages. ACS Appl. Polym. Mater. acsapm.3c01843 (2023). Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6191631","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":430099530,"identity":"bc428fc2-5195-4f25-8311-a0962396cd8b","order_by":0,"name":"Yuta Sawanaka","email":"","orcid":"","institution":"Gunma University","correspondingAuthor":false,"prefix":"","firstName":"Yuta","middleName":"","lastName":"Sawanaka","suffix":""},{"id":430099531,"identity":"38c491d8-e4ca-4caf-aad8-222f75ca64df","order_by":1,"name":"Junko Torii","email":"","orcid":"","institution":"Gunma University","correspondingAuthor":false,"prefix":"","firstName":"Junko","middleName":"","lastName":"Torii","suffix":""},{"id":430099532,"identity":"5adb8aef-c481-4c9a-ab16-3e98bfab6652","order_by":2,"name":"Yuya Tachibana","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYHACNobEBiDF3oAsyINbPQ9cC88BUrQwgrRIJBDpKnv2w88ePNxhl8c/8/k16cIcOwaDA8wPPzDI3MFtC0+auUHimeRiids5ZdIztyUDtbAZSzDwPMPjsBw2icQ25sSG2zlp0rzbmOs3HGAwA4ofxq2F/w1IS33i/JtnQFrqgbawf8OvRQJsy+HEDTfYjwG1HAZq4SFgy41nZhKJZ44nbjyTw2zNu+04g+RhnmKJBDx+Ye9Pfib5c0d14rzjxx/e5t1WzcB3vH3jh489uEMM2UIDCM0MxIk9B4jRwv4AifODKC2jYBSMglEwMgAAgnlSKoNsUJ0AAAAASUVORK5CYII=","orcid":"","institution":"Gunma University","correspondingAuthor":true,"prefix":"","firstName":"Yuya","middleName":"","lastName":"Tachibana","suffix":""},{"id":430099534,"identity":"7cd60fe7-84e0-44f8-a4ba-5f1155609d02","order_by":3,"name":"Ken-ichi Kasuya","email":"","orcid":"","institution":"Gunma University","correspondingAuthor":false,"prefix":"","firstName":"Ken-ichi","middleName":"","lastName":"Kasuya","suffix":""}],"badges":[],"createdAt":"2025-03-10 04:08:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6191631/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6191631/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78738293,"identity":"163df2fd-98c1-4177-914f-068717422eba","added_by":"auto","created_at":"2025-03-18 08:48:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":172663,"visible":true,"origin":"","legend":"\u003cp\u003eBOD biodegradation curves of PHBV (green), PCL (blue), and PBSu (magenta) for \u003cstrong\u003ea\u003c/strong\u003e.6 days and \u003cstrong\u003eb\u003c/strong\u003e.30 days in seawater. The bar indicates the error bar. The average BOD biodegradability was represented in parentheses.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6191631/v1/22384ee15ad49f26be6b91f3.png"},{"id":78738128,"identity":"24665e6d-a92c-4ddb-8b58-872e50a81978","added_by":"auto","created_at":"2025-03-18 08:40:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":134829,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR changes in residual polymers from the culture media of \u003cstrong\u003ea\u003c/strong\u003e. PHBV, \u003cstrong\u003eb\u003c/strong\u003e. PCL, and \u003cstrong\u003ec\u003c/strong\u003e. PBSu during BOD biodegradation testing. The peak intensity was normalized using an internal standard.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6191631/v1/55ccb790049aac54de4cade3.png"},{"id":78738130,"identity":"de993066-763d-4cb7-b1f9-44c471d1151e","added_by":"auto","created_at":"2025-03-18 08:40:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":261928,"visible":true,"origin":"","legend":"\u003cp\u003eSEC curves of the extract from the culture media of \u003cstrong\u003ea\u003c/strong\u003e. PHBV, \u003cstrong\u003eb\u003c/strong\u003e. PCL, \u003cstrong\u003ec\u003c/strong\u003e. PBSu, and the blank medium during BOD biodegradation testing.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6191631/v1/9cd3aa668030130a0250ca1e.png"},{"id":95224770,"identity":"019e2ef5-9955-4780-bfb6-d84c1ab06ae1","added_by":"auto","created_at":"2025-11-05 16:24:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1073781,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6191631/v1/517d6e2c-34ee-4bc8-8e70-ff830b7622b4.pdf"},{"id":78739472,"identity":"758cf9ee-30b1-42d1-aad0-93c786965342","added_by":"auto","created_at":"2025-03-18 08:56:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1332497,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6191631/v1/5bdb4a8bf9dd333b1f298d32.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Feasible Analytical Protocol of Residual polymers in Culture Medium after Biodegradation Testing","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBiodegradable polymers have attracted much attention as a potential solution to the environmental pollution caused by plastic waste\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In particular, plastic pollution in the ocean is a serious problem because the ocean is the final definition of plastics. To develop biodegradable polymers and certify their biodegradability, it is indispensable to establish a feasible and reliable evaluation method\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Although the weight loss method\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e does not provide direct information on biodegradability, it is widely used for monitoring polymer disintegration because of its simplicity. In contrast, methods\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e such as measuring carbon dioxide evolution (CO\u003csub\u003e2\u003c/sub\u003e evolution)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and monitoring O\u003csub\u003e2\u003c/sub\u003e consumption through biochemical oxygen demand (BOD) measurements\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e have been developed to evaluate polymer biodegradability and have been applied in studies of biodegradable polymers. Since polymer degradation involves hydrolysis followed by microbial metabolism of the hydrolysates\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, CO\u003csub\u003e2\u003c/sub\u003e evolution and O\u003csub\u003e2\u003c/sub\u003e consumption serve as suitable indicators. As a result, the International Organization for Standardization (ISO), American Society for Testing and Materials (ASTM), and Organization for Economic Co-operation and Development (OECD) have standardized various testing protocols.\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e These include CO\u003csub\u003e2\u003c/sub\u003e evolution measurements, as outlined in ISO 9439,\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and OECD 301 B\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, as well as BOD measurements detailed in ISO 18830\u003csup\u003e15\u003c/sup\u003e and ISO23977\u003csup\u003e16\u003c/sup\u003e. To gain a comprehensive understanding of the biodegradation process, researchers often combined additional methods, such as total organic carbon (TOC) analysis \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, infrared (IR) spectroscopy \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, and mass spectrometry (MS)\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. These techniques offer deeper insights into the residual polymers, hydrolysates, and biomass.\u003c/p\u003e \u003cp\u003eTOC analysis, which measures the carbon content in a sample, is a standard method for detecting organic substances in the medium after biodegradation testing based on O\u003csub\u003e2\u003c/sub\u003e consumption and CO\u003csub\u003e2\u003c/sub\u003e evolution. However, this method does not provide information on the chemical structures, molecular mass, and composition ratios of the residual polymer, partially degraded compounds, and microbially derived biomass. To obtain these details, some researchers employ IR spectroscopy, liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) as post-biodegradation evaluation methods.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e In addition to the difficulty of analyzing residual polymers during ongoing biodegradation, organic substances from microorganisms can interfere with these analyses and further complicate data interpretation. Furthermore, the inorganic and organic substances in seawater add complexity because seawater contains 35 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e inorganic compounds, 0.9 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e organic compounds.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn this study, we propose a refined analytical approach to examine the culture medium post-BOD biodegradation testing in seawater. First, we confirmed the quantitative recovery of the polymer from the culture medium in seawater using an extraction protocol. Next, we performed BOD biodegradation testing on the biodegradable polymers\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e i.e., poly(3-hydroxybutyrate-\u003cem\u003eco\u003c/em\u003e-3-hydroxyvalerate) (PHBV)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, poly(\u003cem\u003eε\u003c/em\u003e-caprolactone) (PCL)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, and poly(butylene succinate) (PBSu)\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, in seawater. The organic substances were extracted from the culture medium for further analyses. SEC analysis revealed that these extracted organic substances contained the residual polymers with or without hydrolysis. Additionally, a correlation was identified between the degradability estimates derived from \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR analyses and BOD biodegradability.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eExtraction efficiency. To confirm the quantifiability of the extraction protocol from the culture medium of BOD biodegradation testing, a mixture of BOD culture medium using seawater and PHBV, PCL, or PBSu, which can dissolve in chloroform, was prepared. Because the terminal carboxylic groups were in the carboxylate form in the pH 7.8 medium, the medium was acidified to pH 4 to protonate the carboxylate group back to the carboxylic acid form. The organic substances were extracted with chloroform. The extraction efficiency of each polymer was determined from \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR spectra, using nitrobenzene as an internal standard, yielding the average extraction efficiencies of PHBV, PCL, and PBSu were found to be 101\u0026thinsp;\u0026plusmn;\u0026thinsp;4%, 104\u0026thinsp;\u0026plusmn;\u0026thinsp;3%, and 99\u0026thinsp;\u0026plusmn;\u0026thinsp;4%, respectively. The SEC curves of the extracted residue overlapped with those of the corresponding polymers before extraction, as shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, confirming the quantitative recovery without any loss in molecular mass. Therefore, this protocol ensures reliable polymer quantification in BOD biodegradation studied.\u003c/p\u003e \u003cp\u003eBOD biodegradation testing. Next, we used the extraction protocol to evaluate residual polymers in BOD biodegradation tests with seawater. PHBV and PCL are recognized as biodegradable polymers in seawater. On the other hand, PBSu is employed as a hardly degradable polymer in seawater, while it well degraded in soil and compost.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e To recover the residual polymers during the biodegradation in progress, the BOD biodegradation testing of each culture media of PHBV, PCL, and PBSu was stopped at 6 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, Figure S2, and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The BOD biodegradability of PHBV, PCL, and PBSu after 6 days was 62%, 18%, and 4%, respectively. Additionally, the other culture media were incubated for 30 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, Figure S3, and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The biodegradability after 30 days was 84%, 37%, and 6%, respectively. The BOD curve of PHBV reached a plateau after 30 days, indicating near-complete biodegradation. The shortfall from 100% is typically attributed to biomass formation by the microorganisms.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBOD biodegradability and NMR degradability of polymers.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePolymer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncubation\u003c/p\u003e \u003cp\u003eperiod / day\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRun\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBOD\u003c/p\u003e \u003cp\u003ebiodegradability\u003c/p\u003e \u003cp\u003e/ %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAverage BOD\u003c/p\u003e \u003cp\u003edegradability\u003c/p\u003e \u003cp\u003e/ %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNMR\u003c/p\u003e \u003cp\u003edegradability\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e/ %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAverage NMR\u003c/p\u003e \u003cp\u003edegradability\u003c/p\u003e \u003cp\u003e/ %\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003ePHBV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e 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\u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003ePCL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e18\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e25\u0026thinsp;\u0026plusmn;\u0026thinsp;13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e37\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e46\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003ePBSu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e4\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e7\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e Estimated from the \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR spectra with nitrobenzene as an internal standard.\u003c/p\u003e \u003cp\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e BOD biodegradability was negative owing to the significant failure.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo obtain chemical information from the culture medium after BOD biodegradation testing, we analyzed the extract from the culture medium by \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR and SEC measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, and Figure S4\u0026thinsp;\u0026minus;\u0026thinsp;14). In the \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR spectra of the extract from the blank media, the peaks around 0.8\u0026ndash;1.2, and 4.3 ppm were observed (Figure S4). The SEC chart also reveals peaks in the lower molecular region (Figure S5). These peaks were derived from the organic substances present in the seawater and were observed in the spectra of the extract with the polymers. We used the peak of proton \u003cb\u003ea\u003c/b\u003e adjacent to the oxygen (\u0026minus;\u0026thinsp;O\u0026minus;C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003eR\u0026thinsp;\u0026minus;\u0026thinsp;or \u0026minus;\u0026thinsp;O\u0026minus;C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e2\u003c/sub\u003e\u0026minus;) moiety in the polymer to quantitatively evaluate each residual polymer. The changes in the peak of each proton \u003cb\u003ea\u003c/b\u003e of the extracts from the media at 0, 6, and 30 days are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. In PHBV, the peak of proton \u003cb\u003ea\u003c/b\u003e in decreased with ongoing biodegradation and was mostly absent after 30 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In PCL, the peak of proton \u003cb\u003ea\u003c/b\u003e also declined (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In PBSu, the peak of proton \u003cb\u003ea\u003c/b\u003e showed only a slight decrease after 30 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eNMR degradability. The decrease in the peak intensity reflects polymer loss during BOD biodegradation testing. To compare the decrease in peak intensity in \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR spectra and BOD biodegradability, we introduce \u0026ldquo;NMR degradability\u0026rdquo; in Eq.\u0026nbsp;(3), which was derived from the reduction of the polymer in the \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR spectra. Although the culture medium contains numerous organic substances, hydrolysates, e.g. hydroxycarboxylic acid, diol, and dicarboxylic acid, are rapidly metabolized or remain unextractable in chloroform. Some metabolites could be extracted, but their peaks were identified in the blank extract and thus did not interfere with the analysis. Consequently, NMR degradability indicates the degradation of the polymer. The BOD biodegradability and NMR degradability values were listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe NMR degradabilities of PHBV and PCL were marginally higher than their BOD biodegradabilities, suggesting that some of the polymers transformed into other organic substances via microbial assimilation rather than being fully mineralized to a carbon dioxide. Notably, the BOD biodegradability of PHBV after 6 days in Run 2 was negative, while its NMR degradability remained comparable to that of other runs. It is uncertain whether this anomaly reflects a true measurement or an error caused by external factors or instrumentation. Indeed, the BOD equipment employed in this study often produces such large errors.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e The NMR degradability thus helps validate the results. In contrast, the BOD biodegradabilities and NMR degradabilities of PBSu were nearly identical, indicating extremely low biodegradability in seawater and complete recovery of residual PBSu.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSEC analysis of extracts. Molecular mass is crucial for assessing the biodegradation process, because polymer biodegradation initially involves fragmentation into low-molecular mass compounds assimilable by microorganisms. To examine the molecular mass of the residual polymer, SEC analysis of the extracts was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the blank extracts, the peak appeared at 18\u0026thinsp;\u0026minus;\u0026thinsp;20 min, corresponding to the organic substances present in seawater. (Figure S5).\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e For the extracts of PHBV media at 0 day, the peaks of the extracts were observed at 11\u0026thinsp;\u0026minus;\u0026thinsp;18 min, and the average peak molecular weight was 155 kg\u0026middot;mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). After 6 days and 30 days, the SEC curves nearly overlapped with those of the blank extract (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), suggesting a low recovery due to extensive PHBV degradation in the culture media. For PCL on 0 day, the peaks appeared at 13\u0026thinsp;\u0026minus;\u0026thinsp;18 min, with the average peak molecular weight of 50 kg\u0026middot;mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). At 30 days, the average peak molecular weight shifted to 39 kg\u0026middot;mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the peak broadened with the incubation period (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), indicating PCL hydrolysis during the BOD biodegradation testing. For PBSu on 0 day, peaks were observed at 15\u0026thinsp;\u0026minus;\u0026thinsp;20 min and the average peak molecular weight was 12 kg\u0026middot;mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). At 30 days, the average peak molecular weight shifted to 9.6 kg\u0026middot;mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, broadening over time, indicating slight PBSu hydrolysis despite its negligible BOD biodegradability within 30 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). This implies that PBSu underwent further degradation over an extended period.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eUntil now, the direct analysis of residual polymers from incubation media has been challenging due to the lack of established analytical protocols and the interfering effects of seawater. We demonstrated that our chloroform-based extraction method serves as a feasible analytical approach for post-BOD biodegradation testing. The extraction of PHBV, PCL, and PBSu from BOD biodegradation media was quantitative. Subsequently, \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR analysis of the extract not only identified the chemical structures of residual polymers but also allowed NMR degradability to be evaluated, which correlated with BOD biodegradability. Additionally, SEC analysis revealed changes in the molecular mass, clarifying polymer hydrolysis. Hence, the extraction\u0026ndash;NMR\u0026ndash;SEC protocol proposed in this study enables effective analysis of residual polymers after BOD biodegradation. Furthermore, it is both accessible and reliable and requires no specialized equipment or technicians.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eReagents and materials. Poly(hydroxybutyrate-\u003cem\u003eco\u003c/em\u003e-hydroxyvalerate) (PHBV) was purchased from HighChem Co., Ltd. Polycaprolactone (PCL) was kindly supplied from Daicel Co., Ltd. Succinic acid (SA), KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, and NH\u003csub\u003e4\u003c/sub\u003eCl were purchased from FUJIFILM Wako Pure Chemical Co. (Osaka, Japan). Hydrochloric acid (HCl), chloroform (CHCl\u003csub\u003e3\u003c/sub\u003e), chloroform-\u003cem\u003ed\u003c/em\u003e (CDCl\u003csub\u003e3\u003c/sub\u003e), and Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Methanol and 1,4-butanediol (BD) were purchased from Kishida Chemical Co., Ltd. (Osaka, Japan). \u003cem\u003eN\u003c/em\u003e-allylthiourea and titanium tetraisopropoxide (Ti(O\u003csup\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sup\u003ePr)\u003csub\u003e4\u003c/sub\u003e) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All chemicals used were reagent grade. PHBV, PCL and PBSu were purified by reprecipitation before using with chloroform and methanol. Other chemicals were used without further purifications.\u003c/p\u003e \u003cp\u003eRecovery efficiency of polymer from BOD solution with seawater. The polymer i.e., PHBV (8 mg), PCL (6 mg), and PBSu (8 mg), was added to the BOD culture medium (200 mL) with seawater based on ASTMD6691-17 and the mixture was stirred for 1 h. After acidification with 1 M hydrochloric acid (2.0 mL), the culture medium was extracted three times with chloroform (50 mL, 30 mL, and 30 mL). The organic layers were combined and dried over with anhydrous sodium sulfate (Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e). The filtrate was evaporated in vacuo to obtain the residue. After adding nitrobenzene (0.49 mmol) as an internal standard for \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR analysis, all the residues were dissolved in chloroform-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e (2.0 mL). Recovery efficiency (%) was estimated using the following equation:\u003c/p\u003e \u003cp\u003eRecovery efficiency (%)\u0026thinsp;=\u0026thinsp;\u003cem\u003ex\u003c/em\u003e\u003csub\u003ef\u003c/sub\u003e / \u003cem\u003ex\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e \u0026times; 100 (1)\u003c/p\u003e \u003cp\u003ewhere (\u003cem\u003ex\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e) is the initial weight of polymer, (\u003cem\u003ex\u003c/em\u003e\u003csub\u003ef\u003c/sub\u003e) is the residual weight of polymer estimated from the integral ratio between the protons of \u003cem\u003eo\u003c/em\u003e-position of nitro benzene and the proton(s) adjacent to the oxygen (\u0026minus;\u0026thinsp;O\u0026minus;C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003eR\u0026thinsp;\u0026minus;\u0026thinsp;or \u0026minus;\u0026thinsp;O\u0026minus;C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e2\u003c/sub\u003e\u0026minus;) moiety in polymer.\u003c/p\u003e \u003cp\u003eBOD biodegradation testing. The BOD biodegradability of the samples by the aerobic microorganisms from seawater was determined by measuring oxygen consumption with a BOD instrumentation (OxiTop-C measuring head with a 300-mL BOD reactor, WTW GmbH, Weilheim, Germany) referring to ASTM D6691-17. Seawater collected from quay of Yokosuka Headquarters in Japan agency marine-earth science and technology (JAMSTEC), which was filtered through a filter paper (ADVANTEC No. 2), was used as the base medium for a BOD biodegradation testing. Then, the filtrate was allowed to stand for 1 day before the testing. The base medium (1.0 L) was filtrated using a membrane filter (MEMBRANE FILTER mixed cellulose ester, φ\u0026thinsp;=\u0026thinsp;0.45 \u0026micro;m, ADVANTEC) and the filter was added to the base medium (10 mL) to prepare the condensed seawater. The BOD medium contained 199.0 mL of the base medium and KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.1 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, NH\u003csub\u003e4\u003c/sub\u003eCl, 0.5 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, \u003cem\u003eN\u003c/em\u003e-allylthiourea 5.0 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1.0 mL of the concentrated seawater as an additional inoculum. A sample (ca. 4 mg) was placed in the BOD reactor, and 200 mL of the BOD medium was added to the BOD reactor. Two bottles without a sample were also prepared as a blank medium. The OxiTop-C measuring head was attached to the head of a BOD reactor, and the reactor was incubated at 30\u0026deg;C. The BOD biodegradability of BD, SA, and PBSu was defined as follows:\u003c/p\u003e \u003cp\u003eBOD biodegradability (%) = (BOD\u003csub\u003esample\u003c/sub\u003e (mg) \u0026ndash; BOD\u003csub\u003eblank\u003c/sub\u003e (mg)) / ThOD (mg) \u0026times; 100 (2)\u003c/p\u003e \u003cp\u003eHere, BOD\u003csub\u003esample\u003c/sub\u003e and BOD\u003csub\u003eblank\u003c/sub\u003e are the experimentally observed values of oxygen demand of BD, SA, and PBSu media and the blank media, respectively. ThOD is a theoretically calculated value of oxygen demand of a sample, which was obtained by assuming that the sample completely degraded into CO\u003csub\u003e2\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e \u003cp\u003eSynthesis of PBSu. A 100 mL flask was equipped with a nitrogen gas inlet and outlet. BD (92 mmol), SA (20 mmol), and titanium tetraisopropoxide (77 \u0026micro;mol) was added to the flask under a dry nitrogen gas flow. The reaction mixture was stirred at 200 ℃ under a dry nitrogen gas flow to remove the water and BD produced during the esterification reaction. After 1 h, titanium tetraisopropoxide (77 \u0026micro;mol) was added and the mixture was stirred at 200 ℃ under reduced pressure (50 Pa) for 1 h. PBSu was corrected as a white solid. PBSu was dissolved in a chloroform (50 mL) and then the solution was poured into a methanol (700 mL). PBSu (11.5 g) was obtained as a white solid. Before BOD biodegradation testing, the PBSu was reprecipitated with a chloroform and methanol, which was used for the experiments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eAuthor contributions statement\u003c/h2\u003e \u003cp\u003eY. S and Y. T. designed the study. Y. S. wrote original draft and Y. S., Y. T. and K. K. wrote the paper. Y. S. and J. T. conducted all experiments. Y. T. and K. K. are the corresponding authors. All the authors participated in the analysis and discussion of the results.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e \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 \u003c/p\u003e\u003cp\u003e \u003ch2\u003eData and materials availability\u003c/h2\u003e \u003cp\u003eAll data needed to evaluate the conclusions in the paper are present in the paper and the Supplementally Information. Any additional data related to this paper may be requested from Yuya Tachibana ([email protected]) or Ken-ichi Kasuya ([email protected]).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY. S and Y. T. designed the study. Y. S. wrote original draft and Y. S., Y. T. and K. K. wrote the paper. Y. S. and J. T. conducted all experiments. Y. T. and K. K. are the corresponding authors. All the authors participated in the analysis and discussion of the results.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eYuya Tachibana is grateful for financial support from JSPS KAKENHI Grant-in-Aid for Scientific Research (C) Number 20K12233. Ken-ichi Kasuya is grateful for financial support from the New Energy and Industrial Technology Development Organization (NEDO), project code JPNP14004. NMR measurements were performed on a NM-ECS400 NMR spectrometer and JNM-ECA600 NMR spectrometer (JEOL Ltd., Tokyo, Japan) at the Center for Instrumental Analysis of Gunma University. The seawater was collected from the shore of the Japan Agency for Marine -Earth Science and Technology (JAMSTEC) facing the Tokyo Bay of Japan.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data needed to evaluate the conclusions in the paper are present in the paper and the Supplementally Information. Any additional data related to this paper may be requested from Yuya Tachibana ([email protected]) or Ken-ichi Kasuya ([email protected]).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNarancic, T. et al. Biodegradable Plastic Blends Create New Possibilities for End-of-Life Management of Plastics but They Are Not a Panacea for Plastic Pollution. \u003cem\u003eEnviron. Sci. Technol.\u003c/em\u003e \u003cb\u003e52\u003c/b\u003e, 10441\u0026ndash;10452 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFagnani, D. E. et al. 100th Anniversary of Macromolecular Science Viewpoint: Redefining Sustainable Polymers. \u003cem\u003eACS Macro Lett.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 41\u0026ndash;53 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, M. S. et al. A Review of Biodegradable Plastics: Chemistry, Applications, Properties, and Future Research Needs. \u003cem\u003eChem. 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Mater.\u003c/em\u003e acsapm.3c01843 (2023).\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":"","lastPublishedDoi":"10.21203/rs.3.rs-6191631/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6191631/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe development of biodegradable polymers and their certification require an analytical method that is both reliable and practical. Although biochemical oxygen demand (BOD) testing remains a robust method for confirming polymer metabolism, it does not provide precise information about the residual compounds during and after biodegradation. Moreover, direct analysis these residues is challenging, particularly when seawater interferes with analysis. In this study, we propose an extraction/chemical-structure-analysis/molecular-mass-analysis protocol as an enhanced analytical approach for investigating the culture medium post-BOD biodegradation testing in seawater. We conducted BOD biodegradation test of poly(3-hydroxybutyrate-\u003cem\u003eco\u003c/em\u003e-3-hydroxyvalerate), poly(\u003cem\u003eε\u003c/em\u003e-caprolactone), and poly(butylene succinate) in seawater. Following testing, \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR analysis of the extracts identified the chemical structures of the residual polymers and enabled the assessment of NMR degradability, which aligned well with the BOD biodegradability trend. Additionally, molecular-mass-analysis revealed changes in the molecular mass, supporting evaluation of the chain scission of polymer. This study advances analytical methods in the field of biodegradable polymers.\u003c/p\u003e","manuscriptTitle":"Feasible Analytical Protocol of Residual polymers in Culture Medium after Biodegradation Testing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-18 08:40:48","doi":"10.21203/rs.3.rs-6191631/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":"5d150159-3b5d-455a-b288-ff6c770583b4","owner":[],"postedDate":"March 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45808933,"name":"Physical sciences/Chemistry/Green chemistry/Sustainability"},{"id":45808934,"name":"Physical sciences/Materials science/Techniques and instrumentation/Characterization and analytical techniques"}],"tags":[],"updatedAt":"2025-11-04T16:23:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-18 08:40:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6191631","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6191631","identity":"rs-6191631","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}