Comparison of Mechanical and Thermal Properties of Poly(vinyl alcohol)- Bionanocomposite Films Reinforced with Various Modified Cellulose Nanofibrils and Biologically Active Silver Fir Knotwood Extract

Comparison of Mechanical and Thermal Properties of Poly(vinyl alcohol)- Bionanocomposite Films Reinforced with Various Modified Cellulose Nanofibrils and Biologically Active Silver Fir Knotwood Extract | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparison of Mechanical and Thermal Properties of Poly(vinyl alcohol)- Bionanocomposite Films Reinforced with Various Modified Cellulose Nanofibrils and Biologically Active Silver Fir Knotwood Extract Urša Osolnik, Viljem Vek, Miha Humar, Primož Oven, Ida Poljanšek This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6162976/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Graphical Abstract Abstract Unmodified cellulose nanofibrils (CNFs), TEMPO cellulose nanofibrils (TCNFs) and lignocellulose nanofibrils (LCNFs) were used to reinforce a poly(vinyl alcohol) (PVA) matrix. The aim of the study was to compare the performance of these nanofillers with respect to the properties of PVA-based biocomposites. XRD and thermal analyses showed that CNFs were the most crystalline and thermally stable, followed by LCNFs and TCNFs. All nanofillers improved the mechanical properties of PVA, with TCNFs providing the greatest reinforcement. The PVA biocomposite with 6% TCNF showed a 55% higher modulus of elasticity and 58% higher tensile strength than the reference film. The most thermally stable bionanocomposite among the PVA-nanocellulose biocomposites was PVA with 6 % CNF; thermal degradation (Tonset) started at a temperature 4 °C higher than the reference. A hydrophilic extract of silver fir knotwood was added to the PVA/nanocellulose system to produce biologically active biocomposites, and its effects on mechanical and thermal properties were evaluated. The extract shifted the Tonset to higher temperatures, with the higher content having an even greater effect. While the extract slightly decreased the tensile strength of the biocomposite, the addition of 4% extract to the LCNF-reinforced biocomposite increased the tensile strength by 10% compared to the PVA-LCNF biocomposite. bionanocomposite films (ligno)cellulose nanofibrils poly(vinyl alcohol) reagent TEMPO silver fir extract antioxidant properties Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction In recent years, interest in and demand for environmentally friendly biocomposite materials has increased. These are based on naturally occurring building blocks and can provide an alternative to environmentally chalanging plastics - petroleum-based, non-biodegradable polymers. Polymer composites are biocomposites if at least one component of the composite is biobased or biodegradable. The biocomposites, based on a biodegradable polymer matrix (PLA, PHA, PVA) with the addition of natural fibers, are more environmentally friendly and are often referred to as green composites (John and Thomas 2008 ). The use of green bionanocomposites is highly desirable in the food and packaging industry, agriculture and other applications, beacuse it can reduce the amount of waste products and pollution (Darder et al. 2007 ) contributing to sustainable development goals. Natural fibers, especially plant cellulose fibers, can be a possible substitute for environmentaly questionable synthetic fibers, which are traditionally used as reinforcing components for various polymer matrices in the production of polymer composites. Cellulose fibers have several advantages over petroleum-based synthetic ones: renewability, biodegradability, low cost, large specific surface area, high tensile strength and flexibility (Zhang et al. 2020 ). Nanomaterials are extremely interesting because of their extensive and active surface area, with naturally occurring nanofibers occupying a special place (Saito et al. 2007 ). Nanofibrils and microfibrils obtained from cellulose fibers and are of particular importance for the production of polymer composites with improved relevant properties because of their exceptional mechanical properties (high elastic modulus ̴140 GPa) (Fujisawa et al. 2012 ). Including cellulose nanofibrils (CNFs) in biopolymer matrices opens up many possibilities for replacing conventional composite materials based partially or entirely on petroleum derivatives (Kalia et al. 2011 ; Wu et al. 2019 ). CNFs are generally isolated by delamination of wood pulp using mechanical and/or chemical and enzymatic methods (Lin et al. 2012 ; Abdul Khalil et al. 2014 ). Tetramethylpiperidine-1-oxyl radical (TEMPO) mediated oxidation is one of the possible chemical pretreatments of cellulosic material, in which primary OH groups (C6 atom of cellulose) are selectively oxidized to carboxyl groups under aqueous conditions at pH 10. In this reaction, NaClO acts as the primary oxidizing agent, and TEMPO and NaBr as catalysts (Saito and Isogai 2004 ; Dufresne 2017 ; Isogai and Zhou 2019 ). The product of this oxidation is β-(1,4)-polyglucuronic acid sodium salt (cellouronic acid Na salt). Electrostatic repulsion between C6 carboxylate anions on cellulose fibrils enables less energy-intensive mechanical processing of oxidized cellulose in water to produce TEMPO cellulose nanofibrils (TCNFs) (Saito and Isogai 2004 ). This electrostatic repulsion disrupts the formation of interfibrillar H-bonds, enabling the production of more individualized cellulose nanofibrils (Saito et al. 2007 ). In the production of CNFs, the lignin is usually removed before nanofibrillation, whereas lignocellulosic nanofibrils (LCNFs) are cellulose nanofibrils that contain lignin (Liu et al. 2023 ). LCNFs production is more environmentally friendly than CNFs production, since LCNFs can be produced directly from raw lignocellulosic biomass without delignification. The residual lignin in LCNFs may contribute to some of the advantages of LCNFs over CNFs, such as greater hydrophobicity, UV blocking and antioxidant activity (Iwamoto and Endo 2014 ; Liu et al. 2021 ; Zhang et al. 2022 ). With various pretreatments of lignocellulosic materials, it is possible to apply a surface charge to the fibrils that causes repulsion between them, which further facilitates the mechanical defibrillation of material to produce nanofibril LCNFs (Iwamoto and Endo 2014 ). Nanocellulose, particularly nanofibrils, offers exceptional barrier properties, strength, sustainability, and potential for functionalization, making it an ideal material for high-performance, environmentally friendly food and pharmaceutical packaging. Thus, the incorporation of unmodified and modified cellulose nanofibrils into various polymer matrices for the production of various packaging films is an area in which many research groups around the world are working. Biodegradable polymer matrices for the production of cellulose-polymer composites can be PVA (Espinosa et al. 2019 ; Lee et al. 2020 ; Liu et al. 2022 ; Osolnik et al. 2024 ), PLA (Žepič et al. 2016 ; Jacob et al. 2024 ) and chitosan (Talebi et al. 2021 ; Qin et al. 2023 ) and others. Due to its biodegradability, PVA is an exciting candidate for the production of bio-based packaging films. PVA is a synthetic, water-soluble polymer with good chemical stability and excellent film-forming properties, good mechanical and oxygen barrier properties (Tretinnikov and Zagorskaya 2012 ; Espinosa et al. 2019 ; Kassab et al. 2019 ). The main disadvantage of PVA films is the hydrophilicity, which is an issue when packaging moist products (Arun et al. 2022 ). When PVA films are exposed to an environment with higher humidity (above 60%), water molecules penetrate the PVA structure and act as plasticizers, causing the mechanical properties of the PVA to deteriorate (Jain et al. 2017 ; Lee et al. 2020 ; Chou et al. 2021 ). One of the solutions for the production of less hydrophilic PVA films is the inclusion of nanocellulose. Due to the hydrogen bonds formed between the PVA and nanocellulose, the number of free OH groups on the PVA chains is decreased, which also contributes to a reduction of hydrophilicity, possibly also resulting in a more hydrophobic character of the PVA biocomposite film (Lee et al. 2020 ; Osolnik et al. 2024 ). Literatue review rewealed that individual types of nanostructured cellulose objects have been used for development of PVA matrix based biocomposite films in separate investigations, CNFs in a PVA matrix (Rowe et al. 2016 ; Fahma et al. 2017 ; Liu et al. 2022 ; Osolnik et al. 2024 ), TCNFs in a PVA matrix (Choo et al. 2016 ; Sánchez-Gutiérrez et al. 2021 ) and LCNFs in a PVA matrix (Espinosa et al. 2019 ; Bascón-Villegas et al. 2021 ), but the comparative study on properties of PVA films with addition of different types of nanocellulose is still missing. By adding biologically active compounds, such as some polyphenolic wood extractives, biocomposites with antibacterial, fungicidal and antioxidant effects can be produced. Biologically active extractives include low and high-molecular weight polyphenolic compounds, which are characterized by antioxidant, antiviral and antibacterial activity (Papuc et al. 2017 ; Guo et al. 2019 ; Poljanšek et al. 2019 ; Vek et al. 2021 ; Vek et al. 2023 ). Biologically active biocomposites are extremely interesting in the field of packaging since they can replace the use of ecologically questionable conventional packaging (PP, PET, PE) and take their place in the “active packaging” field. "Active packaging" is defined in European Regulation (EC) No. 450/2009 as a packaging system that interacts with the packaged product (food) through the components incorporated into the packaging system. These components can release substances to the packaged product or into the area of the packaged product or absorb substances from the packaged product or the area around the product to increase the product's shelf life (Yildirim and Röcker 2018 ). In addition to inertness and suitable barrier and thermal properties, packaging films can also have biological activity since they can extend the shelf life of the packaged product by adding suitable antioxidant and antimicrobial compounds (Missio et al. 2018 ; Missio et al. 2019 ). In our study, the wood extractives of silver fir ( Abies alba ) knotwood were used as a biologically active agent for the production of PVA biocomposites, since hydrophilic extractives of silver fir knotwood have been shown to have high antioxidant activity (Vek et al. 2021 ). To our knowledge, no study has yet been conducted in which PVA biocomposite films were produced with the addition of various cellulose nanofibrils and wood extractives of silver fir knotwood. The aims of our study was to compare the influence of different types of cellulose nanofibrils (CNFs, LCNFs, TCNFs) on the reinforcement of the PVA polymer matrix and to investigate the compatibility between cellulose nanofibrils (CNFs, LCNFs, TCNFs) and the biologically active silver fir knotwood extract (EX) in the PVA polymer matrix. A further aim of the present study was to investigate which of the cellulose nanofibrils used (CNFs, LCNFs, TCNFs) proved to be the best filler for the PVA matrix in terms of the mechanical and thermal properties of the final PVA bionanocomposite films. We also aimed to examine the influence of the added silver fir knotwood extract (EX) in the PVA/CNF, PVA/TCNF and PVA/LCNF system on the final mechanical and thermal properties of the three-component PVA biocomposite films produced. 2. Materials and methods 2.1. Materials Cellulose nanofibrils (CNFs) were provided by the Centre for Biocomposite and Biomaterial Processing at the University of Toronto, Canada. The CNFs were a CNF-water suspension with a solid content of 1.40% and served as unmodified CNFs. The CNF suspension was obtained by mechanical homogenization of sulfite coniferous wood pulp and consisted of cellulose nanofibrils with diameters ranging from 20 nm to 60 nm (Žepič et al. 2015 ). LCNF, TCNF and EX were produced at Department of wood science and technology of the University of Ljubljana. The raw material for LCNFs production was Norway spruce wood ( Picea abies ) and silver fir knotwood for EX production, both originating from the forest of Kočevska reka, Slovenia. The starting material for TCNFs production was PÖLS cellulose paper, obtained from Papirnica Vevče, d.o.o., Ljubljana, Slovenia. Maleic anhydride, TEMPO ((2, 2, 6, 6 – tetramethylpiperidine – 1 – yl) oxyl), sodium bromide, sodium hydroxide, hydrochloric acid, polyvinyl alcohol (PVA) – Mw ~ 47000, tannic acid (TA), methanol, cyclohexane, acetone and formic acid were purchased from Merck (Sigma-Aldrich Chemie, Taufkirchen, Germany). Sodium hypochlorite was purchased from Acros Organics (Thermo Fisher Scientific, UK). 2.2. Preparation of active hydrophilic extract Sequential extraction of silver fir knotwood samples was performed in an ASE 350 accelerated solvent extraction system (Thermo Scientific Dionex). Before extraction, samples were milled by a Retsch SM 2000 cutting mill with a sieve opening of 1 mm and then freeze-dried in a Telstar LyoQuest CC1930 freeze dryer for 24 hours at − 85°C and 4.5 Pa. Extraction was performed as described by (Vek et al. 2021 ), first extracting the lipophilic extractives with cyclohexane and then the hydrophilic extractives with an acetone/water mixture (95:5, v/v), both extractions at 100°C and 10.34 MPa.(2 × 5 min static cycles). The freeze-dried hydrophilic extract of the silver fir knotwood (EX) was further used as the biologically active component of the PVA biocomposite film. 2.3. TCNFs production The TEMPO oxidation of PÖLS cellulose paper was carried out according to a method proven by Isogai (Isogai et al. 2011 ; Levanič et al. 2020 ). Six g of dry cellulose paper was soaked in 600 mL of distilled water. The soaked cellulose was transferred to a three-neck flask, and 0.096 g TEMPO reagent and 0.96 g NaBr were added, then 30 mL NaClO. The reaction took place under constant stirring and pH measurement. The pH was initially above 11; with the addition of HCl, it was lowered to pH 10.2. The pH was kept between 10 and 10.2 for 2 hours with 0.5 M NaOH. Finally, 30 mL ethanol was added to the reaction mixture. The resulting TEMPO-oxidized cellulose was washed with large amounts of distilled water. A suspension of TEMPO-oxidized cellulose with an approximate dry matter content of 1% was then prepared. The suspension was first treated with an Ultraturrax at 12,500 min − 1 . Mechanical fibrillation of the mentioned product to produce TEMPO-oxidized cellulose nanofibrils (TCNFs) was performed using a PandaPLUS 2000 high pressure homogenizer (GEA NS, Parma, Italy) − 1 pass at 50 MPa and 1 pass at 69 MPa. A TCNF water suspension with a solids content of 0.74% was obtained. 2.4. LCNFs production LCNFs was isolated from spruce wood (particle size between 0.25 and 0.63 mm) using the method described in a study by Iwamoto (Iwamoto and Endo 2014 ), with slight modifications. Ten g of milled (Retsch SM 2000 cutting mill) spruce wood was weighed into an Erlenmeyer flask and 50 g of maleic anhydride was added. The Erlenmeyer flask with its contents was covered with a watch glass and placed in a heated oil bath − 130°C. The esterification reaction lasted 3.5 hours in a melt of maleic anhydride. After 3 hours, the product was washed with acetone and distilled water. The esterification product was soaked in a 0.5 M NaOH solution for 5 minutes to swell the fibers and deprotonate the introduced COOH groups to COO-. The product was then washed again with distilled water. The prepared aqueous suspension of esterified fibers was first treated with an Ultraturrax at 12,500 min − 1 . Mechanical fibrillation was then carried out using a PandaPLUS 2000 high-pressure homogenizer (GEA NS, Parma, Italy). The fiber suspension was passed through the homogenizer three times at 80 MPa and an LCNF water suspension with 0.70% solid content was obtained. 2.5. Reversed-Phase High-performance Liquid Chromatography (RP-HPLC) Reversed-Phase High-performance Liquid Chromatography (RP-HPLC) was performed on a Thermo Scientific high-performance liquid chromatography system (Accela HPLC, Waltham, Massachusetts, USA) equipped with a photodiode array detector (PDA); the mobile phase consisted of water and methanol with the addition of 0.1% formic acid. Separation of the components was performed on a Thermo Accucore ODS column (4.6 id × 150 mm, 2.6 µm), as described in detail in (Vek et al. 2021 ). 2.6. Preparation of PVA biocomposite films PVA biocomposite films with added CNFs/TCNFs/LCNFs were produced by solvent casting as described in a previous study (Osolnik et al. 2024 ). A 10% PVA solution was first prepared. The calculated amount of cellulose nanofibrils (CNFs, TCNFs, LCNFs) was added to the 10% PVA solution so that the weight fraction of nanocellulose relative to PVA was 6% in all of the two-component, PVA/nanocellulose films. Our previous studies (Osolnik et al. 2024 ; Osolnik et al. 2025 ) revealed that this content of reinforcing component gave us PVA bionanocomposite films with best mechanical properties. In the production of three-component biocomposite films PVA/nanocellulose/EX, the EX was added in a proportion of 2% and 4% in relation to PVA in the PVA/nanocellulose system. These biocomposites will be referred to as three-component PVA/nanocellulose/EX films. The resulting suspensions were mixed for 2 days and then treated with an ultrasonic probe (US) and Ultraturrax. The resulting suspensions were then poured into polystyrene Petri dishes. The films were dried for one week under ambient conditions. The compositions of the films and film labels are listed in Table 1 . In addition, two reference films were prepared: a PVA film (P) cast from a 10% PVA base solution and a PVA-US film (P-US) cast from a 10% PVA base solution and treated with an ultrasonic probe (US) and Ultraturrax. Table 1 shows the film labels and compositions of the PVA reference and PVA biocomposite films produced. Table 1 Film labels and composition Film type Film label % CNFs* % TCNFs* % LCNFs* % EX* PVA P 0 0 0 0 PVA + US P-US 0 0 0 0 PVA + 6%CNF + US P6CNF-US 6 0 0 0 PVA + 6%TCNF + US P6TCNF-US 0 6 0 0 PVA + 6%LCNF + US P6LCNF-US 0 0 6 0 PVA + 6%CNF + 2%EX + US P6CNF2EX-US 6 0 0 2 PVA + 6%CNF + 4%EX + US P6CNF4EX-US 6 0 0 4 PVA + 6%TCNF + 2%EX + US P6TCNF2EX-US 0 6 0 2 PVA + 6%TCNF + 4%EX + US P6TCNF4EX-US 0 6 0 4 PVA + 6%LCNF + 2%EX + US P6LCNF2EX-US 0 0 6 2 PVA + 6%LCNF + 4%EX + US P6LCNF4EX-US 0 0 6 4 *% of nanocellulose (CNFs, TCNFs, LCNFs) and % of silver fir knotwood extract (EX) on dry weight of PVA 2.7. FT-IR analysis To compare the chemical structure of the reinforcing fillers CNFs, TCNFs and LCNFs as well as the chemical structure of the produced PVA biocomposite films and possible interactions between the building blocks of the biocomposite, Fourier transform infrared spectroscopy (FTIR) was performed in ATR (attenuated total reflectance) mode. The freeze-dried samples of CNFs, TCNFs, LCNFs and EX, as well as the prepared PVA biocomposite films, were scanned in the spectral range of 4000 to 450 cm − 1 with 16 scans and a resolution of 4 cm − 1 using a Spectrum Two UATR FT-IR instrument (Perkin Elmer, Waltham, MA, USA). All spectra were ATR and baseline corrected and normalized. 2.8. X-ray diffraction analysis X-ray diffraction analysis was performed to analyse the crystallinity of the different nanocelluloses (CNFs, LCNFs, TCNFs). For the XRD measurements, the nanocellulose (CNFs, LCNFs, TCNFs) was freeze-dried using a ModulyoD freeze dryer (Thermo Fisher Scientific, Waltham, MA, USA) for 72 hours (pressure 7 Pa, temperature of the plates 22°C and temperature of the condenser − 50°C). Dried samples were pressed uniaxially into tablets using a hydraulic press. XRD analysis was performed using an X'Pert PRO MPD high-resolution X-ray powder diffractometer from PANalytical (Netherlands). Measurements were performed in the 2θ range between 5° and 60° with a step size of 0.034° and an integration time of 250 s. Light corresponding to the electronic transition of CuKα with a wavelength of 0.154 nm was used as the radiation source. The crystallinity index was calculated using the Eq. (1), as described in the study by (Segal et al. 1959 ; Zepic et al. 2014 ): \(\:{I}_{c}=\left(\frac{{I}_{002}-\:{I}_{am}}{{I}_{002}}\right)\times\:\:\) 100 ( 1 ) where I c is the crystallinity index, I 002 is the intensity of the 002 peak (I 002 , 2θ = approx. 22 °) and represents both crystalline and amorphous materials, and I am is the intensity of the minimum between the 002 and 101 peaks (I am , 2θ = approx. 18 °) and represents amorphous material (Segal et al. 1959 ; Zepic et al. 2014 ). 2.9. Tensile test Modulus of elasticity (E t ), tensile strength (σ M ) and elongation at break (ε tb ) of all films were tested on a Zwick/Roell Z005 (Zwick GmbH & Co. KG, Ulm, Germany) universal machine under ambient conditions. The preparation of the film samples for testing and the tensile test were performed as in a previous study (Osolnik et al. 2024 ) using the ASTM D882–02 standard (Standard Test Method for tensile properties of thin plastic sheeting), with ten samples tested for each film type. 2.10. Thermal analysis Thermogravimetric analysis (TGA) The thermal stability of freeze-dried CNFs, TCNFs, LCNFs and biocomposite films was determined by TGA analysis using a TG Mettler Toledo TGA/DSC1 instrument (Mettler Toledo, Greifensee, Switzerland). Approximately 5 mg of the sample was weighed into Pt crucibles and then heated in a temperature program from 25°C to 600°C at a heating rate of 10 K/min in an Ar atmosphere with a flow rate of 50 mL/min. The baseline was subtracted for all measurements. Differential scanning calorimetry (DSC) The thermal transitions of all films were evaluated with a DSC Mettler Toledo DSC1 instrument (Mettler Toledo, Greifensee, Switzerland). Approximately 5 mg of the sample was heated in aluminum crucibles with perforated lids in a temperature program from 0°C to 230°C at a heating rate of 10 K/min, cooled from 230°C to 0°C at a cooling rate of 5 K/min and heated again from 0°C to 230°C at a heating rate of 10 K/min in an Ar atmosphere at a flow rate of 50 ml/min. The degree of crystallinity of the PVA was calculated using the Eq. ( 2 ) as described in previous studies (Idris et al. 2021 ; Osolnik et al. 2024 ): $$\:{X}_{c}^{DSC}\:\left[\%\right]=\:\frac{{\varDelta\:H}_{f}}{{\varDelta\:H}_{f}^{◦}}\times\:100$$ 2 where ΔH f and \(\:{\varDelta\:H}_{f}^{◦}\) are the enthalpy of melting of pure, reference PVA films and the PVA biocomposite films and PVA with 100% crystallinity in J/g, as described in a study by Idris et al. The melting enthalpy for PVA with 100% crystallinity (ΔH◦f) is 138.7 J/g (Idris et al. 2021 ). 2.11. Statistics The results obtained were subjected to basic statistical analysis using Statgraphics Centurion 17. software. The analysis of variance (ANOVA) and the least significant difference (LSD) method of Fisher with a confidence level of 95% were performed. 3. Results 3.1. HPLC analysis The results of the chromatographic quality control of the EX, are shown in Fig. 1 . EX was characterized by large amounts of polyphenols, most of which were lignans isolariciresinol, lariciresinol, secoisolariciresinol, pinoresinol and matairesinol (Figs. 1 and 2 ), with secoisolariciresinol and lariciresinol being the predominant ones. Other phenolic compounds in the EX were phenolic acids, with homovanillic acid, coumaric acid and ferulic acid, as well as the flavonoids epicatechin, taxifolin and quercetin. Compared to the identified lignans, these were present in lower quantities in the EX. Phenolic compounds of silver fir have already been shown to be natural antioxidants and biocidal compounds (Willför et al. 2004 ; Benković et al. 2014 ; Vek et al. 2024 ). 3.2. FTIR analysis Figure 3 shows the FTIR spectra of CNFs, LCNFs and TCNFs. The FTIR spectra contain characteristic peaks for the vibrations of the bonds in the cellulose molecules, the broad band between 3600 and 3000 cm − 1 corresponds to the stretching vibration of O-H, 2892 cm − 1 due to the symmetric vibration of CH 2 groups, 1408 cm − 1 due to the m –CH 2 scissoring motion, 1370 cm − 1 represents the C-H bending, 1060 cm − 1 due to the C–O–C stretching vibration of the pyranose ring (Široký et al. 2010 ; Soni et al. 2015 ), but they differ from each other. The vibrational signal associated with the OH groups at a wavenumber of about 3300 cm − 1 is broadest for LCNFs (aromatic OH groups) (Osolnik et al. 2025 ) and least broad for CNFs. After the esterification of the lignocellulose surface with maleic anhydride, a vibration of C = O in the ester group at 1714 cm − 1 and a vibration of C = O in the carboxylate anion at 1637 cm − 1 were observed. The FTIR spectra of LCNFs contain peaks at 1575 cm − 1 and 1510 cm − 1 , which are not found in the FTIR spectra of CNFs and TCNFs. These peaks belong to C = C vibrations in the lignin structure (Horseman et al. 2017 ; Osolnik et al. 2025 ). The FTIR spectra of TCNFs contains a signal at 1600 cm − 1 , which belongs to vibration of the carboxylate anion on the cellulose chain as a result of the TEMPO-mediated oxidation of the starting cellulose (Karim et al. 2017 ; Poyraz et al. 2018 ). Figure 4 shows the FTIR spectra of the neat PVA film, the EX, the two-component PVA/nanocellulose films and the three-component PVA/nanocellulose/EX films. The FTIR spectra of the neat PVA film contain characteristic peaks typical of PVA, which are described in detail in a study by (Osolnik et al. 2024 ). These peaks also appeared in the FTIR spectra of the PVA biocomposite films. The inclusion of LCNFs in the P6LCNF-US film is confirmed by the fact that a peak at 1575 cm − 1 appears in both the FTIR spectra of LCNFs (Fig. 3 ) and the spectra of P6LCNF-US (Fig. 4 C), indicating the presence of C = C in the lignin. This is also true for TCNFs, which was successfully incorporated into the P6TCNF-US biocomposite, since the peak at 1600 cm − 1 , which is due to the vibration of the carboxylate anion on TCNFs (Fig. 3 ), also appears in the FTIR spectra of P6TCNF-US. The FTIR spectra of the EX contain some main peaks belonging to –OH stretching (3319 cm − 1 ), CH stretching (2933 cm − 1 ), CH2 stretching (2854 cm − 1 ), C = O stretching (1751 cm − 1 , 1696 cm − 1 ), C = C stretching in the aromatic ring (1601 cm − 1 , 1513 cm − 1 ), asymmetric C–H deformation (1449 cm − 1 ) and C–O stretching (1030 cm − 1 ) (Popescu et al. 2007 ; Morar et al. 2024 ). EX is also successfully included in the three-component biocomposites since a peak at 1513 cm − 1 appears in the FTIR spectra of P6CNF4EX-US (Fig. 4 A), P6LTNF4EX-US (Fig. 4 B) and P6LCNF4EX-US (Fig. 4 C). 3.3. X-ray diffraction analysis Figure 5 shows the X-ray diffraction patterns of CNFs, LCNFs and TCNFs. Two prominent crystalline peaks are present in all the diffractograms, the first at about 2θ = 16° and the second at about 2θ = 22°, corresponding to the 101- and 002- reflection planes of the structure of cellulose I; the region around 2θ = 18° or the minimum between the mentioned crystalline peaks is typical of the amorphous part of cellulose (Zepic et al. 2014 ; Ji et al. 2019 ; Morcillo et al. 2022 ; Zhang et al. 2024 ). CNFs showed the highest crystallinity (Ic = 84%) among the analyzed samples, followed by LCNFs (Ic = 75%) and TCNFs (Ic = 69%). The crystallinity of the nanofibrils depends on the proportion of amorphous structures present (hemicellulose, lignin) and on the nanofibril production process (bleaching) (Demuner et al. 2020 ). The lower crystallinity of LCNFs is probably due to the amorphous structure of lignin in LCNFs. The lowest crystallinity of TCNF is attributed to the presence of a carboxylate anion on the cellulose chains, and due to this relatively large substituent, the arrangement of the chains is restricted so that the crystallinity is lower. Another possible cause is the cleavage of covalent bonds (C-O-C) by TEMPO-mediated oxidation, which leads to cellulose chains with a lower molecular weight (Milanovic et al. 2013 ; Hiraoki et al. 2015 ). 3.4. Tensile properties Table 2 shows the average values and standard deviations for elastic modulus (E t ), tensile strength (σ M ) and elongation at break (ε tb ) for the reference films P and P-US, for the two-component PVA/nanocellulose films and the three-component PVA/nanocellulose/EX films. Figure 6 A shows the average stress-strain curves for the reference films P and P-US and for two-component PVA/nanocellulose films, each ending with the lowest elongation at break of ten measured samples for each film. The modulus of elasticity of the P-US film is 57% higher, and the tensile strength is 21% higher than that of the P film. The elongation at break is 10% lower than that of the P film. The influence of ustrasound treatment on all measured mechanical parametrs was significant. It could be concluded that the ultrasonic treatment brings the PVA chains into closer contact with each other, which increases the number of H-bonds between the PVA chains, i.e., the physical cross-linking is more potent than in the case in which the solution was not treated with US (P). When processed with an ultrasonic probe, the amorphous parts are probably destroyed to a certain extent, the crystallinity of P-US is slightly higher (Table 4 ) and therefore, the mechanical properties of P-US are better. Table 2 Tensile properties of PVA reference films and PVA biocomposite films with different nanocellulose (CNFs; TCNFs, LCNFs) and different weight percentages of silver fir knotwood extract (EX); (ANOVA, p < 0.0001) E t [MPa] σ M [MPa] ε tb [%] P 2136 ± 406 61.0 ± 7.7 79.2 ± 24.6 P-US 3352 ± 489 73.9 ± 3.6 71.3 ± 9.1 P6CNF-US 4889 ± 569 100.4 ± 9.0 15.7 ± 2.0 P6TCNF-US 5201 ± 640 116.5 ± 15.5 17.3 ± 2.0 P6LCNF-US 4648 ± 640 98.3 ± 24.4 14.1 ± 3.1 P6CNF2EX-US 4370 ± 794 93.9 ± 5.8 13.5 ± 3.7 P6CNF4EX-US 4535 ± 734 98.0 ± 9.7 12.6 ± 2.5 P6TCNF2EX-US 5002 ± 1175 109.2 ± 5.1 16.3 ± 2.5 P6TCNF4EX-US 4693 ± 791 106.5 ± 6.9 15.5 ± 2.2 P6LCNF2EX-US 3795 ± 639 94.1 ± 6.9 14.8 ± 1.6 P6LCNF4EX-US 4919 ± 1507 109.3 ± 16.8 10.6 ± 2.2 Ultrasonic treatment was chosen according to the studies (Osolnik et al. 2024 ; Osolnik et al. 2025 ) because the results of the mechanical properties of PVA/CNF biocomposite films were better when the suspensions for film production were treated with ultrasound. Based on a study (Osolnik et al. 2025 ), it was decided that the optimum CNF addition concerning dry PVA was 6%. In the present study, all PVA biocomposite films were prepared with with 6% addition of nanocellulose (CNFs, TCNFs and LCNFs). In all three cases, the biocomposite films' modulus of elasticity and tensile strength were significantly improved compared to the two reference films. This was expected based on the available literature (Choo et al. 2016 ; Zhang et al. 2020 ; Liu et al. 2022 ; Xu et al. 2022 ; Majumdar et al. 2023 ; Osolnik et al. 2024 ). The addition of TCNFs proved to be the best; the elastic modulus value was 143% and 55% higher for P6TCNF-US than for the reference films P and P-US, and the tensile strength was 91% and 58% higher than for the reference films P and P-US. The elongation at break can be expected to be lower than that of the reference films, accompanied by a significant increase in tensile strength. To improve the mechanical properties of the final nanocomposite, a large contact surface between the nanofiller and the polymer matrix, as well as a good distribution of the nanofiller (nanocellulose) in the matrix, are important (Oksman et al. 2016 ; Mondal 2018 ). From the results of the mechanical properties, it can be concluded that CNFs, TCNFs and LCNFs were all well distributed in the PVA matrix. Among the investigated nanocelluloses, TCNFs have the most hydrophilic character, so its distribution in the hydrophilic PVA matrix was probably the most homogeneous. Moreover, due to the presence of functional groups (COO – and OH groups) on TCNFs, many intermolecular forces probably formed between these groups and the OH groups on the PVA chains, which further improved the mechanical strength of the final P6TCNF-US composite. Despite the highest tensile strength, it is interesting to note that the elongation at break of the P6TCNF-US biocomposite was higher than the elongation at break of P6CNF-US and P6LCNF-US. Figure 6 B represents the average stress-strain curves for the P-US reference film and the three-component PVA/nanocellulose/EX films, with each average curve ending at the lowest elongation at break among 10 parallel samples for each film. Our results showed that the addition of he EX variously affected the mechanical properties of PVA biocomposite films. The addition of 4% EX to the PVA/LCNF and PVA/CNF system was more effective in terms of increasing tensile strength and elastic modulus than in the case of TCNFs. The tensile strength of P6CNF4EX –US was comparable to the tensile strength of the P6CNF-US biocomposite film. In contrast, the modulus of elasticity was slightly lower. In the case of P6LCNF4EX-US the tensile strength and modulus of elasticity were even improved compared to the values of the P6LCNF-US biocomposite film. Adding EX (2% or 4%) to the PVA/TCNF system slightly lowers the values for tensile strength and modulus of elasticity, but these values are still much higher than the values for the polymer itself. Among the three-component composite films, the PVA/TCNF/EX films proved to be the best according to the values of the measured mechanical parameters. Similar values for the modulus of elasticity and the tensile strength value were observed for the P6LCNF4EX-US film, but with a lower value for elongation at break compared to P6TCNF2EX and P6TCNF4EX-US. TCNFs are the most polar of all the cellulose nanofibrils used to reinforce the PVA matrix, which means that the distribution of TCNFs in the hydrophilic polymer matrix was probably the most homogeneous compared to the other two types of nanofibrils used. The contact area between the TCNFs and the PVA matrix was likely larger than in the other two cases, allowing better transfer of stress from the matrix to the fiber. In addition, the hydrophilic extract, which is able to form hydrogen bonds with both PVA and TCNFs, also has a positive effect on the mechanical properties of the three-component films. .3.4. Thermal analysis Table 3 shows Tonset and Tmax for the neat PVA film (P, P-US), CNFs, TCNFs, LCNFs and PVA biocomposite films. Figure 7 shows Among the nanofillers investigated, CNFs were the most thermally stable, followed by LCNFs and TCNFs (Table 3 ). The biocomposites reinforced with these nanofillers and the three-component biocomposites with EX additive follow the same trend of thermal degradation as CNFs, LCNFs and TCNFs.the TG - A and DTG - B curves of freeze-dried samples of nanocellulose – CNFs, TCNFs and LCNFs. Table 3 T onset and T max for the neat PVA film, CNF, TCNF, LCNF and PVA biocomposite films Film type T onset [°C] T max [°C] P 299 364 P-US 281 362 CNF 319 346 TCNF 218 237 LCNF 261 296 P6CNF-US 285 363 P6TCNF-US 268 327 P6LCNF-US 277 331 P6CNF2EX-US 289 363 P6CNF4EX-US 291 361 P6TCNF2EX-US 269 325 P6TCNF4EX-US 272 325 P6LCNF2EX-US 280 332 P6LCNF4EX-US 284 333 As mentioned, TCNFs showed the lowest thermal stability among the different nanocelluloses, T onset was the lowest among the investigated nanocelluloses (T onset = 218°C). This was probably due to the decarboxylation of carboxyl groups on the surface of TCNFs (Lichtenstein and Lavoine 2017 ), which were introduced into the surface of cellulose nanofibrils during TEMPO-mediated oxidation, in which C6 hydroxyl groups of cellulose were converted to sodium carboxylate groups (Fukuzumi et al. 2010 ). The reason for the poor thermal stability of TCNFs could also be that the cellulose chains were cleaved during the TEMPO-mediated oxidation of cellulose to produce TCNFs, suggesting that the molecular weight of the TEMPO-oxidized cellulose decreased, also making TCNFs less thermally stable (Mahendra et al. 2019 ). This observation agrees with the values for crystallinity, the lower the crystallinity of the material, the lower the temperature at which thermal decomposition begins. The TG and DTG curves for CNFs, TCNFs, and LCNFs are different. Still, the first mass loss region, in which there is a minor mass loss and which ends at about 130°C, is due to the elimination of water (Zepic et al. 2014 ; Zhang et al. 2019 ) then thermal decomposition starts at 218°C for TCNFs, 261°C for LCNFs and 319°C for CNFs. From here on, there is a more significant loss of mass, probably initially at the expense of the formation of dehydrogenated cellulose and further strong depolymerization of cellulose molecules, producing methane, carbon monoxide and carbon dioxide, as described by Zhang et al. (Zhang et al. 2019 ). As reported by Lichtenstein and Lavoine 2017 , thermal degradation of TCNFs in which sodium carboxylate groups were present occurred in three stages of degradation (Lichtenstein and Lavoine 2017 ), which was also observed in the DTG curve of TCNFs in the present study (Fig. 7 B; T max1 =235°C, T max2 =281°C and T max3 =approx. 420°C). In a study on the thermal stability of TEMPO-oxidized cellulose (Fukuzumi et al. 2010 ), the authors reported in relation to the DTG curve of TEMPO-oxidized CNFs that the first DTG peak at 233°C belongs to the decomposition of anhydrous sodium glucuronate units and the second DTG peak at 282°C belongs to the decomposition of crystalline cellulose chains. Our results of the thermal decomposition of TCNFs are consistent with the study of Fukuzumi et al. 2010 , since the main DTG peaks occurred at almost the same temperatures (235 and 281°C). The thermal decomposition of TCNFs thus takes place in a wider temperature range and in three stages, while the thermal decomposition of CNFs and LCNFs takes place in a narrower temperature range and in two stages (Fig. 7 ). As with TCNFs, there were also carboxyl groups on the surface of LCNFs, which resulted from the pretreatment (esterification) of the lignocellulosic feedstock with maleic anhydride. The presence of carboxyl groups on the surface of LCNFs could be a reason for the lower thermal stability (Lichtenstein and Lavoine 2017 ) of LCNF (T onset = 261°C) compared to CNFs (T onset = 319°C), which also agrees well with the values for crystallinity. Thermal degradation of the neat PVA film started at 299°C (P) and at 281°C (P-US) (Table 3 ). Both films were produced to investigate the influence of treatment with an ultrasonic probe on the thermal properties of the neat PVA film. The treatment of the PVA solution with the ultrasonic probe probably caused a shortening of the PVA chains and a further deterioration of the thermal properties. The shortening of the chains probably starts first in the amorphous parts of the chains, which means that the amorphous parts partially break up, and thus, the crystallinity of the film increases slightly (Table 4 ). A slight increase in crystallinity is also associated with better mechanical strength and lower elasticity (lower elongation at break) of the P-US film compared to the P-film (table of mechanical properties). Figures 8 A and 8 B show the TG and DTG curves of the reference films (P, P-US) and the three-component PVA/nanocellulose/EX films. In the thermal decomposition of PVA films, there are three zones of mass loss. The first mass loss, a small weight loss that started at about 100°C and stabilized up to 200°C, is due to the evaporation of water. The first decomposition stage, which started at 281°C (T onset for P-US) or 299°C (T onset for P), is due to the cleavage of the polymer chains, resulting in smaller fragments. The main chain cleavage occurred in the second decomposition stage, above approx. 400°C (Thomas et al. 2001 ; Liu et al. 2013 ; Wu et al. 2019 ; Suleiman et al. 2024 ). The thermal degradation for the two-component and three-component PVA biocomposite films produced was very similar. The addition of 6% CNFs to the PVA matrix improved the thermal stability of the P6CNF-US film compared to the P-US film. The T onset of the biocomposite film shifted from 281°C for P-US to 285°C for P6CNF-US. The addition of the EX to the PVA/CNF system resulted in a shift of the T onset to a higher T, in the case of P6CNF2EX-US T onset = 289°C and in the case of P6CNF4EX-US T onset = 291°C. The addition of TCNFs and LCNFs to the PVA matrix did not lead to an improvement in the thermal stability of the biocomposite films. EX contains hydrophilic extractives, with the largest proportion being lignans (Vek et al. 2021 ), which probably contribute to or influence cross-linking between the PVA, nanocellulose and extract components. Of all the biocomposite films, P6TCNF-US had the lowest T onset value, but the highest value for modulus of elasticity and tensile strength (Table 2 ). The poorer thermal stability of the biocomposite is probably related to the least thermally stable nanocellulose – TCNFs. The thermal stability of the three-component PVA/nanocellulose/EX films is better than that of the two-component biocomposites (P6TCNF-US, P6LCNF-US). A 4% addition of EX to the PVA/nanocellulose system had a more significant influence on the T onset shift to a higher temperature than a 2% addition of EX. This was probably because more interactions were formed in the biocomposite at a higher EX addition, which contributed to a higher thermal stability. Table 4 Summary of glass transition temperature (T g ), peak of melting temperature (T m ) and crystallinity by DSC (X C DSC ) of the neat PVA film and PVA biocomposite films Film type T g [°C] T m [°C] X C DSC [%] P 76 221 38 P-US 77 222 40 P6CNF-US 79 220 37 P6TCNF-US 81 219 37 P6LCNF-US 80 217 36 P6CNF2EX-US 77 219 39 P6CNF4EX-US 76 221 37 P6TCNF2EX-US 79 218 40 P6TCNF4EX-US 77 218 38 P6LCNF2EX-US 80 217 36 P6LCNF4EX-US 79 216 38 Table 4 shows the glass transition temperature (T g ), the peak of melting temperature (T m ) and crystallinity by DSC (X C DSC ) of the neat PVA film and PVA biocomposite films. Adding any type of cellulose nanofibrils to the PVA matrix increased the glass transition temperature of the formed PVA biocomposite films compared to the reference PVA film (P-US). The glass transition temperature is related to the flexibility of the polymer segments; since the cross-linking of the structure increases at the expense of the interactions between the components of the composite; the mobility of the macromolecules decreases, which increases the T g of the biocomposite films (Wu et al. 2019 ; Parit et al. 2022 ; Osolnik et al. 2024 ). Of all types of cellulose nanofibrils studied, TCNFs had the greatest influence on the T g value. The reason for the highest T g value is probably due to the presence of hydroxyl and carboxyl functional groups on the TCNFs surface, which probably form more interactions with the OH groups on the PVA chains (Fig. 4 ) than there were interactions between CNFs and PVA and between LCNFs and PVA. Another reason for the highest T g of P6TCNF-US is also that TCNFs are the most polar cellulose fibrils and are probably also the most homogeneously distributed in the hydrophilic PVA matrix, and thus the contact area between PVA and TCNFs was larger. The P6LCNF-US film had just 1°C lower T g compared to P6TCNF-US. LCNFs also contain carboxyl groups, hydroxyl groups and more double bonds, which also leads to interaction between LCNFs and PVA. The addition of EX had a slightly negative effect on T g , which is in contrast to our recent study (Osolnik et al. 2024 ) in which tannic acid was used as a biologically active component and crosslinker for PVA and CNFs, in which a 10% addition of tannic acid increased the T g by 10°C. The structure of the EX was less defined than the structure of the tannic acid, so the EX's distribution in the PVA/nanocellulose system was probably less homogeneous than the distribution of the tannic acid in the PVA/CNF system. This meant that the contact area between PVA, nanocellulose and the EX was also smaller, fewer interactions were formed with all components of the composition and therefore, the movement of the macromolecules was not as restricted, which is probably why the T g for the three-component biocomposite did not shift to a higher temperature. The crystallinity of PVA in PVA biocomposite films was determined by DSC measurements. As Table 4 shows, the crystallinity of PVA in the PVA biocomposite films decreased compared to that of the P-US reference film. The nanocellulose and the components of EX interact with PVA, which probably restricts the arrangement of PVA molecules and the formation of crystalline parts, resulting in a lower crystallinity of PVA biocomposite films (Parit et al. 2022 ; Osolnik et al. 2024 ; Suleiman et al. 2024 ). The crystallinity determined from the DSC data showed the lowest value for PVA biocomposite films to which LCNFs was added. In addition to the probably favorable interactions between the building blocks of the PVA-LCNF composite and the PVA-LCNF-EX, which confirm the improved mechanical properties of the composite compared to the PVA reference film, the reason for the lower crystallinity may also be the lowest crystallinity of LCNFs among the investigated nanocelluloses (XRD crystallinity, Fig. 5 ). The introduction of aromatic groups (LCNFs, EX) into the PVA matrix may also disrupt the regular arrangement of the PVA molecules and thus reduce the crystallinity of PVA (Suleiman et al. 2024 ). As mentioned before, P-US had a higher crystallinity than P, which was probably due to the influence of treating the PVA solution with an ultrasonic probe, and apparently the amorphous parts of PVA had disintegrated to a certain extent, resulting in a slight increase in the crystallinity of PVA in P-US film compared to P film. A decrease in crystallinity was also accompanied by a decrease in melting temperature, since the structure was less ordered – the amorphous parts of the biocomposite in which the polymer chains are not ordered - resulting in weaker intermolecular interactions and thus lower melting temperatures (Suleiman et al. 2024 ). 4. Conclusion The chemical structure of the different reinforcing components (CNFs, LCNFs and TCNFs) varies, which is clearly demonstrated by the FTIR spectra. We believe that the different chemical structure influences the different distribution of the reinforcing components in the hydrophilic polymer PVA matrix. The crystallinity results obtained from the XRD analysis also varied for the reinforcing nanofillers used. The unmodified CNFs were the most crystalline, followed by the chemically modified LCNFs and the oxidized TCNFs. FTIR spectroscopy confirmed not only the successful incorporation of nanocellulose, but also the successful incorporation of silver fir knotwood extract (EX). The results of the tensile tests showed that CNFs, LCNFs and TCNFs were all suitable for reinforcing the PVA polymer, since the tensile strength and modulus of elasticity of the PVA-nanocellulose biocomposites clearly exceeded those of the reference polymer films. The addition of TCNFs proved to be the most effective reinforcement for PVA, probably because its more hydrophilic structure distributes most homogeneously in the PVA matrix, thereby improving the mechanical properties. In the study, a comparison was also made between the ultrasonically sonicated and non-sonicated base solutions of the PVA polymer (P, P-US) and their subsequent mechanical and thermal properties. The P-US film exhibited a higher tensile strength and modulus of elasticity than the P film, which is probably because the ultrasonic treatment breaks up some of the amorphous regions and the crystallinity of the P-US film is slightly higher than that of the P film, resulting in better mechanical properties. By incorporating EX into the PVA/nanocellulose system, three-component PVA biocomposite films were produced, with the TCNF-containing biocomposite film again exhibiting the best mechanical strength. The results of thermal analysis showed that TCNFs was the least thermally stable of the investigated nanofillers, since thermal degradation started at the lowest temperature. Comparison of the P and P-US reference films showed that ultrasonic treatment of the polymer solution probably caused chain shortening in PVA, resulting in a deterioration of the thermal properties. The P6CNF-US film showed better thermal stability than the P-US film. Furthermore, the addition of EX in both investigated weight fractions in the PVA/CNF system resulted in a shift of the onset temperature (T onset ) to higher temperatures. However, the addition of LCNFs and TCNFs to the PVA matrix did not contribute to improving the thermal stability of the PVA/LCNF and PVA/TCNF biocomposites. The addition of EX to the PVA/nanocellulose system had a positive effect on shifting the T onset to higher temperatures compared to the two-component PVA/nanocellulose biocomposite films, with the higher weight fraction of EX having an even greater effect. However, EX had a slightly negative effect on the Tg of the biocomposite films due to the chemical structure of the EX and the fact that EX was probably not homogeneously distributed in the PVA/nanocellulose system. Of the nanocelluloses studied, TCNFs had the greatest effect on the Tg value due to the structure of TCNFs – the presence of hydroxyl and carboxyl groups on the cellulose chains - which probably led to more interactions between PVA and TCNF compared to PVA with LCNF or CNF. Declarations Author Contributions: Conceptualization, I.P., U.O. and P.O.; methodology, V.V., U.O. and I.P.; software, I.P., V.V. and U.O.; validation, I.P., U.O. and V.V.; formal analysis, I.P. and U.O.; investigation, I.P., U.O., V.V., M. H. and P.O.; figures, U.O. and I.P.; writing—original draft preparation, U.O. and I.P.; writing—review and editing, I.P., V.V., M.H. and P.O. and supervision, P.O. and I.P. All authors reviewed the manuscript. Funding Declaration: The authors would like to thank the Slovenian Research and Innovation Agency (ARIS) for financial support within the research program P4-0015, projects L4-2623 and V4-2017, as well as the University of Ljubljana, the Ministry of Higher Education, Science and Innovation and the EU within the NOO project "UL for a sustainable society - ULTRA" and the University of Ljubljana for supporting project BAPUR in the frame of M.ERA-Net. Institutional Review Board Statement: This study did not include any studies conducted by any author on human participants or animals. The authors declare compliance with ethical standards. Data Availability Statement: The data presented in this study are available on request from the corresponding authors. Acknowledgments: Special thanks to Mr. Martin Cregeen for language editing. Conflicts of Interest: The authors declare no conflicts of interest. 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Holzforschung 70. Žepič, V., Poljanšek, I., Oven, P., Škapin, A., Hančič, A. (2015) Effect of Drying Pretreatment on the Acetylation of Nanofibrillated Cellulose. Bioresources 10. Additional Declarations No competing interests reported. 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-6162976","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":425465151,"identity":"6f4d3af3-0a3c-4591-84cf-cee80a74efac","order_by":0,"name":"Urša Osolnik","email":"","orcid":"","institution":"University of Ljubljana","correspondingAuthor":false,"prefix":"","firstName":"Urša","middleName":"","lastName":"Osolnik","suffix":""},{"id":425465152,"identity":"6daa09e7-3a37-4a03-b3d8-cf9d48e6d3ef","order_by":1,"name":"Viljem Vek","email":"","orcid":"","institution":"University of Ljubljana","correspondingAuthor":false,"prefix":"","firstName":"Viljem","middleName":"","lastName":"Vek","suffix":""},{"id":425465153,"identity":"9343f2d3-f965-4780-9528-8e49907d2d17","order_by":2,"name":"Miha Humar","email":"","orcid":"","institution":"University of Ljubljana","correspondingAuthor":false,"prefix":"","firstName":"Miha","middleName":"","lastName":"Humar","suffix":""},{"id":425465154,"identity":"1cca81e9-33f9-4803-94f8-47923fa3f751","order_by":3,"name":"Primož Oven","email":"","orcid":"","institution":"University of Ljubljana","correspondingAuthor":false,"prefix":"","firstName":"Primož","middleName":"","lastName":"Oven","suffix":""},{"id":425465155,"identity":"780b6ae0-2358-4c04-9cf4-38886b1221f3","order_by":4,"name":"Ida Poljanšek","email":"data:image/png;base64,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","orcid":"","institution":"University of Ljubljana","correspondingAuthor":true,"prefix":"","firstName":"Ida","middleName":"","lastName":"Poljanšek","suffix":""}],"badges":[],"createdAt":"2025-03-05 13:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6162976/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6162976/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78154559,"identity":"666282be-b40d-4f28-96f7-eee8e38aa7e7","added_by":"auto","created_at":"2025-03-10 12:17:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":77181,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC–PDA chromatogram of EX measured at 280 nm.\u003c/p\u003e","description":"","filename":"floatimage210.png","url":"https://assets-eu.researchsquare.com/files/rs-6162976/v1/458e5852720f38a17956adff.png"},{"id":78155518,"identity":"17940a56-5ec7-4f49-b905-3411eefa932f","added_by":"auto","created_at":"2025-03-10 12:25:55","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":91021,"visible":true,"origin":"","legend":"\u003cp\u003eStructural formulas of the most abundant lignans in silver fir knotwood extract (EX)\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6162976/v1/af0d3616d03f2e37a7046476.jpeg"},{"id":78154057,"identity":"eadeebfd-5743-4843-bf26-661f4be48b5e","added_by":"auto","created_at":"2025-03-10 12:09:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":87856,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of CNF, LCNF and TCNF\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6162976/v1/e268d464201f5fcff95b20ef.png"},{"id":78154056,"identity":"813fe860-bbb2-495e-902b-cd4d0d2c5a64","added_by":"auto","created_at":"2025-03-10 12:09:54","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":623477,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of the neat PVA film, EX and PVA biocomposite films with 6 % CNF – A, TCNF – B, LCNF – C and 4 % EX\u003c/p\u003e","description":"","filename":"floatimage54.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6162976/v1/993e78503adcacefc2021241.jpeg"},{"id":78154065,"identity":"2eca0125-636b-419e-a097-86c3e034bf21","added_by":"auto","created_at":"2025-03-10 12:09:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":80852,"visible":true,"origin":"","legend":"\u003cp\u003eX-ray diffraction patterns of CNF, LCNF and TCNF\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6162976/v1/158dbdb4dcccc2812df903a2.png"},{"id":78154068,"identity":"78604bbd-c326-44ef-9d63-62181e8c3438","added_by":"auto","created_at":"2025-03-10 12:09:55","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":498932,"visible":true,"origin":"","legend":"\u003cp\u003eAverage stress-strain curves of the PVA reference films (P, P-US) and the PVA biocomposite films with 6 % nanocellulose (CNFs, TCNFs, LCNFs) – A and average stress-strain curves of the reference film P-US and the three-component PVA biocomposite films with 6 % nanocellulose (CNFs, TCNFs, LCNFs) and 2 % EX or 4 % EX – B; (ANOVA, p \u0026lt; 0.0001)\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6162976/v1/15bd576e081e5f5ad25f110d.jpeg"},{"id":78155519,"identity":"eae030ee-5985-43b8-9152-04aba8108135","added_by":"auto","created_at":"2025-03-10 12:25:55","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":478979,"visible":true,"origin":"","legend":"\u003cp\u003eTG curves – A and DTG curves – B of CNF, LCNF and TCNF.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6162976/v1/028ecbd17c2cdfb0e9e8a5b6.jpeg"},{"id":78155515,"identity":"5d49dae8-b389-446b-a0ee-38038cdc6d50","added_by":"auto","created_at":"2025-03-10 12:25:54","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":531709,"visible":true,"origin":"","legend":"\u003cp\u003eTG curves – A and DTG curves – B for the reference films – P, P-US and biocompostie films – P6CNF-US, P6CNF2EX-US and P6CNF4EX-US\u003c/p\u003e","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6162976/v1/aee80156eec19d21af651e70.jpeg"},{"id":78154562,"identity":"86d9d722-7bbc-4962-a655-4003e5960cfe","added_by":"auto","created_at":"2025-03-10 12:17:55","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"graphical-abstract","size":147737,"visible":true,"origin":"","legend":"Unmodified cellulose nanofibrils (CNFs), TEMPO cellulose nanofibrils (TCNFs) and lignocellulose nanofibrils (LCNFs) were used to reinforce a poly(vinyl alcohol) (PVA) matrix. The aim of the study was to compare the performance of these nanofillers with respect to the properties of PVA-based biocomposites. XRD and thermal analyses showed that CNFs were the most crystalline and thermally stable, followed by LCNFs and TCNFs. All nanofillers improved the mechanical properties of PVA, with TCNFs providing the greatest reinforcement. The PVA biocomposite with 6% TCNF showed a 55% higher modulus of elasticity and 58% higher tensile strength than the reference film. The most thermally stable bionanocomposite among the PVA-nanocellulose biocomposites was PVA with 6% CNF; thermal degradation (T) started at a temperature 4\u0026deg;C higher than the reference. A hydrophilic extract of silver fir knotwood was added to the PVA/nanocellulose system to produce biologically active biocomposites, and its effects on mechanical and thermal properties were evaluated. The extract shifted the T to higher temperatures, with the higher content having an even greater effect. While the extract slightly decreased the tensile strength of the biocomposite, the addition of 4% extract to the LCNF-reinforced biocomposite increased the tensile strength by 10% compared to the PVA-LCNF biocomposite.","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6162976/v1/4d3254ce0587dcf2b801cb90.png"},{"id":79221218,"identity":"274d5228-6485-4ec1-a176-6257401dc83a","added_by":"auto","created_at":"2025-03-25 21:16:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3427127,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6162976/v1/2c321649-791b-4ed6-a6d1-77bf5b79f534.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparison of Mechanical and Thermal Properties of Poly(vinyl alcohol)- Bionanocomposite Films Reinforced with Various Modified Cellulose Nanofibrils and Biologically Active Silver Fir Knotwood Extract","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn recent years, interest in and demand for environmentally friendly biocomposite materials has increased. These are based on naturally occurring building blocks and can provide an alternative to environmentally chalanging plastics - petroleum-based, non-biodegradable polymers. Polymer composites are biocomposites if at least one component of the composite is biobased or biodegradable. The biocomposites, based on a biodegradable polymer matrix (PLA, PHA, PVA) with the addition of natural fibers, are more environmentally friendly and are often referred to as green composites (John and Thomas \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The use of green bionanocomposites is highly desirable in the food and packaging industry, agriculture and other applications, beacuse it can reduce the amount of waste products and pollution (Darder et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) contributing to sustainable development goals.\u003c/p\u003e \u003cp\u003eNatural fibers, especially plant cellulose fibers, can be a possible substitute for environmentaly questionable synthetic fibers, which are traditionally used as reinforcing components for various polymer matrices in the production of polymer composites. Cellulose fibers have several advantages over petroleum-based synthetic ones: renewability, biodegradability, low cost, large specific surface area, high tensile strength and flexibility (Zhang et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Nanomaterials are extremely interesting because of their extensive and active surface area, with naturally occurring nanofibers occupying a special place (Saito et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Nanofibrils and microfibrils obtained from cellulose fibers and are of particular importance for the production of polymer composites with improved relevant properties because of their exceptional mechanical properties (high elastic modulus ̴140 GPa) (Fujisawa et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Including cellulose nanofibrils (CNFs) in biopolymer matrices opens up many possibilities for replacing conventional composite materials based partially or entirely on petroleum derivatives (Kalia et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCNFs are generally isolated by delamination of wood pulp using mechanical and/or chemical and enzymatic methods (Lin et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Abdul Khalil et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Tetramethylpiperidine-1-oxyl radical (TEMPO) mediated oxidation is one of the possible chemical pretreatments of cellulosic material, in which primary OH groups (C6 atom of cellulose) are selectively oxidized to carboxyl groups under aqueous conditions at pH 10. In this reaction, NaClO acts as the primary oxidizing agent, and TEMPO and NaBr as catalysts (Saito and Isogai \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Dufresne \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Isogai and Zhou \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The product of this oxidation is β-(1,4)-polyglucuronic acid sodium salt (cellouronic acid Na salt). Electrostatic repulsion between C6 carboxylate anions on cellulose fibrils enables less energy-intensive mechanical processing of oxidized cellulose in water to produce TEMPO cellulose nanofibrils (TCNFs) (Saito and Isogai \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). This electrostatic repulsion disrupts the formation of interfibrillar H-bonds, enabling the production of more individualized cellulose nanofibrils (Saito et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the production of CNFs, the lignin is usually removed before nanofibrillation, whereas lignocellulosic nanofibrils (LCNFs) are cellulose nanofibrils that contain lignin (Liu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). LCNFs production is more environmentally friendly than CNFs production, since LCNFs can be produced directly from raw lignocellulosic biomass without delignification. The residual lignin in LCNFs may contribute to some of the advantages of LCNFs over CNFs, such as greater hydrophobicity, UV blocking and antioxidant activity (Iwamoto and Endo \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). With various pretreatments of lignocellulosic materials, it is possible to apply a surface charge to the fibrils that causes repulsion between them, which further facilitates the mechanical defibrillation of material to produce nanofibril LCNFs (Iwamoto and Endo \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNanocellulose, particularly nanofibrils, offers exceptional barrier properties, strength, sustainability, and potential for functionalization, making it an ideal material for high-performance, environmentally friendly food and pharmaceutical packaging. Thus, the incorporation of unmodified and modified cellulose nanofibrils into various polymer matrices for the production of various packaging films is an area in which many research groups around the world are working. Biodegradable polymer matrices for the production of cellulose-polymer composites can be PVA (Espinosa et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Osolnik et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), PLA (Žepič et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Jacob et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and chitosan (Talebi et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Qin et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and others.\u003c/p\u003e \u003cp\u003eDue to its biodegradability, PVA is an exciting candidate for the production of bio-based packaging films. PVA is a synthetic, water-soluble polymer with good chemical stability and excellent film-forming properties, good mechanical and oxygen barrier properties (Tretinnikov and Zagorskaya \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Espinosa et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kassab et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The main disadvantage of PVA films is the hydrophilicity, which is an issue when packaging moist products (Arun et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). When PVA films are exposed to an environment with higher humidity (above 60%), water molecules penetrate the PVA structure and act as plasticizers, causing the mechanical properties of the PVA to deteriorate (Jain et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Chou et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). One of the solutions for the production of less hydrophilic PVA films is the inclusion of nanocellulose. Due to the hydrogen bonds formed between the PVA and nanocellulose, the number of free OH groups on the PVA chains is decreased, which also contributes to a reduction of hydrophilicity, possibly also resulting in a more hydrophobic character of the PVA biocomposite film (Lee et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Osolnik et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLiteratue review rewealed that individual types of nanostructured cellulose objects have been used for development of PVA matrix based biocomposite films in separate investigations, CNFs in a PVA matrix (Rowe et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Fahma et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Osolnik et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), TCNFs in a PVA matrix (Choo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; S\u0026aacute;nchez-Guti\u0026eacute;rrez et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and LCNFs in a PVA matrix (Espinosa et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Basc\u0026oacute;n-Villegas et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), but the comparative study on properties of PVA films with addition of different types of nanocellulose is still missing.\u003c/p\u003e \u003cp\u003eBy adding biologically active compounds, such as some polyphenolic wood extractives, biocomposites with antibacterial, fungicidal and antioxidant effects can be produced. Biologically active extractives include low and high-molecular weight polyphenolic compounds, which are characterized by antioxidant, antiviral and antibacterial activity (Papuc et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Guo et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Poljanšek et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Vek et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Vek et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Biologically active biocomposites are extremely interesting in the field of packaging since they can replace the use of ecologically questionable conventional packaging (PP, PET, PE) and take their place in the \u0026ldquo;active packaging\u0026rdquo; field. \"Active packaging\" is defined in European Regulation (EC) No. 450/2009 as a packaging system that interacts with the packaged product (food) through the components incorporated into the packaging system. These components can release substances to the packaged product or into the area of the packaged product or absorb substances from the packaged product or the area around the product to increase the product's shelf life (Yildirim and R\u0026ouml;cker \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In addition to inertness and suitable barrier and thermal properties, packaging films can also have biological activity since they can extend the shelf life of the packaged product by adding suitable antioxidant and antimicrobial compounds (Missio et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Missio et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In our study, the wood extractives of silver fir (\u003cem\u003eAbies alba\u003c/em\u003e) knotwood were used as a biologically active agent for the production of PVA biocomposites, since hydrophilic extractives of silver fir knotwood have been shown to have high antioxidant activity (Vek et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To our knowledge, no study has yet been conducted in which PVA biocomposite films were produced with the addition of various cellulose nanofibrils and wood extractives of silver fir knotwood.\u003c/p\u003e \u003cp\u003eThe aims of our study was to compare the influence of different types of cellulose nanofibrils (CNFs, LCNFs, TCNFs) on the reinforcement of the PVA polymer matrix and to investigate the compatibility between cellulose nanofibrils (CNFs, LCNFs, TCNFs) and the biologically active silver fir knotwood extract (EX) in the PVA polymer matrix. A further aim of the present study was to investigate which of the cellulose nanofibrils used (CNFs, LCNFs, TCNFs) proved to be the best filler for the PVA matrix in terms of the mechanical and thermal properties of the final PVA bionanocomposite films. We also aimed to examine the influence of the added silver fir knotwood extract (EX) in the PVA/CNF, PVA/TCNF and PVA/LCNF system on the final mechanical and thermal properties of the three-component PVA biocomposite films produced.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eCellulose nanofibrils (CNFs) were provided by the Centre for Biocomposite and Biomaterial Processing at the University of Toronto, Canada. The CNFs were a CNF-water suspension with a solid content of 1.40% and served as unmodified CNFs. The CNF suspension was obtained by mechanical homogenization of sulfite coniferous wood pulp and consisted of cellulose nanofibrils with diameters ranging from 20 nm to 60 nm (Žepič et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLCNF, TCNF and EX were produced at Department of wood science and technology of the University of Ljubljana. The raw material for LCNFs production was Norway spruce wood (\u003cem\u003ePicea abies\u003c/em\u003e) and silver fir knotwood for EX production, both originating from the forest of Kočevska reka, Slovenia.\u003c/p\u003e \u003cp\u003eThe starting material for TCNFs production was P\u0026Ouml;LS cellulose paper, obtained from Papirnica Vevče, d.o.o., Ljubljana, Slovenia. Maleic anhydride, TEMPO ((2, 2, 6, 6 \u0026ndash; tetramethylpiperidine \u0026ndash; 1 \u0026ndash; yl) oxyl), sodium bromide, sodium hydroxide, hydrochloric acid, polyvinyl alcohol (PVA) \u0026ndash; Mw\u0026thinsp;~\u0026thinsp;47000, tannic acid (TA), methanol, cyclohexane, acetone and formic acid were purchased from Merck (Sigma-Aldrich Chemie, Taufkirchen, Germany). Sodium hypochlorite was purchased from Acros Organics (Thermo Fisher Scientific, UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of active hydrophilic extract\u003c/h2\u003e \u003cp\u003eSequential extraction of silver fir knotwood samples was performed in an ASE 350 accelerated solvent extraction system (Thermo Scientific Dionex). Before extraction, samples were milled by a Retsch SM 2000 cutting mill with a sieve opening of 1 mm and then freeze-dried in a Telstar LyoQuest CC1930 freeze dryer for 24 hours at \u0026minus;\u0026thinsp;85\u0026deg;C and 4.5 Pa. Extraction was performed as described by (Vek et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), first extracting the lipophilic extractives with cyclohexane and then the hydrophilic extractives with an acetone/water mixture (95:5, v/v), both extractions at 100\u0026deg;C and 10.34 MPa.(2 \u0026times; 5 min static cycles). The freeze-dried hydrophilic extract of the silver fir knotwood (EX) was further used as the biologically active component of the PVA biocomposite film.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. TCNFs production\u003c/h2\u003e \u003cp\u003eThe TEMPO oxidation of P\u0026Ouml;LS cellulose paper was carried out according to a method proven by Isogai (Isogai et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Levanič et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Six g of dry cellulose paper was soaked in 600 mL of distilled water. The soaked cellulose was transferred to a three-neck flask, and 0.096 g TEMPO reagent and 0.96 g NaBr were added, then 30 mL NaClO. The reaction took place under constant stirring and pH measurement. The pH was initially above 11; with the addition of HCl, it was lowered to pH 10.2. The pH was kept between 10 and 10.2 for 2 hours with 0.5 M NaOH. Finally, 30 mL ethanol was added to the reaction mixture. The resulting TEMPO-oxidized cellulose was washed with large amounts of distilled water. A suspension of TEMPO-oxidized cellulose with an approximate dry matter content of 1% was then prepared. The suspension was first treated with an Ultraturrax at 12,500 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Mechanical fibrillation of the mentioned product to produce TEMPO-oxidized cellulose nanofibrils (TCNFs) was performed using a PandaPLUS 2000 high pressure homogenizer (GEA NS, Parma, Italy) \u0026minus;\u0026thinsp;1 pass at 50 MPa and 1 pass at 69 MPa. A TCNF water suspension with a solids content of 0.74% was obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. LCNFs production\u003c/h2\u003e \u003cp\u003eLCNFs was isolated from spruce wood (particle size between 0.25 and 0.63 mm) using the method described in a study by Iwamoto (Iwamoto and Endo \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), with slight modifications. Ten g of milled (Retsch SM 2000 cutting mill) spruce wood was weighed into an Erlenmeyer flask and 50 g of maleic anhydride was added. The Erlenmeyer flask with its contents was covered with a watch glass and placed in a heated oil bath \u0026minus;\u0026thinsp;130\u0026deg;C. The esterification reaction lasted 3.5 hours in a melt of maleic anhydride. After 3 hours, the product was washed with acetone and distilled water. The esterification product was soaked in a 0.5 M NaOH solution for 5 minutes to swell the fibers and deprotonate the introduced COOH groups to COO-. The product was then washed again with distilled water. The prepared aqueous suspension of esterified fibers was first treated with an Ultraturrax at 12,500 min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Mechanical fibrillation was then carried out using a PandaPLUS 2000 high-pressure homogenizer (GEA NS, Parma, Italy). The fiber suspension was passed through the homogenizer three times at 80 MPa and an LCNF water suspension with 0.70% solid content was obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Reversed-Phase High-performance Liquid Chromatography (RP-HPLC)\u003c/h2\u003e \u003cp\u003eReversed-Phase High-performance Liquid Chromatography (RP-HPLC) was performed on a Thermo Scientific high-performance liquid chromatography system (Accela HPLC, Waltham, Massachusetts, USA) equipped with a photodiode array detector (PDA); the mobile phase consisted of water and methanol with the addition of 0.1% formic acid. Separation of the components was performed on a Thermo Accucore ODS column (4.6 id \u0026times; 150 mm, 2.6 \u0026micro;m), as described in detail in (Vek et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Preparation of PVA biocomposite films\u003c/h2\u003e \u003cp\u003ePVA biocomposite films with added CNFs/TCNFs/LCNFs were produced by solvent casting as described in a previous study (Osolnik et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A 10% PVA solution was first prepared. The calculated amount of cellulose nanofibrils (CNFs, TCNFs, LCNFs) was added to the 10% PVA solution so that the weight fraction of nanocellulose relative to PVA was 6% in all of the two-component, PVA/nanocellulose films. Our previous studies (Osolnik et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Osolnik et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) revealed that this content of reinforcing component gave us PVA bionanocomposite films with best mechanical properties. In the production of three-component biocomposite films PVA/nanocellulose/EX, the EX was added in a proportion of 2% and 4% in relation to PVA in the PVA/nanocellulose system. These biocomposites will be referred to as three-component PVA/nanocellulose/EX films. The resulting suspensions were mixed for 2 days and then treated with an ultrasonic probe (US) and Ultraturrax. The resulting suspensions were then poured into polystyrene Petri dishes. The films were dried for one week under ambient conditions. The compositions of the films and film labels are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In addition, two reference films were prepared: a PVA film (P) cast from a 10% PVA base solution and a PVA-US film (P-US) cast from a 10% PVA base solution and treated with an ultrasonic probe (US) and Ultraturrax. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the film labels and compositions of the PVA reference and PVA biocomposite films produced.\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\u003eFilm labels and composition\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFilm type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFilm label\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e% CNFs*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e% TCNFs*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e% LCNFs*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e% EX*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePVA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePVA\u0026thinsp;+\u0026thinsp;US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePVA\u0026thinsp;+\u0026thinsp;6%CNF\u0026thinsp;+\u0026thinsp;US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP6CNF-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePVA\u0026thinsp;+\u0026thinsp;6%TCNF\u0026thinsp;+\u0026thinsp;US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP6TCNF-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePVA\u0026thinsp;+\u0026thinsp;6%LCNF\u0026thinsp;+\u0026thinsp;US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP6LCNF-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePVA\u0026thinsp;+\u0026thinsp;6%CNF\u0026thinsp;+\u0026thinsp;2%EX\u0026thinsp;+\u0026thinsp;US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP6CNF2EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePVA\u0026thinsp;+\u0026thinsp;6%CNF\u0026thinsp;+\u0026thinsp;4%EX\u0026thinsp;+\u0026thinsp;US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP6CNF4EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePVA\u0026thinsp;+\u0026thinsp;6%TCNF\u0026thinsp;+\u0026thinsp;2%EX\u0026thinsp;+\u0026thinsp;US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP6TCNF2EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePVA\u0026thinsp;+\u0026thinsp;6%TCNF\u0026thinsp;+\u0026thinsp;4%EX\u0026thinsp;+\u0026thinsp;US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP6TCNF4EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePVA\u0026thinsp;+\u0026thinsp;6%LCNF\u0026thinsp;+\u0026thinsp;2%EX\u0026thinsp;+\u0026thinsp;US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP6LCNF2EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePVA\u0026thinsp;+\u0026thinsp;6%LCNF\u0026thinsp;+\u0026thinsp;4%EX\u0026thinsp;+\u0026thinsp;US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP6LCNF4EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e*% of nanocellulose (CNFs, TCNFs, LCNFs) and % of silver fir knotwood extract (EX) on dry weight of PVA\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. FT-IR analysis\u003c/h2\u003e \u003cp\u003eTo compare the chemical structure of the reinforcing fillers CNFs, TCNFs and LCNFs as well as the chemical structure of the produced PVA biocomposite films and possible interactions between the building blocks of the biocomposite, Fourier transform infrared spectroscopy (FTIR) was performed in ATR (attenuated total reflectance) mode. The freeze-dried samples of CNFs, TCNFs, LCNFs and EX, as well as the prepared PVA biocomposite films, were scanned in the spectral range of 4000 to 450 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with 16 scans and a resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e using a Spectrum Two UATR FT-IR instrument (Perkin Elmer, Waltham, MA, USA). All spectra were ATR and baseline corrected and normalized.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. X-ray diffraction analysis\u003c/h2\u003e \u003cp\u003eX-ray diffraction analysis was performed to analyse the crystallinity of the different nanocelluloses (CNFs, LCNFs, TCNFs). For the XRD measurements, the nanocellulose (CNFs, LCNFs, TCNFs) was freeze-dried using a ModulyoD freeze dryer (Thermo Fisher Scientific, Waltham, MA, USA) for 72 hours (pressure 7 Pa, temperature of the plates 22\u0026deg;C and temperature of the condenser \u0026minus;\u0026thinsp;50\u0026deg;C). Dried samples were pressed uniaxially into tablets using a hydraulic press. XRD analysis was performed using an X'Pert PRO MPD high-resolution X-ray powder diffractometer from PANalytical (Netherlands). Measurements were performed in the 2θ range between 5\u0026deg; and 60\u0026deg; with a step size of 0.034\u0026deg; and an integration time of 250 s. Light corresponding to the electronic transition of CuKα with a wavelength of 0.154 nm was used as the radiation source.\u003c/p\u003e \u003cp\u003eThe crystallinity index was calculated using the Eq.\u0026nbsp;(1), as described in the study by (Segal et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1959\u003c/span\u003e; Zepic et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2014\u003c/span\u003e):\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:{I}_{c}=\\left(\\frac{{I}_{002}-\\:{I}_{am}}{{I}_{002}}\\right)\\times\\:\\:\\)\u003c/span\u003e \u003c/span\u003e100 (\u003cem\u003e1\u003c/em\u003e)\u003c/p\u003e \u003cp\u003ewhere I\u003csub\u003ec\u003c/sub\u003e is the crystallinity index, I\u003csub\u003e002\u003c/sub\u003e is the intensity of the 002 peak (I\u003csub\u003e002\u003c/sub\u003e, 2θ\u0026thinsp;=\u0026thinsp;approx. 22 \u0026deg;) and represents both crystalline and amorphous materials, and I\u003csub\u003eam\u003c/sub\u003e is the intensity of the minimum between the 002 and 101 peaks (I\u003csub\u003eam\u003c/sub\u003e, 2θ\u0026thinsp;=\u0026thinsp;approx. 18 \u0026deg;) and represents amorphous material (Segal et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1959\u003c/span\u003e; Zepic et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Tensile test\u003c/h2\u003e \u003cp\u003eModulus of elasticity (E\u003csub\u003et\u003c/sub\u003e), tensile strength (σ\u003csub\u003eM\u003c/sub\u003e) and elongation at break (ε\u003csub\u003etb\u003c/sub\u003e) of all films were tested on a Zwick/Roell Z005 (Zwick GmbH \u0026amp; Co. KG, Ulm, Germany) universal machine under ambient conditions. The preparation of the film samples for testing and the tensile test were performed as in a previous study (Osolnik et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) using the ASTM D882\u0026ndash;02 standard (Standard Test Method for tensile properties of thin plastic sheeting), with ten samples tested for each film type.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Thermal analysis\u003c/h2\u003e \u003cp\u003e \u003cem\u003eThermogravimetric analysis (TGA)\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe thermal stability of freeze-dried CNFs, TCNFs, LCNFs and biocomposite films was determined by TGA analysis using a TG Mettler Toledo TGA/DSC1 instrument (Mettler Toledo, Greifensee, Switzerland). Approximately 5 mg of the sample was weighed into Pt crucibles and then heated in a temperature program from 25\u0026deg;C to 600\u0026deg;C at a heating rate of 10 K/min in an Ar atmosphere with a flow rate of 50 mL/min. The baseline was subtracted for all measurements.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDifferential scanning calorimetry (DSC)\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe thermal transitions of all films were evaluated with a DSC Mettler Toledo DSC1 instrument (Mettler Toledo, Greifensee, Switzerland). Approximately 5 mg of the sample was heated in aluminum crucibles with perforated lids in a temperature program from 0\u0026deg;C to 230\u0026deg;C at a heating rate of 10 K/min, cooled from 230\u0026deg;C to 0\u0026deg;C at a cooling rate of 5 K/min and heated again from 0\u0026deg;C to 230\u0026deg;C at a heating rate of 10 K/min in an Ar atmosphere at a flow rate of 50 ml/min.\u003c/p\u003e \u003cp\u003eThe degree of crystallinity of the PVA was calculated using the Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e2\u003c/span\u003e) as described in previous studies (Idris et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Osolnik et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e):\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:{X}_{c}^{DSC}\\:\\left[\\%\\right]=\\:\\frac{{\\varDelta\\:H}_{f}}{{\\varDelta\\:H}_{f}^{◦}}\\times\\:100$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eΔH\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varDelta\\:H}_{f}^{◦}\\)\u003c/span\u003e\u003c/span\u003e are the enthalpy of melting of pure, reference PVA films and the PVA biocomposite films and PVA with 100% crystallinity in J/g, as described in a study by Idris et al. The melting enthalpy for PVA with 100% crystallinity (ΔH◦f) is 138.7 J/g (Idris et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Statistics\u003c/h2\u003e \u003cp\u003eThe results obtained were subjected to basic statistical analysis using Statgraphics Centurion 17. software. The analysis of variance (ANOVA) and the least significant difference (LSD) method of Fisher with a confidence level of 95% were performed.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. HPLC analysis\u003c/h2\u003e \u003cp\u003eThe results of the chromatographic quality control of the EX, are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. EX was characterized by large amounts of polyphenols, most of which were lignans isolariciresinol, lariciresinol, secoisolariciresinol, pinoresinol and matairesinol (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), with secoisolariciresinol and lariciresinol being the predominant ones. Other phenolic compounds in the EX were phenolic acids, with homovanillic acid, coumaric acid and ferulic acid, as well as the flavonoids epicatechin, taxifolin and quercetin. Compared to the identified lignans, these were present in lower quantities in the EX. Phenolic compounds of silver fir have already been shown to be natural antioxidants and biocidal compounds (Willf\u0026ouml;r et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Benković et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Vek et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. FTIR analysis\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the FTIR spectra of CNFs, LCNFs and TCNFs. The FTIR spectra contain characteristic peaks for the vibrations of the bonds in the cellulose molecules, the broad band between 3600 and 3000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to the stretching vibration of O-H, 2892 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e due to the symmetric vibration of CH\u003csub\u003e2\u003c/sub\u003e groups, 1408 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e due to the m \u0026ndash;CH\u003csub\u003e2\u003c/sub\u003e scissoring motion, 1370 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represents the C-H bending, 1060 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e due to the C\u0026ndash;O\u0026ndash;C stretching vibration of the pyranose ring (Širok\u0026yacute; et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Soni et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), but they differ from each other.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe vibrational signal associated with the OH groups at a wavenumber of about 3300 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is broadest for LCNFs (aromatic OH groups) (Osolnik et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and least broad for CNFs. After the esterification of the lignocellulose surface with maleic anhydride, a vibration of C\u0026thinsp;=\u0026thinsp;O in the ester group at 1714 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a vibration of C\u0026thinsp;=\u0026thinsp;O in the carboxylate anion at 1637 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were observed. The FTIR spectra of LCNFs contain peaks at 1575 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1510 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which are not found in the FTIR spectra of CNFs and TCNFs. These peaks belong to C\u0026thinsp;=\u0026thinsp;C vibrations in the lignin structure (Horseman et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Osolnik et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The FTIR spectra of TCNFs contains a signal at 1600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which belongs to vibration of the carboxylate anion on the cellulose chain as a result of the TEMPO-mediated oxidation of the starting cellulose (Karim et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Poyraz et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the FTIR spectra of the neat PVA film, the EX, the two-component PVA/nanocellulose films and the three-component PVA/nanocellulose/EX films. The FTIR spectra of the neat PVA film contain characteristic peaks typical of PVA, which are described in detail in a study by (Osolnik et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These peaks also appeared in the FTIR spectra of the PVA biocomposite films. The inclusion of LCNFs in the P6LCNF-US film is confirmed by the fact that a peak at 1575 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e appears in both the FTIR spectra of LCNFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and the spectra of P6LCNF-US (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), indicating the presence of C\u0026thinsp;=\u0026thinsp;C in the lignin. This is also true for TCNFs, which was successfully incorporated into the P6TCNF-US biocomposite, since the peak at 1600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which is due to the vibration of the carboxylate anion on TCNFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), also appears in the FTIR spectra of P6TCNF-US. The FTIR spectra of the EX contain some main peaks belonging to \u0026ndash;OH stretching (3319 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), CH stretching (2933 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), CH2 stretching (2854 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), C\u0026thinsp;=\u0026thinsp;O stretching (1751 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1696 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), C\u0026thinsp;=\u0026thinsp;C stretching in the aromatic ring (1601 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1513 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), asymmetric C\u0026ndash;H deformation (1449 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and C\u0026ndash;O stretching (1030 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Popescu et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Morar et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). EX is also successfully included in the three-component biocomposites since a peak at 1513 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e appears in the FTIR spectra of P6CNF4EX-US (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), P6LTNF4EX-US (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) and P6LCNF4EX-US (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. X-ray diffraction analysis\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the X-ray diffraction patterns of CNFs, LCNFs and TCNFs. Two prominent crystalline peaks are present in all the diffractograms, the first at about 2θ\u0026thinsp;=\u0026thinsp;16\u0026deg; and the second at about 2θ\u0026thinsp;=\u0026thinsp;22\u0026deg;, corresponding to the 101- and 002- reflection planes of the structure of cellulose I; the region around 2θ\u0026thinsp;=\u0026thinsp;18\u0026deg; or the minimum between the mentioned crystalline peaks is typical of the amorphous part of cellulose (Zepic et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ji et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Morcillo et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCNFs showed the highest crystallinity (Ic\u0026thinsp;=\u0026thinsp;84%) among the analyzed samples, followed by LCNFs (Ic\u0026thinsp;=\u0026thinsp;75%) and TCNFs (Ic\u0026thinsp;=\u0026thinsp;69%). The crystallinity of the nanofibrils depends on the proportion of amorphous structures present (hemicellulose, lignin) and on the nanofibril production process (bleaching) (Demuner et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The lower crystallinity of LCNFs is probably due to the amorphous structure of lignin in LCNFs. The lowest crystallinity of TCNF is attributed to the presence of a carboxylate anion on the cellulose chains, and due to this relatively large substituent, the arrangement of the chains is restricted so that the crystallinity is lower. Another possible cause is the cleavage of covalent bonds (C-O-C) by TEMPO-mediated oxidation, which leads to cellulose chains with a lower molecular weight (Milanovic et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hiraoki et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Tensile properties\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the average values and standard deviations for elastic modulus (E\u003csub\u003et\u003c/sub\u003e), tensile strength (σ\u003csub\u003eM\u003c/sub\u003e) and elongation at break (ε\u003csub\u003etb\u003c/sub\u003e) for the reference films P and P-US, for the two-component PVA/nanocellulose films and the three-component PVA/nanocellulose/EX films. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA shows the average stress-strain curves for the reference films P and P-US and for two-component PVA/nanocellulose films, each ending with the lowest elongation at break of ten measured samples for each film. The modulus of elasticity of the P-US film is 57% higher, and the tensile strength is 21% higher than that of the P film. The elongation at break is 10% lower than that of the P film. The influence of ustrasound treatment on all measured mechanical parametrs was significant. It could be concluded that the ultrasonic treatment brings the PVA chains into closer contact with each other, which increases the number of H-bonds between the PVA chains, i.e., the physical cross-linking is more potent than in the case in which the solution was not treated with US (P). When processed with an ultrasonic probe, the amorphous parts are probably destroyed to a certain extent, the crystallinity of P-US is slightly higher (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and therefore, the mechanical properties of P-US are better.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTensile properties of PVA reference films and PVA biocomposite films with different nanocellulose (CNFs; TCNFs, LCNFs) and different weight percentages of silver fir knotwood extract (EX); (ANOVA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eE\u003csub\u003et\u003c/sub\u003e [MPa]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eσ\u003csub\u003eM\u003c/sub\u003e [MPa]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eε\u003csub\u003etb\u003c/sub\u003e [%]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2136\u0026thinsp;\u0026plusmn;\u0026thinsp;406\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e61.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e79.2\u0026thinsp;\u0026plusmn;\u0026thinsp;24.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3352\u0026thinsp;\u0026plusmn;\u0026thinsp;489\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e73.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e71.3\u0026thinsp;\u0026plusmn;\u0026thinsp;9.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6CNF-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4889\u0026thinsp;\u0026plusmn;\u0026thinsp;569\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e100.4\u0026thinsp;\u0026plusmn;\u0026thinsp;9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e15.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6TCNF-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5201\u0026thinsp;\u0026plusmn;\u0026thinsp;640\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e116.5\u0026thinsp;\u0026plusmn;\u0026thinsp;15.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e17.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6LCNF-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4648\u0026thinsp;\u0026plusmn;\u0026thinsp;640\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e98.3\u0026thinsp;\u0026plusmn;\u0026thinsp;24.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e14.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6CNF2EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4370\u0026thinsp;\u0026plusmn;\u0026thinsp;794\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e93.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e13.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6CNF4EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4535\u0026thinsp;\u0026plusmn;\u0026thinsp;734\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e98.0\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6TCNF2EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5002\u0026thinsp;\u0026plusmn;\u0026thinsp;1175\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e109.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e16.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6TCNF4EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4693\u0026thinsp;\u0026plusmn;\u0026thinsp;791\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e106.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e15.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6LCNF2EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3795\u0026thinsp;\u0026plusmn;\u0026thinsp;639\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e94.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e14.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6LCNF4EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4919\u0026thinsp;\u0026plusmn;\u0026thinsp;1507\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e109.3\u0026thinsp;\u0026plusmn;\u0026thinsp;16.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e10.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\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\u003e \u003c/p\u003e \u003cp\u003eUltrasonic treatment was chosen according to the studies (Osolnik et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Osolnik et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) because the results of the mechanical properties of PVA/CNF biocomposite films were better when the suspensions for film production were treated with ultrasound. Based on a study (Osolnik et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), it was decided that the optimum CNF addition concerning dry PVA was 6%. In the present study, all PVA biocomposite films were prepared with with 6% addition of nanocellulose (CNFs, TCNFs and LCNFs). In all three cases, the biocomposite films' modulus of elasticity and tensile strength were significantly improved compared to the two reference films. This was expected based on the available literature (Choo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Majumdar et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Osolnik et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The addition of TCNFs proved to be the best; the elastic modulus value was 143% and 55% higher for P6TCNF-US than for the reference films P and P-US, and the tensile strength was 91% and 58% higher than for the reference films P and P-US. The elongation at break can be expected to be lower than that of the reference films, accompanied by a significant increase in tensile strength. To improve the mechanical properties of the final nanocomposite, a large contact surface between the nanofiller and the polymer matrix, as well as a good distribution of the nanofiller (nanocellulose) in the matrix, are important (Oksman et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mondal \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). From the results of the mechanical properties, it can be concluded that CNFs, TCNFs and LCNFs were all well distributed in the PVA matrix. Among the investigated nanocelluloses, TCNFs have the most hydrophilic character, so its distribution in the hydrophilic PVA matrix was probably the most homogeneous. Moreover, due to the presence of functional groups (COO\u003csup\u003e\u0026ndash;\u003c/sup\u003e and OH groups) on TCNFs, many intermolecular forces probably formed between these groups and the OH groups on the PVA chains, which further improved the mechanical strength of the final P6TCNF-US composite. Despite the highest tensile strength, it is interesting to note that the elongation at break of the P6TCNF-US biocomposite was higher than the elongation at break of P6CNF-US and P6LCNF-US.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB represents the average stress-strain curves for the P-US reference film and the three-component PVA/nanocellulose/EX films, with each average curve ending at the lowest elongation at break among 10 parallel samples for each film. Our results showed that the addition of he EX variously affected the mechanical properties of PVA biocomposite films. The addition of 4% EX to the PVA/LCNF and PVA/CNF system was more effective in terms of increasing tensile strength and elastic modulus than in the case of TCNFs. The tensile strength of P6CNF4EX \u0026ndash;US was comparable to the tensile strength of the P6CNF-US biocomposite film. In contrast, the modulus of elasticity was slightly lower. In the case of P6LCNF4EX-US the tensile strength and modulus of elasticity were even improved compared to the values of the P6LCNF-US biocomposite film. Adding EX (2% or 4%) to the PVA/TCNF system slightly lowers the values for tensile strength and modulus of elasticity, but these values are still much higher than the values for the polymer itself.\u003c/p\u003e \u003cp\u003eAmong the three-component composite films, the PVA/TCNF/EX films proved to be the best according to the values of the measured mechanical parameters. Similar values for the modulus of elasticity and the tensile strength value were observed for the P6LCNF4EX-US film, but with a lower value for elongation at break compared to P6TCNF2EX and P6TCNF4EX-US. TCNFs are the most polar of all the cellulose nanofibrils used to reinforce the PVA matrix, which means that the distribution of TCNFs in the hydrophilic polymer matrix was probably the most homogeneous compared to the other two types of nanofibrils used. The contact area between the TCNFs and the PVA matrix was likely larger than in the other two cases, allowing better transfer of stress from the matrix to the fiber. In addition, the hydrophilic extract, which is able to form hydrogen bonds with both PVA and TCNFs, also has a positive effect on the mechanical properties of the three-component films.\u003c/p\u003e \u003cp\u003e.3.4. Thermal analysis\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows Tonset and Tmax for the neat PVA film (P, P-US), CNFs, TCNFs, LCNFs and PVA biocomposite films. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows Among the nanofillers investigated, CNFs were the most thermally stable, followed by LCNFs and TCNFs (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The biocomposites reinforced with these nanofillers and the three-component biocomposites with EX additive follow the same trend of thermal degradation as CNFs, LCNFs and TCNFs.the TG - A and DTG - B curves of freeze-dried samples of nanocellulose \u0026ndash; CNFs, TCNFs and LCNFs.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eT\u003csub\u003eonset\u003c/sub\u003e and T\u003csub\u003emax\u003c/sub\u003e for the neat PVA film, CNF, TCNF, LCNF and PVA biocomposite films\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFilm type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT\u003csub\u003eonset\u003c/sub\u003e [\u0026deg;C]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT\u003csub\u003emax\u003c/sub\u003e [\u0026deg;C]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e299\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e364\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e281\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e362\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCNF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e319\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e346\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTCNF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e237\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLCNF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e296\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6CNF-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e285\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e363\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6TCNF-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e268\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e327\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6LCNF-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e331\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6CNF2EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e289\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e363\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6CNF4EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e291\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e361\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6TCNF2EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e269\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e325\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6TCNF4EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e272\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e325\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6LCNF2EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e280\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e332\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6LCNF4EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e333\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\u003e \u003c/p\u003e \u003cp\u003eAs mentioned, TCNFs showed the lowest thermal stability among the different nanocelluloses, T\u003csub\u003eonset\u003c/sub\u003e was the lowest among the investigated nanocelluloses (T\u003csub\u003eonset\u003c/sub\u003e = 218\u0026deg;C). This was probably due to the decarboxylation of carboxyl groups on the surface of TCNFs (Lichtenstein and Lavoine \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), which were introduced into the surface of cellulose nanofibrils during TEMPO-mediated oxidation, in which C6 hydroxyl groups of cellulose were converted to sodium carboxylate groups (Fukuzumi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The reason for the poor thermal stability of TCNFs could also be that the cellulose chains were cleaved during the TEMPO-mediated oxidation of cellulose to produce TCNFs, suggesting that the molecular weight of the TEMPO-oxidized cellulose decreased, also making TCNFs less thermally stable (Mahendra et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This observation agrees with the values for crystallinity, the lower the crystallinity of the material, the lower the temperature at which thermal decomposition begins.\u003c/p\u003e \u003cp\u003eThe TG and DTG curves for CNFs, TCNFs, and LCNFs are different. Still, the first mass loss region, in which there is a minor mass loss and which ends at about 130\u0026deg;C, is due to the elimination of water (Zepic et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) then thermal decomposition starts at 218\u0026deg;C for TCNFs, 261\u0026deg;C for LCNFs and 319\u0026deg;C for CNFs. From here on, there is a more significant loss of mass, probably initially at the expense of the formation of dehydrogenated cellulose and further strong depolymerization of cellulose molecules, producing methane, carbon monoxide and carbon dioxide, as described by Zhang et al. (Zhang et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs reported by Lichtenstein and Lavoine \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, thermal degradation of TCNFs in which sodium carboxylate groups were present occurred in three stages of degradation (Lichtenstein and Lavoine \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), which was also observed in the DTG curve of TCNFs in the present study (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB; T\u003csub\u003emax1\u003c/sub\u003e=235\u0026deg;C, T\u003csub\u003emax2\u003c/sub\u003e=281\u0026deg;C and T\u003csub\u003emax3\u003c/sub\u003e=approx. 420\u0026deg;C). In a study on the thermal stability of TEMPO-oxidized cellulose (Fukuzumi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), the authors reported in relation to the DTG curve of TEMPO-oxidized CNFs that the first DTG peak at 233\u0026deg;C belongs to the decomposition of anhydrous sodium glucuronate units and the second DTG peak at 282\u0026deg;C belongs to the decomposition of crystalline cellulose chains. Our results of the thermal decomposition of TCNFs are consistent with the study of Fukuzumi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, since the main DTG peaks occurred at almost the same temperatures (235 and 281\u0026deg;C). The thermal decomposition of TCNFs thus takes place in a wider temperature range and in three stages, while the thermal decomposition of CNFs and LCNFs takes place in a narrower temperature range and in two stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs with TCNFs, there were also carboxyl groups on the surface of LCNFs, which resulted from the pretreatment (esterification) of the lignocellulosic feedstock with maleic anhydride. The presence of carboxyl groups on the surface of LCNFs could be a reason for the lower thermal stability (Lichtenstein and Lavoine \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) of LCNF (T\u003csub\u003eonset\u003c/sub\u003e= 261\u0026deg;C) compared to CNFs (T\u003csub\u003eonset\u003c/sub\u003e= 319\u0026deg;C), which also agrees well with the values for crystallinity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThermal degradation of the neat PVA film started at 299\u0026deg;C (P) and at 281\u0026deg;C (P-US) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Both films were produced to investigate the influence of treatment with an ultrasonic probe on the thermal properties of the neat PVA film. The treatment of the PVA solution with the ultrasonic probe probably caused a shortening of the PVA chains and a further deterioration of the thermal properties. The shortening of the chains probably starts first in the amorphous parts of the chains, which means that the amorphous parts partially break up, and thus, the crystallinity of the film increases slightly (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A slight increase in crystallinity is also associated with better mechanical strength and lower elasticity (lower elongation at break) of the P-US film compared to the P-film (table of mechanical properties).\u003c/p\u003e \u003cp\u003eFigures \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB show the TG and DTG curves of the reference films (P, P-US) and the three-component PVA/nanocellulose/EX films. In the thermal decomposition of PVA films, there are three zones of mass loss. The first mass loss, a small weight loss that started at about 100\u0026deg;C and stabilized up to 200\u0026deg;C, is due to the evaporation of water. The first decomposition stage, which started at 281\u0026deg;C (T\u003csub\u003eonset\u003c/sub\u003e for P-US) or 299\u0026deg;C (T\u003csub\u003eonset\u003c/sub\u003e for P), is due to the cleavage of the polymer chains, resulting in smaller fragments. The main chain cleavage occurred in the second decomposition stage, above approx. 400\u0026deg;C (Thomas et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Suleiman et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The thermal degradation for the two-component and three-component PVA biocomposite films produced was very similar.\u003c/p\u003e \u003cp\u003eThe addition of 6% CNFs to the PVA matrix improved the thermal stability of the P6CNF-US film compared to the P-US film. The T\u003csub\u003eonset\u003c/sub\u003e of the biocomposite film shifted from 281\u0026deg;C for P-US to 285\u0026deg;C for P6CNF-US. The addition of the EX to the PVA/CNF system resulted in a shift of the T\u003csub\u003eonset\u003c/sub\u003e to a higher T, in the case of P6CNF2EX-US T\u003csub\u003eonset\u003c/sub\u003e = 289\u0026deg;C and in the case of P6CNF4EX-US T\u003csub\u003eonset\u003c/sub\u003e = 291\u0026deg;C. The addition of TCNFs and LCNFs to the PVA matrix did not lead to an improvement in the thermal stability of the biocomposite films. EX contains hydrophilic extractives, with the largest proportion being lignans (Vek et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which probably contribute to or influence cross-linking between the PVA, nanocellulose and extract components. Of all the biocomposite films, P6TCNF-US had the lowest T\u003csub\u003eonset\u003c/sub\u003e value, but the highest value for modulus of elasticity and tensile strength (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The poorer thermal stability of the biocomposite is probably related to the least thermally stable nanocellulose \u0026ndash; TCNFs.\u003c/p\u003e \u003cp\u003eThe thermal stability of the three-component PVA/nanocellulose/EX films is better than that of the two-component biocomposites (P6TCNF-US, P6LCNF-US). A 4% addition of EX to the PVA/nanocellulose system had a more significant influence on the T\u003csub\u003eonset\u003c/sub\u003e shift to a higher temperature than a 2% addition of EX. This was probably because more interactions were formed in the biocomposite at a higher EX addition, which contributed to a higher thermal stability.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of glass transition temperature (T\u003csub\u003eg\u003c/sub\u003e), peak of melting temperature (T\u003csub\u003em\u003c/sub\u003e) and crystallinity by DSC (X\u003csub\u003eC\u003c/sub\u003e \u003csup\u003eDSC\u003c/sup\u003e) of the neat PVA film and PVA biocomposite films\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFilm type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT\u003csub\u003eg\u003c/sub\u003e [\u0026deg;C]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT\u003csub\u003em\u003c/sub\u003e [\u0026deg;C]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eX\u003csub\u003eC\u003c/sub\u003e\u003csup\u003eDSC\u003c/sup\u003e [%]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e221\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e222\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6CNF-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6TCNF-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e219\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6LCNF-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e217\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6CNF2EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e219\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6CNF4EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e221\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6TCNF2EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6TCNF4EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6LCNF2EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e217\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6LCNF4EX-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e216\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c5\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the glass transition temperature (T\u003csub\u003eg\u003c/sub\u003e), the peak of melting temperature (T\u003csub\u003em\u003c/sub\u003e) and crystallinity by DSC (X\u003csub\u003eC\u003c/sub\u003e\u003csup\u003eDSC\u003c/sup\u003e) of the neat PVA film and PVA biocomposite films. Adding any type of cellulose nanofibrils to the PVA matrix increased the glass transition temperature of the formed PVA biocomposite films compared to the reference PVA film (P-US). The glass transition temperature is related to the flexibility of the polymer segments; since the cross-linking of the structure increases at the expense of the interactions between the components of the composite; the mobility of the macromolecules decreases, which increases the T\u003csub\u003eg\u003c/sub\u003e of the biocomposite films (Wu et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Parit et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Osolnik et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Of all types of cellulose nanofibrils studied, TCNFs had the greatest influence on the T\u003csub\u003eg\u003c/sub\u003e value. The reason for the highest T\u003csub\u003eg\u003c/sub\u003e value is probably due to the presence of hydroxyl and carboxyl functional groups on the TCNFs surface, which probably form more interactions with the OH groups on the PVA chains (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) than there were interactions between CNFs and PVA and between LCNFs and PVA. Another reason for the highest T\u003csub\u003eg\u003c/sub\u003e of P6TCNF-US is also that TCNFs are the most polar cellulose fibrils and are probably also the most homogeneously distributed in the hydrophilic PVA matrix, and thus the contact area between PVA and TCNFs was larger. The P6LCNF-US film had just 1\u0026deg;C lower T\u003csub\u003eg\u003c/sub\u003e compared to P6TCNF-US. LCNFs also contain carboxyl groups, hydroxyl groups and more double bonds, which also leads to interaction between LCNFs and PVA.\u003c/p\u003e \u003cp\u003eThe addition of EX had a slightly negative effect on T\u003csub\u003eg\u003c/sub\u003e, which is in contrast to our recent study (Osolnik et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) in which tannic acid was used as a biologically active component and crosslinker for PVA and CNFs, in which a 10% addition of tannic acid increased the T\u003csub\u003eg\u003c/sub\u003e by 10\u0026deg;C. The structure of the EX was less defined than the structure of the tannic acid, so the EX's distribution in the PVA/nanocellulose system was probably less homogeneous than the distribution of the tannic acid in the PVA/CNF system. This meant that the contact area between PVA, nanocellulose and the EX was also smaller, fewer interactions were formed with all components of the composition and therefore, the movement of the macromolecules was not as restricted, which is probably why the T\u003csub\u003eg\u003c/sub\u003e for the three-component biocomposite did not shift to a higher temperature.\u003c/p\u003e \u003cp\u003eThe crystallinity of PVA in PVA biocomposite films was determined by DSC measurements. As Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows, the crystallinity of PVA in the PVA biocomposite films decreased compared to that of the P-US reference film. The nanocellulose and the components of EX interact with PVA, which probably restricts the arrangement of PVA molecules and the formation of crystalline parts, resulting in a lower crystallinity of PVA biocomposite films (Parit et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Osolnik et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Suleiman et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The crystallinity determined from the DSC data showed the lowest value for PVA biocomposite films to which LCNFs was added. In addition to the probably favorable interactions between the building blocks of the PVA-LCNF composite and the PVA-LCNF-EX, which confirm the improved mechanical properties of the composite compared to the PVA reference film, the reason for the lower crystallinity may also be the lowest crystallinity of LCNFs among the investigated nanocelluloses (XRD crystallinity, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The introduction of aromatic groups (LCNFs, EX) into the PVA matrix may also disrupt the regular arrangement of the PVA molecules and thus reduce the crystallinity of PVA (Suleiman et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs mentioned before, P-US had a higher crystallinity than P, which was probably due to the influence of treating the PVA solution with an ultrasonic probe, and apparently the amorphous parts of PVA had disintegrated to a certain extent, resulting in a slight increase in the crystallinity of PVA in P-US film compared to P film.\u003c/p\u003e \u003cp\u003eA decrease in crystallinity was also accompanied by a decrease in melting temperature, since the structure was less ordered \u0026ndash; the amorphous parts of the biocomposite in which the polymer chains are not ordered - resulting in weaker intermolecular interactions and thus lower melting temperatures (Suleiman et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe chemical structure of the different reinforcing components (CNFs, LCNFs and TCNFs) varies, which is clearly demonstrated by the FTIR spectra. We believe that the different chemical structure influences the different distribution of the reinforcing components in the hydrophilic polymer PVA matrix. The crystallinity results obtained from the XRD analysis also varied for the reinforcing nanofillers used. The unmodified CNFs were the most crystalline, followed by the chemically modified LCNFs and the oxidized TCNFs. FTIR spectroscopy confirmed not only the successful incorporation of nanocellulose, but also the successful incorporation of silver fir knotwood extract (EX). The results of the tensile tests showed that CNFs, LCNFs and TCNFs were all suitable for reinforcing the PVA polymer, since the tensile strength and modulus of elasticity of the PVA-nanocellulose biocomposites clearly exceeded those of the reference polymer films. The addition of TCNFs proved to be the most effective reinforcement for PVA, probably because its more hydrophilic structure distributes most homogeneously in the PVA matrix, thereby improving the mechanical properties. In the study, a comparison was also made between the ultrasonically sonicated and non-sonicated base solutions of the PVA polymer (P, P-US) and their subsequent mechanical and thermal properties. The P-US film exhibited a higher tensile strength and modulus of elasticity than the P film, which is probably because the ultrasonic treatment breaks up some of the amorphous regions and the crystallinity of the P-US film is slightly higher than that of the P film, resulting in better mechanical properties. By incorporating EX into the PVA/nanocellulose system, three-component PVA biocomposite films were produced, with the TCNF-containing biocomposite film again exhibiting the best mechanical strength. The results of thermal analysis showed that TCNFs was the least thermally stable of the investigated nanofillers, since thermal degradation started at the lowest temperature. Comparison of the P and P-US reference films showed that ultrasonic treatment of the polymer solution probably caused chain shortening in PVA, resulting in a deterioration of the thermal properties. The P6CNF-US film showed better thermal stability than the P-US film. Furthermore, the addition of EX in both investigated weight fractions in the PVA/CNF system resulted in a shift of the onset temperature (T\u003csub\u003eonset\u003c/sub\u003e) to higher temperatures. However, the addition of LCNFs and TCNFs to the PVA matrix did not contribute to improving the thermal stability of the PVA/LCNF and PVA/TCNF biocomposites. The addition of EX to the PVA/nanocellulose system had a positive effect on shifting the T\u003csub\u003eonset\u003c/sub\u003e to higher temperatures compared to the two-component PVA/nanocellulose biocomposite films, with the higher weight fraction of EX having an even greater effect. However, EX had a slightly negative effect on the Tg of the biocomposite films due to the chemical structure of the EX and the fact that EX was probably not homogeneously distributed in the PVA/nanocellulose system. Of the nanocelluloses studied, TCNFs had the greatest effect on the Tg value due to the structure of TCNFs \u0026ndash; the presence of hydroxyl and carboxyl groups on the cellulose chains - which probably led to more interactions between PVA and TCNF compared to PVA with LCNF or CNF.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eConceptualization, I.P., U.O. and P.O.; methodology, V.V., U.O. and I.P.; software, I.P., V.V. and U.O.; validation, I.P., U.O. and V.V.; formal analysis, I.P. and U.O.; investigation, I.P., U.O., V.V., M. H. and P.O.; figures, U.O. and I.P.; writing\u0026mdash;original draft preparation, U.O. and I.P.; writing\u0026mdash;review and editing, I.P., V.V., M.H. and P.O. and supervision, P.O. and I.P. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration:\u0026nbsp;\u003c/strong\u003eThe authors would like to thank the Slovenian Research and Innovation Agency (ARIS) for financial support within the research program P4-0015, projects L4-2623 and V4-2017, as well as the University of Ljubljana, the Ministry of Higher Education, Science and Innovation and the EU within the NOO project \u0026quot;UL for a sustainable society - ULTRA\u0026quot; and the University of Ljubljana for supporting project BAPUR in the frame of M.ERA-Net.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eThis study did not include any studies conducted by any author on human participants or animals. The authors declare compliance with ethical standards.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eThe data presented in this study are available on request from the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eSpecial thanks to Mr. Martin Cregeen for language editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdul Khalil, H.P.S., Davoudpour, Y., Islam, M.N., Mustapha, A., Sudesh, K., Dungani, R., Jawaid, M. 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(2015) Effect of Drying Pretreatment on the Acetylation of Nanofibrillated Cellulose. Bioresources 10.\u003c/span\u003e\u003c/li\u003e\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":"bionanocomposite films, (ligno)cellulose nanofibrils, poly(vinyl alcohol), reagent TEMPO, silver fir, extract, antioxidant, properties","lastPublishedDoi":"10.21203/rs.3.rs-6162976/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6162976/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Unmodified cellulose nanofibrils (CNFs), TEMPO cellulose nanofibrils (TCNFs) and lignocellulose nanofibrils (LCNFs) were used to reinforce a poly(vinyl alcohol) (PVA) matrix. The aim of the study was to compare the performance of these nanofillers with respect to the properties of PVA-based biocomposites. XRD and thermal analyses showed that CNFs were the most crystalline and thermally stable, followed by LCNFs and TCNFs. All nanofillers improved the mechanical properties of PVA, with TCNFs providing the greatest reinforcement. The PVA biocomposite with 6% TCNF showed a 55% higher modulus of elasticity and 58% higher tensile strength than the reference film. The most thermally stable bionanocomposite among the PVA-nanocellulose biocomposites was PVA with 6 % CNF; thermal degradation (Tonset) started at a temperature 4 °C higher than the reference. A hydrophilic extract of silver fir knotwood was added to the PVA/nanocellulose system to produce biologically active biocomposites, and its effects on mechanical and thermal properties were evaluated. The extract shifted the Tonset to higher temperatures, with the higher content having an even greater effect. While the extract slightly decreased the tensile strength of the biocomposite, the addition of 4% extract to the LCNF-reinforced biocomposite increased the tensile strength by 10% compared to the PVA-LCNF biocomposite.","manuscriptTitle":"Comparison of Mechanical and Thermal Properties of Poly(vinyl alcohol)- Bionanocomposite Films Reinforced with Various Modified Cellulose Nanofibrils and Biologically Active Silver Fir Knotwood Extract","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-10 12:09:49","doi":"10.21203/rs.3.rs-6162976/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":"c71343a0-6ee3-41a9-8e1d-4d68f04e908a","owner":[],"postedDate":"March 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-25T21:08:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-10 12:09:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6162976","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6162976","identity":"rs-6162976","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}