Ionic liquid, ultrasound-assisted synthesis of lignin nanoparticles for barrier-enhanced all-cellulose nanocomposite films

The cellulose source used was highly purified bleached fibre of cotton linter pulp supplied by Celsur (Granada, Spain). The pulp contained more than 90% \(\alpha\)-cellulose as calculated according to TAPPI T203. The ionic liquid, 1-ethyl-3-methylimidazolium acetate (EmimAc), was supplied in 98% purity by Sigma-Aldrich (Darmstadt, Germany) and removed with distilled water when needed. Sulphonated kraft lignin powder of molecular weight (\(M_{w}\))= 46500 g/mol and a total sulphur content of 6.9% was obtained from Borregaard (Sarpsborg, Norway).

The original paper was obtained from cotton linter pulp containing more than 90% \(\alpha\)-cellulose according to ISO 5269-2 on a Rapid-Köthen machine. Moist paper was then sandwiched between two stacks of standard filter paper and compressed at 0.1 MPa at 90 \(^\circ\)C for 10 min. Based on preliminary research and previous reports (Zou et al. 2022; Garcia Gonzalez et al. 2017), variable amounts of lignin (3, 5 and 7 wt% of paper) were dispersed in 3 mL of ionic liquid and sonicated at room temperature (25 \(^\circ\)C), a frequency of 20 kHz and a power of 130 W for 30 min to obtain homogeneous, stable nanodispersions. The starting cellulosic material and the glassware used were carefully dried to avoid any adverse effects of water on cellulose dissolution.

The required amount of dried paper (aerial weight 40 g/m\(^{2}\), area 64 cm\(^{2}\)) was placed in a glass Petri dish after preparing the nanolignin-IL solution to be used as nanowelding and UV-blocking agent for application to the paper surface. In order to boost IL-assisted partial dissolution of cellulose, the process was allowed to continue in an oven at 80 \(^\circ\)C for 24 h. After dissolution, the resulting gel nanocomposite films were removed from the oven and allowed to cool at room temperature. Then, each film was washed by coagulation in de-ionized water to obtain a transparent nanocomposite gel, residual ionic liquid present in the regenerated sample being removed by washing several times. Next, each film was sandwiched between two glass plates and oven-dried at 60 \(^\circ\)C for 1 h. Finally, the nanocomposite was blotted between filter paper and glass plate to dry at 50% relative humidity (RH) at 25 \(^\circ\)C for at least 3 days. The all-cellulose composite film used as control sample was obtained by following the above-described procedure in the absence of lignin nanoparticles. The nanocomposite films obtained from 3, 5 and 7 wt% LNPs were coded ACNC3%LNPs, ACNC5%LNPs and ACNC7%LNPs, respectively.

The lignin nanoparticles obtained with sonication in the ionic liquid were examined by high-resolution transmission electron microscopy. Figure 2 shows the TEM images for an all-cellulose nanocomposite (ACNC) film containing 7 wt% nanoparticles of average size 5.133 ± 0.003 nm. The images confirm that sonication enabled the formation of nanosized lignin particles that were unevenly spherical in shape. Therefore, as previously found by Aditya et al. (2018), the ionic liquid provided an excellent medium for their preparation. Thus, it enhanced electronic and steric stabilization of the nanoparticles, and reduced particle growth by effect of its constituent anion and cation forming a protective electrostatic shell that prevented agglomeration (Verma et al. 2019; Montalbán et al. 2018).

FTIR spectroscopy was used to examine structural changes in cellulose from the raw material to the ACC and ACNC films. As can be seen from Fig. 3, all exhibited the typical broad bands over the wavenumber range 3000–3650 cm\(^{-1}\) due to stretching vibrations of OH groups in cellulose. O–H vibrations in the base paper appeared at 3336 cm\(^{-1}\), whereas those in the nanocomposite films containing some or no LNPs were observed at 3440 and 3490 cm\(^{-1}\), respectively.

According to Xu et al. (2010), the shift in the O–H vibration bands results mainly from the cleavage of hydrogen bonds. The peak for OH vibrations in regenerated cellulose containing no LNPs was higher than those for the films containing nanoparticles.

Lignin nanoparticles probably blocked interactions between cellulose and EmimAc to some extent, thereby increasing the ability of the ionic liquid to break intermolecular and intramolecular hydrogen bonds relative to the IL/LNPs solvent system (Yang et al. 2019). Conversion of cellulose I into cellulose II caused the bands at 1430 and 1110 cm\(^{-1}\) for the former in ACC and ACNC reinforced with dissolved cellulose to be flattened, which suggests a prevalence of crystalline cellulose (cellulose II; Zhu et al. (2018)). Also, the spectra exhibited similar characteristic bands, so the macromolecular structure of cellulose was not destroyed by dissolution or regeneration (Amini et al. 2023a, 2023b; Yang et al. 2019). Overall, no new absorption peaks were observed in the FTIR spectra for the ACNC films, which suggests that no new chemical bonds were formed even after adding LNPs in a proportion of 3–5%. This in turn suggests that the nanoparticles were compatible with the cellulose matrix. Similar results were previously reported for chitosan films containing LNPs (Zou et al. 2022).

Figure 4a shows the XRD patterns for the original paper, ACC film and ACNC film with the highest content in lignin nanoparticles, and Fig. 4b their changes in crystallinity index and crystallite size. As can be seen, the XRD spectrum for the base paper exhibited lines for cellulose I at the \(2\theta\) values 22.8\(^\circ\), 34.6\(^\circ\), 14.8\(^\circ\) and 16.6\(^\circ\) on the 200, 004, – 110 and 110 crystal plane, respectively. After dissolution and regeneration, the spectrum exhibited additional lines for regenerated cellulose at 12.2\(^\circ\) (– 110) and 20.7\(^\circ\) (110), which correspond to cellulose II (Yang et al. 2019).

As can be seen, the crystal structure of cellulose was altered by partial dissolution and regeneration. The ACC films with and without LNPs had the same crystal structure as cellulose II. This result suggests that LNPs did not alter the crystal structure of the regenerated cellulose matrix. The crystallinity index (CrI) and crystallite size of base paper were considerably lower in the ACC film (46.58% and 1.50 nm, respectively, versus 89.36% and 6.75 nm, respectively). Therefore, the crystal structure of cellulose I changed to amorphous cellulose upon dissolution in the ionic liquid. These results suggest that the solvent penetrated gaps and partially dissolved cellulose crystallites Amini et al. (2023a, 2023b). The crystallinity index and crystallite size of the film containing 7% LNPs were considerably lower than those of the control film, a result that can be ascribed to weaker interaction of LNPs with cellulose chains leading to a less cohesive structure.

The surface of the ACNC films was examined in SEM images. The original paper had a very open, porous structure consisting of randomly intertwined fibers (Fig. 5a and b). On the other hand, the ACC film, which was partially dissolved, exhibited a uniform, compact, relatively smooth surface without aggregates (Fig. 5c and d).

Interfacial compatibility between the reinforcing phase (undissolved cellulose fibres) and the reinforced matrix in the ACC film was excellent because both consisted of cellulose. The matrix provides partial mechanical strength and acts as a binder to weld the reinforcing phase together. The surface of the ACC film contained micro- and nanoholes suitable for embedding LNPs. The distribution of LNPs in the regenerated cellulose matrix of the ACNC film with the highest LNP content was examined. Increasing the amount of LNPs in the ACC film was found to cause filling of most microholes and cavities in the cellulose matrix (Fig. 5e). At the nanoscale level, the welding agent successfully caused nanofibres to bind to one another by penetrating deeply into the nanogaps between nanofibrils (Fig. 5f and g). This result is consistent with previous reports of Yousefi et al. (2011) and Amini et al. (2023b) on the nanowelding/joining effect of partial dissolution of nano/microcellulose in an ionic liquid.

Antioxidants, which are free-radical scavengers that neutralize reactive oxygen species (ROS) produced during aerobic cell metabolism, have gained growing interest as film components. Free radicals can be harmful to both biotic and abiotic entities (Găman et al. 2020). Antioxidant activity in the ACNC films was assessed with the ABTS radical scavenging method. As can be seen from Fig. 6, the antioxidant activity of the nanocomposite films was substantially improved by incorporation of lignin nanoparticles. In contrast, the paper and the ACC film exhibited little antioxidant activity by effect of exposed electron-releasing hydroxyl groups in the cellulose matrix reacting with free radicals over time (Priyadarshi et al. 2021).

A prior study showed a cellulose-based film to possess limited antioxidant activity (Priyadarshi et al. 2021). However, other reports suggest that lignin possesses antioxidant activity by effect of its containing phenolic hydroxyl groups that effectively reduce or inhibit free radicals by virtue of their hydrogen-releasing ability which makes lignin a useful antioxidant for nanocomposite films (Tian et al. 2017). In fact, increasing the proportion of LNPs in the nanocomposite films boosted antioxidant activity. Thus, the film containing 3 wt% LNPs had a considerably increased activity (30.46%) and that containing 7% LNPs an even higher one (68.97%). Therefore, LNPs seemingly provide a safe, inexpensive way of enhancing the antioxidant activity of films with a view to producing materials with improved properties.

S. aureus and E. coli are among the most common bacterial contaminants encountered in foodstuff. By targeting these bacteria, antibacterial packaging can be freed of major sources of foodborne contamination and the shelf life of the food it contains extended (Gutiérrez et al. 2012).

Table 1 shows the antibacterial activity against S. aureus and E. coli of the ACC film, as control sample, and that of ACNC films containing variable amounts of LNPs. As can be seen, antibacterial activity in the ACNC films increased with increasing content in LNPs. In fact, LNPs introduced further active functional groups (e.g., aliphatic OH, carbonyl CO, COOH, sulphur) in the film structure, which likely contributed to enhancing the antibacterial properties of the film (Alzagameem et al. 2019). The results also indicate that the nanocomposite films were effective against both bacteria. The control sample exhibited little antibacterial activity, with microbe counts less than 5% in both species. Adding 3% LNPs to the films increased reduction of S. aureus and E. coli to 15.71% and 17.80%, respectively. However, such small reductions suggest that the film ACNC3%LNPs was inefficient against both bacteria. Antibacterial activity was considerably higher in ACNC7%LNPs, which exhibited a reduction of 42.85% in S. aureus and of 63.88% in E. coli.

Previous studies showed microbial growth to be inhibited by phenolic groups in lignin. Also, the presence of lignin nanoparticles further increased antimicrobial activity by effect of their high surface area providing better contact with microbes (Wang et al. 2019; Yang et al. 2021). Growth of Gram-negative bacteria such as E. coli is usually easier to stop than is that of Gram-positive bacteria such as S. aureus, probably as a result of the difference in the cell wall structure between the two. Thus, Gram-negative bacteria have more complex cell walls consisting of lipids, proteins and lipopolysaccharides (LPS), which can make them more susceptible to some antimicrobial agents. On the other hand, Gram-positive bacteria possess an additional protective layer in the form of a dense peptidoglycan cell wall that provides effective defense against some biocides and makes them generally more resistant to antimicrobial treatments (Amini et al. 2016; Babaee et al. 2022). For instance, Yang et al. (2018) found LNPs from alkaline lignin to be effective against Gram-negative bacteria, possibly as a result of (1) polyphenols damaging (lysing) bacterial cell walls; and (2) small nanoparticles penetrating bacterial cells and depleting adenosine triphosphate (ATP) by infiltration (Yang et al. 2018; Huang et al. 2020).

As can be seen from Fig. 7, EmimAc is one of the best ILs for homogeneous dissolution of cellulosic materials and production of highly transparent films. The substantial transparency of the ACC and ACNC films may have resulted from mobilization of cellulose fibres, and plasticization at surface level reducing light scattering and widening the band gap of the films (Reyes et al. 2018).

ACNC films additionally exhibited a visually uniform distribution of lignin. Consumers tend to favor transparent food packaging. ACNC films are transparent but gradually develop a light brown colour that alters their optical properties (see Table 2). The color parameters compared here included.

\(L^{*}\) (lightness), \(a^{*}\) (green–red), \(b^{*}\) (blue–yellow) and \({\bigtriangleup }E\) (total color difference). The ACC film had an \(L^{*}\), \(a^{*}\) and \(b^{*}\) value of 88.75, \(-\)1.32 and 3.25, respectively. \(L^{*}\) decreased slightly with increasing lignin content, whereas \(b^{*}\) was greater than the value for the ACC film. The nanocomposite films containing 5% and 7% LNPs exhibited similar \(L^{*}\) values (86.11 and 86.78, respectively). The \({\bigtriangleup }E\) values for the ACNC films containing 3, 5, and 7% LNPs were 4.24%, 6.20%, 7.82% and 9.78%, respectively. Therefore, ACNC7%LNPs was the film with the highest \({\bigtriangleup }E\) value as a result of the typical brown colour of lignin (Haqiqi et al. 2021; Hararak et al. 2021).

A number of organic compounds are degraded by UV. As a result, UV-blocking films are appealing as packaging materials, and the conjugated structure of lignin imparts a substantial UV-blocking ability to cellulose films (Ma et al. 2022). Transmittance of UV and visible light through the ACNC films was assessed here at 280 and 660 nm, respectively.

As can be seen from Table 2, the ACC film exhibited poor UV blocking performance. In contrast, the films containing LNPs showed excellent UV-blocking properties, their performance increasing with increase in LNP content. In fact, UV-blocking by ACNC3%LNPs, ACNC5%LNPs and ACNC7%LNPs was 57.33%, 70.43% and 93.31%, respectively. Similarly to a neat ACC film, ACNC3%NLPs exhibited low UV protection with high transmittance in the visible region. Moreover, visible light transmittance was sacrificed with higher proportions of LNPs owing to the colour of lignin itself (Hararak et al. 2021). The light blocking ability of the films can thus seemingly be ascribed to the presence of substantial amounts of unsaturated structures (phenol and ketone groups in addition to other chromophores) in lignin nanoparticles. These results are consistent with those of previous studies that demonstrated the UV-shielding ability of LNPs, particularly when incorporated into cellulose-based films (Posoknistakul et al. 2020; Cao et al. 2021). In general, the nanoparticles provided an effective barrier against UV light while maintaining excellent transparency in the films, which is essential for packaging foods and light-sensitive products.

Knowing the surface properties of cellulosic materials can help develop packaging materials for specific purposes from them. The water contact angle (WCA) is a measure of the affinity of lignin for an ACC surface. We thus made WCA measurements of the ACNC films to assess their hydrophilicity or hydrophobicity. The results are shown in Fig. 8. A film with WCA > 90\(^\circ\) is deemed hydrophobic (Haqiqi et al. 2021). All films studied here had WCA < 90\(^\circ\), so they were classified as hydrophilic. The highest WCA (69\(^\circ\)) was that for ACNC7%LNPs and the lowest one (36.4\(^\circ\)) that for ACC (i.e., the film containing no lignin). WCA increased gradually with increase in LNP content of the ACNC films, which can be ascribed to the hydrophobic nature of lignin.

The hydrophobicity of lignin is imparted mainly by its ester groups by contrast, the hydrophilicity of pure cellulose is a result of its abundant hydroxyl groups. Lignin nanoparticles can be made more hydrophobic through formation of reverse micelles and exposure of their aromatic surface skeletons, thereby further increasing WCA (Qian et al. 2015; Bai et al. 2022). Therefore, consistent with the results of previous studies by Wang et al. (2021) and Teh et al. (2021), incorporating LNPs into an ACC film can be expected to hinder access of water to regenerated cellulose fibres and broaden the scope of ACC films in food contact packaging.

The influence of the LNP content of the films on their water vapour and oxygen barrier properties was investigated. The water vapor permeability (WVP) of the ACC film was 8.67 \(\times\) \(10^{-9}\) g\(\cdot\) m\(^{-1}\) \(\cdot\) h\(^{-1}\).Pa\(^{-1}\) and lower than that of the paper. This result suggests that the ionic liquid enhanced the water barrier properties of the films (see Fig. 9). The remarkable barrier properties of ACC can be ascribed to the multilayered, highly packed nanostructures it contains. During its formation, polymer chains entangle with other, similarly disintegrated chains by forming cohesive bonds throughout the rinsing and drying stages (Yousefi et al. 2011, 2015). Adding 7% LNPs to the regenerated cellulose matrix substantially decreased WVP (from 8.67 to 6.25\(\times\) \(10^{-9}\) g\(\cdot\) m\(^{-1}\) \(\cdot\) h\(^{-1}\).Pa\(^{-1}\), which was better than the value for the ACNC film containing 3% LNPs (Michelin et al. 2020). Shankar et al. (2015) previously found adding LNPs to agar composite films to reduce their WVP, possibly because of strong intermolecular interactions between agar and lignin molecules leading to a more tortuous path for diffusion of water vapour and fewer water molecules permeating through the polymer film as a result.

Figure 10 shows the oxygen permeability (OTR) at 0% and 90% RH of the ACNC films. Clearly, partial dissolution in the ionic liquid considerably decreased OTR. Moreover, incorporating lignin into the film formulation resulted in a significant difference. Thus, at 0% RH, both ACC and ACNC3%LNPs exhibited an identical OTR value (38.25); also, ACNC5%LNPs and ACNC7%LNPs provided a completely impenetrable barrier for oxygen, thus confirming that using lignin as a small molecular filler successfully increases the density of composite films (Ma et al. 2022). At 90% RH, the films exhibited poorer barrier properties. This was a result of cellulose fibrils easily absorbing water and swelling at a high relative humidity, thereby causing further breakage of fibril-fibril bonds in the films by effect of the high polarity of the fibrils (Luo et al. 2021). At 90% RH, increasing the LNP content to 7 wt% decreased OTR by 84.38% relative to the ACC film. Similar results were previously reported by Zou et al. (2022), who found adding nanolignin to polymer matrices to reduce oxygen and water vapour permeability by effect of the films being more compact and containing fewer voids.

As can be seen from Table 2, the welding effect of the ionic liquid reduced film thickness relative to the original paper (from 122 ± 4.08 to 48 ± 2.05µm in the ACC film). This was a result of the welding agent (IL) providing a more compact surface for macrofibres in the original paper. The thickness of the ACC films increased with increasing content in LNPs.

Figure 11 summarizes the mechanical properties of ACNC films with variable amounts of lignin nanoparticles in terms of Young’s modulus (YM), tensile strength (TS) and elongation at break (EB). TS, which was 0.6 MPa for untreated paper, increased 134-fold after dissolution in the ionic liquid. The increase can be ascribed to the ionic liquid acting as a nanowelding agent and causing the formation of a tighter network with more junction points by connecting smaller fibres in the nanostructures. Cellulose fibre surfaces were most likely plasticized by effect of the ionic liquid also acting as a plasticizer and increasing ductility as a result. This outcome is consistent with previous conclusions of Ghaderi et al. (2014) and Amini et al. (2023a,b ). Here, TS for the ACC film was altered by addition of LNPs. In fact, TS and YM for the ACNC films containing up to 5%LNPs were substantially greater (113.59 MPa and 22.51 GPa, respectively). An et al. (2021) previously found TS for cellulose-based films to increase upon incorporation of lignin. However, further addition of LNPs to our films (to a 7 wt% content) dramatically decreased TS and YM (to 92.19 MPa and 12.56 GPa, respectively). The decrease in both parameters may have resulted from non-uniform dispersion and agglomeration of LNPs in the regenerated cellulose matrix (Shankar et al. 2015). These results are consistent with those of the WVP test. In addition, elongation at break was smaller in the LNP-containing ACNC films than in the ACC film. The decreased elongation break resulting from the addition of LNPs can be ascribed to the aromaticity of lignin and its increased structural stiffness relative to the cellulose matrix hindering motion of macromolecular chains in cellulose (Shankar et al. 2015; Rukmanikrishnan et al. 2020a). An et al. (2021) also found lignin to reduce EB in cellulose-based films.

Biodegradability in the ACC and ACNC films was assessed by burying them in soil for 6 weeks. Weight loss is a major proxy for the ease with which biocomposite films can be degraded in the environment. Figure 12 shows the weight loss of the films as measured at regular intervals. Degradation was very slow in all samples after the first week. Thus, the loss of the ACNC film in the second week was 4–6% greater than those of the ACC film (3.57%) and the paper (only 2.5%). ACNC7%LNPs exhibited the greatest weight loss (almost 12 wt%) after 4 weeks. The ACC film also experienced a marked loss after the fourth week, possibly as a consequence of its porous structure allowing water to penetrate into the polymer network and weaken bonds as a result. After 6 weeks, the weight loss of the ACC film and the paper was about 12.5% and 11%, respectively. These values exceeded those for the ACNC3%LNPs and ACNC5%LNPs (9.25% and 10%, respectively). By contrast, the loss of ACNC7%LNPs was essentially similar to that of the ACC film.

Lignin has been ascribed antimicrobial properties by virtue of its containing aromatic alcohol structures (Dudeja et al. 2023). Such properties were enhanced by a high lignin content in the films, which is consistent with the weaker tendency of ACNC films to degrade after 5 weeks of burial in soil. Also, the ACC film exhibited a higher biodegradation rate relative to the paper sample as the likely result of its degradability being strongly influenced by the crystallinity of cellulose.  Crystalline zones are more difficult to degrade. The crystallinity indices found were consistent with biodegradation of the samples. Thus, the cellophane weight loss after 5 weeks was only 0.47%, so degradation of this material was negligible probably because of the additives it typically contains hindering the process (Zhao et al. 2019).