Sustainable Production of Cellulose-Based Hydrogels with Superb Absorbing Potential in Physiological Saline.

Nowadays, most of the commonly used superabsorbent polymers (SAPs) are derived from synthetic polymers, particularly acrylic acid and its copolymers made with acrylamide. Here, we describe a novel and environmentally friendly aqueous-based process for fabrication of a new, natural, cellulose-based SAP (hydrogel). In this two-step process, cellulose was first reacted with sodium monochloroacetate (MCA) to obtain carboxymethyl cellulose (CMC) and then cross-linked with epichlorohydrin (ECH). In distilled water (d-water), the water retention value (WRV) of the newly fabricated hydrogels reached 725 g d-water/g gel, which is significantly greater than any other commercially available superabsorbent cellulose-based material (WRV of 10-100 g/g) and comparable to the commercial synthetic (polyacrylate) SAP gels (WRV of up to 1000 g/g). In saline water (s-water; 0.9% NaCl), the maximum WRV attained was 118 g s-water/g gel, which exceeds more than 2-fold the WRV of commercial gels (40-50 g/g). Compositional analysis was carried out to determine the amount of carboxyl groups and average molecular mass, and the parameters for hydrogel preparation were optimized. The natural SAP was characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The hydrogels showed good re-swelling properties losing only 5-10% of their capabilities to reabsorb d-water when reused in four consecutive cycles. Because of their superior swelling properties in physiological saline, the new hydrogels can compete with their synthetic counterparts in applications such as high-value hygiene and biomedical products.

Introduction

Cross-linked networks of polymer chains with a three-dimensional linear or branched structure, which can absorb and retain significant amounts of water, are termed superabsorbent polymers (SAPs) or hydrogels.[1,2] The high water absorption potential of hydrogels is due to the presence of hydrophilic functional groups, such as hydroxyl and carboxyl groups. Because of their hydrophilic nature and ability to swell, hydrogels are broadly used in personal hygiene products,[3,4] agriculture,[5] biomedical applications,[6,7] heavy metal removal,[8,9] and drug delivery.[10,11] Currently, most of the commercially available hydrogels are cross-linked synthetic polymers, such as polyacrylic acid and acrylic acid-acrylamide-derived copolymers.[12−16] The water intake potential of these SAPs is up to 1000 times of their weight (1000 g water/g SAP).[17]

Hydrogels are normally insoluble in water due to their cross-linked structure and large molecular weight. However, water absorption takes place within the hydrogel matrix, which causes water retention and swelling that are controlled to a great extent by the cross-linking density and the charge groups resided in the hydrogel molecules.[18] The greater the charge of the SAPs, the greater their water absorption capacity in both s-water and pure d-water.

Biodegradability and biocompatibility along with insignificant toxicity are the prime characteristics of renewable bio-based hydrogels such as cellulose-derived superabsorbents.[19−22] However, most of the hydrogels currently available on the market are mainly based on petroleum-derived acrylic acid and acrylamide, which are of oil-based nonrenewable nature. In addition, some natural polymers, such as carboxyalkyl cellulose, gum, carboxyalkyl starch, and cellulose sulfate may contain a portion of grafted polymeric particles from polyacrylates, sulfonated polystyrene, and poly(vinyl alcohol).[23,24] These types of “mixed” superabsorbent materials of the renewable/nonrenewable origin are capable of absorbing 10–100 g d-water/g hydrogel, which is significantly lower than the water intake potential of synthetic SAPs (1000 g d-water/g SAP).[17,23,24]

In search for new and fully bio-based hydrogels, renewable and biodegradable polymers such as cellulose have been extensively studied as a raw material for SAPs.[25−28] Cellulose-based hydrogels prepared by cross-linking with succinic anhydride via etherification were shown to absorb 400 times of the gel’s dry weight.[29] A SAP prepared by cross-linking co-dissolved carboxymethyl hydroxyethyl cellulose with divinyl sulfone denoted a maximum water absorbency of 425 g d-water/g material.[30] Alam and Christopher[31] reported a natural cellulose-chitosan cross-linked hydrogel that was able to absorb up to 610 g d-water/g gel. However, all cellulose-based superabsorbent materials reported to date suffer from inferior water absorbency and production costs compared to their synthetic counterparts.

The objective of this work was to develop a new and completely renewable cellulose-based hydrogel with improved swelling and re-swelling capabilities that could compete with synthetic SAPs of environmental concern. The new hydrogel was prepared using two inexpensive and biodegradable materials: CMC and ECH. The two-step fabrication process is aqueous-based, eco-friendly, and catalyst-free and does not require any energy-intensive post-purification step rather than a simple water washing of the hydrogel. Here, we described the process and its advantages and the properties of the newly prepared hydrogel and its potential applications.

Results and Discussion

Chemical Cross-linking of Hydrogels

The CMC coupling reaction with ECH is presented in Figure . Cross-linking occurred between the epoxy group of ECH and the hydroxyl group in CMC (at any position) via an ether bond. This reaction takes place in an alkaline condition (in the presence of NaOH) without a catalyst, with NaCl and water that are the only co-products. As NaCl is highly soluble in water, it can be easily washed out from the hydrogel once the reaction is complete.

Figure 1

Cross-linking reaction of CMC with ECH used for hydrogel preparation.

The composition and properties of the new hydrogels are shown in Table . The first two columns of Table provide information about the initial concentrations of CMC and ECH in weight percent used for the hydrogel preparation. Although the weight ratio of CMC to ECH in the resulting hydrogels was kept at 0.75, the initial concentrations of CMC and ECH had a profound impact on the gel’s composition (carboxyl content) and properties, such as average molar mass (Mc) and WRV (Table ). Best results in terms of water absorbency were obtained with 3 wt % CMC and 4 wt % ECH: the WRV of the hydrogel reached 725 g d-water/g gel water and 118 g s-water/g gel (Table ). The gels having higher water absorbency are better suited for water absorption applications. Increasing the CMC concentration above 3 wt % resulted in reduction of gel’s WRV, Mc, and carboxyl content. This can be explained with the state of the macromolecular network formed at different initial cellulose concentrations. The gel’s cross-linked network is characterized by the availability of capillary spaces and the cross-linking density (CD). The use of higher CMC concentrations increased the CD (decreased Mc) of the forming gel, which resulted in a decrease of the absorption potential and WRV values. The denser cross-linked structure effectively screened the gel’s internal matrix from the flux of incoming water, which limited cellulose hydration. On the other hand, cross-linking of CMC at concentrations lower than 3 wt % produced very weak gels with a changeable physical condition that could not keep water, hence lowering the WRV (Table ). Therefore, cross-linking plays an important role in the formation and manipulation of the properties of superabsorbent gels. The cross-linking density and swelling capacity of gels can be modified by varying the amount of cross-linker. Moreover, the degree of cross-linking impacts the integrity, strength, swelling, and hygroscopicity of gels.[32] Different weight ratios of CMC to ECH in the resulting hydrogels have also been studied, and the corresponding obtained results are provided in Table .

Table 1

Composition and Properties of Cellulose-Based Superabsorbent Hydrogels

CMC conc (wt %)a	ECH conc (wt %)	CMC/ECH (wt ratio)	COOH groups (mmol/g)	Mc (g/mol)	WRV (g d-H2O/g)	WRV (g s-H2O/g)
2.0	4.0	0.5	1.58	1.23 × 106	413	71
2.0	2.67	0.75	1.69	1.08 × 106	381	65
3.0	4.00		1.68	3.16 × 106	725	118
4.0	5.33		1.52	2.54 × 106	636	93
5.0	6.67		1.52	7.7 × 105	311	47
5.0	3.33	1.5	1.60	1.45 × 106	455	69

CMC containing 3.5 mmol of COOH groups/g cellulose.

The occurrence of the carboxymethylation reaction was confirmed by 13C solid-state NMR and FTIR investigations. Figure a displays the 13C NMR spectra of unmodified and modified cellulose. The unmodified cellulose exhibited carbon peaks at C1 (105 ppm), C4 (85 ppm), C2, C3, and C5 (74 ppm), and C6 (65 ppm), which are all typical of cellulose I.[33] On the other hand, a new peak at 173 ppm appeared in the 13C NMR spectra of the CMC samples confirmed the presence of the newly introduced COOH groups.[33] The FTIR spectra of unmodified cellulose, CMC, and the new cross-linked hydrogel are exhibited in Figure . In the case of unmodified cellulose, the characteristic absorption peaks at 3300 and 2900 cm–1 correspond to O–H and C–H stretching vibrations, respectively.[34] The same peaks can be seen for CMC and the CMC–ECH cross-linked gel. In addition, the presence of the carbonyl (C=O) group is evident from the peak at 1740 cm–1, which confirms the successful introduction of carbonyl groups (Figure ). This result also confirms the presence of COOH groups in the CMC and hydrogel. A new peak at 1328 cm–1 in the cross-linked gel demonstrates the successful ether-based cross-linking of CMC with ECH.[35]

Figure 2

(a) 13C NMR spectra of CMC and (b) FTIR spectra of modified cellulose cross-linked hydrogels.

Swelling Properties of Hydrogels

The time course of the WRV of the cellulose hydrogel obtained from 3 g/L CMC cross-linked with 4 g/L ECH (Table ) in d- and s-water is shown in Figure . The WRV in d-water increased rapidly and reached to 400 g d-water/g gel in 2 h. Afterward, the water absorption rate became slower, and the equilibrium WRV of 725 g d-water/g gel was attained at 40 h (2400 min). The WRV of the modified cellulose hydrogel in s-water also increased sharply to reach 74 g s-water/g gel in the first 40 min. Thereafter, the absorption rate levelled off, with an equilibrium WRV of 118 g s-water/g gel obtained after 2400 min. The obtained WRV values for this modified cellulose hydrogel (cross-linked by ECH) in d- and s-water are larger than any other cellulose-derived hydrogels reported in the literature (400 g d-water/g cellulose-based hydrogels cross-linked with succinic anhydride;[29] 425 g d-water/g cross-linking co-dissolved carboxymethyl hydroxyethyl cellulose with divinyl sulfone;[30] 610 g d-water/g cellulose-chitosan cross-linked hydrogel[31]). One disadvantage of the cellulose-based absorbent materials is their lower water absorbency (in d-water) compared to that of the commercial synthetic SAP (∼1000 g d-water/g gel).[12,36] This work reports the WRVs of cellulose-based hydrogels in d-water that are comparable to the water absorbencies of commercial SAPs. Furthermore, our fabricated cellulose-based hydrogel demonstrated a significantly better WRV in s-water (118 g/g gel) compared to the commercial SAP (40–50 g/g).[12,29,31]

Figure 3

WRV of hydrogel obtained from 3 g/L CMC cross-linked with 4 g/L ECH as a function of time.

The water absorption capacity of the fabricated hydrogel following four consecutives re-swelling cycles is exhibited in Figure . The WRV of the hydrogel in the second cycle decreased only 5–10% as compared to the first cycle, which is probably due to the formation of hydrogen bonds between −COOH and −OH groups during the first cycle. However, the water absorbencies of the hydrogel in cycles 3 and 4 did not change further and remained similar to the WRV for the cycle 2.

Figure 4

WRV of best absorbing hydrogel (obtained from 3 g/L CMC cross-linked with 4 g/L ECH) following four consecutive re-swelling cycles.

The water absorbency of the gel strongly depends on the type of salt present in the water. The effect of the ion types on the swelling ability of the modified cellulose hydrogel cross-linked by ECH is shown in Table . As shown here, the equilibrium swelling capacity Ws of the gel in the presence of monovalent cations decreased in the following descending order: NH4+ > K+ > Na+ (Table ). This can be explained by the fact that the charge screening effect of these cations on the Ws values decreased in the same order. In the presence of salts with different cationic charges, the absorption capacity of the hydrogel declined in the order monovalent > divalent > trivalent cations.

Table 2

Effect of Salt Type on Salt Sensitivity Factor (f) and Equilibrium Swelling Capacity (W) of Best Absorbing Hydrogel (WRV of 725 g d-Water/g Gel)

salt	salt concentration (mol/L)	swelling period (h)	Ws (g s-water/g gel)	f
NaCl	0.08	40	257 ± 2	0.646
	0.11		168 ± 2	0.768
	0.15		118 ± 1	0.838
KCl	0.08		284 ± 2	0.608
	0.11		183 ± 1.5	0.748
	0.15		128 ± 1	0.824
NH4Cl	0.08		312 ± 2	0.570
	0.11		200 ± 1	0.724
	0.15		138 ± 1.5	0.810
CaCl2	0.08		141 ± 1	0.806
	0.11		93 ± 0.8	0.872
	0.15		66 ± 0.7	0.909
AlCl3	0.08		31 ± 0.4	0.957
	0.11		20 ± 0.6	0.972
	0.15		14 ± 0.5	0.980

Apparently, the degree of cross-linking in the gel was influenced by the cation charge of the salt dissolved in water. Additional cross-linking points were formed due to the physicochemical interaction of the divalent (Ca2+) and trivalent (Al3+) cations with the carboxylate groups present in the hydrogel matrix.[37] This caused a decrease in the gel swelling capacity. Table also demonstrates that the salt sensitivity factor f was in reverse proportion to Ws as lower f values corresponded to higher Ws values.

Crystalline Properties of Hydrogels

The crystallinity of the best absorbing hydrogel (WRV of 725 g d-water/g gel and 118 g s-water/g gel, Table ) was investigated by XRD, and the results are displayed in Figure . The typical peaks of cellulose were assigned according to Nishiyama et al.[38] and Isogai et al.[39] The 2θ angles of 15.3°, 16.5°, and 22.6° correspond to the (110), (110), and (200) peaks, which are typical of cellulose Iβ. Using eq , the C.I. of the unmodified cellulose was determined to be 73%. Following cellulose modification to CMC and hydrogels, the C.I. decreased to 52 and 56%. This suggests that some crystalline regions in cellulose were also modified in addition to the amorphous region. The peaks for modified (CMC) and cross-linked cellulose (hydrogel) look very similar (Figure ). The slight shift of both peaks toward left from the peak for unmodified cellulose may be due to a partial conversion of cellulose I to cellulose II during the carboxymethylation reaction.

Figure 5

XRD of cross-linked cellulose hydrogels.

Morphological Properties of Hydrogels

The optical microscopic images of the unmodified and modified CMC cellulose fibers are exhibited in Figure a,b, respectively. The fiber diameter is enlarged due to the electrostatic repulsions caused by the carboxylate anions (−COO−) obtained from the ionization of the carboxyl groups (−COOH) following carboxymethylation (Figure a,b). The plain (control) fibers presented in Figure c are the unmodified cellulose fibers. The carboxymethylation reaction did not convert all hydroxyl groups (−OH) to carboxylmethyl groups (−OCH2–COOH), and some −OH groups remained intact in the modified cellulose. This can explain the two types of fibers that can be seen from Figure d: (1) fibers that have a lower degree of carboxymethylation (similar to the unmodified control fibers—images on Figure a,c) and (2) modified fibers that are highly carboxymethylated (see images for unmodified cellulose fibers from Figure b).

Figure 6

Optical microscopic images of (a) unmodified and (b) modified CMC fibers; SEM images of (c) unmodified and (d) modified CMC fibers; SEM image of (e) 50-fold and (f) 500-fold magnified best absorbing cross-linked hydrogel (WRVs of 725 g d-water/g gel and 118 g s-water/g gel).

The magnified cross-sectional SEM images of the best absorbing hydrogel can be observed in Figure e,f. The hydrogel is highly porous with a macroporous architecture. This suggests that the electrostatic repulsions caused by the ionic charge of the carboxylate anions (COO–) in the hydrogel have increased the space of the cross-linked hydrogel network. The SEM image shows that the gel has an open porous geometry with a pore size in the range of 300–600 μm, which is separated by sheet-like walls and ultrathin structures, as revealed in Figure f. The high porosity of the hydrogels would facilitate rapid mass penetration, which is useful in superabsorbent applications.

Potential Applications of Hydrogels

The new hydrogels hold promise for use as “green superabsorbents” owing to their biodegradable constituents and the eco-friendly cross-linking process employed for their fabrication. The hydrogel was fabricated by a straightforward reaction between CMC and ECH that are both biodegradable, relatively inexpensive, and largely available. This will have a positive impact on the hydrogel production cost. Because of the high absorption capacity of the new hydrogels and in particular, their superb absorbing potential in s-water, they could be suitable for applications such as diapers, feminine hygiene products, wound dressings, meat soaking pads, wiping papers, etc. This material could also serve as a feedstock for development of advanced bioabsorbents with new properties and applications.

Conclusions

A novel hydrogel has been prepared from cellulose as a renewable material with the objective of improving the inferior liquid absorption performance of bio-based superabsorbents currently available on the market. The process for fabrication of the new hydrogels is eco-friendly as it utilizes two inexpensive and biodegradable organic compounds, CMC and ECH. The raw material for CMC, bleached kraft pulp, was used without prior removal of the hemicellulose, which translates into a significant yield and cost advantages. In addition, no catalyst, pre- or post-purification, was required to produce the hydrogel. Although a maximum d-water absorbency of 725 g d-water/g gel attained was still lower than that of the commercial synthetic SAPs (1000 g d-water/g SAP), it was significantly larger than any other superabsorbent cellulosic material reported in the literature. However, the maximum absorbency of 118 g s-water/g gel of the gel in s-water exceeded approximately 2.5 times the WRV of commercially available synthetic SAPs (40–50 g s-water/g SAP). Furthermore, the new hydrogels demonstrated excellent re-swelling potential as only 5–10% of the maximum water absorption capacity was lost after four consecutive times of gel recycling and reuse. The swelling properties of the gel can be controlled by CD, Mc, carboxyl groups, and cationic charge. Because of their green nature and superior absorption capacity, especially in physiological saline solution, the newly developed hydrogels may be able to compete with synthetic SAPs in personal hygiene, pharmaceuticals, food applications, and other novel applications.

Materials and Methods

Materials

A kraft pulp mill in Canada supplied bleached softwood kraft pulp. Sodium monochloroacetate (MCA), epichlorohydrin (ECH), and hydrochloric acid (HCl) were purchased from Sigma-Aldrich (Mississauga, Ontario, Canada). Sodium hydroxide (NaOH) and ethanol (C2H5OH) were supplied by Thermo Fisher Scientific (Whitby, Ontario, Canada). All chemicals were used as received.

Preparation of CMC

The carboxymethylation reaction was carried out in aqueous (water) media according to a previously reported method.[40] Air-dried BSWK pulp (50 g) was mixed with 230 g of MCA solution (100 g of MCA in 130 g of water) in a Hobart mixer at room temperature for 10–15 min and then transferred into a Nalgene bottle. The Nalgene bottle was placed in a 50 °C preheated water bath for 4 h to allow impregnation of MCA into the fiber interior. Thereafter, a solution of NaOH (62.5 g of NaOH in 100 g of water) was added to the reaction mixture and agitated for 10–15 min in the Hobart mixer. The reaction was allowed to proceed overnight (12 h) at room temperature. After completing the reaction, the pulp was washed by 60 vol % ethanol, which assisted in coagulation and filtration of the cellulose fibers out of the pulp mixture. Finally, the CMC fibers were washed with 70% ethanol twice and dried at 50 °C in an oven.

Preparation of Hydrogels

Three weight-based CMC/ECH ratios of 0.5, 0.75, and 1.5 were maintained during hydrogel preparation. Different weight percent concentrations of CMC and ECH (Table ) were added to a 6 wt % NaOH solution to form a cellulose suspension. For example, to prepare a hydrogel that had WRVs of 118 g s-water/g gel and 725 g d-water/g gel, 3 wt % CMC (containing 3.5 mmol of COOH groups/g cellulose) was mixed with 4 wt % ECH and 93 wt % of NaOH (at 6 wt % concentration). The suspension was stirred until the CMC fibers were fully mixed with the NaOH solution. ECH (Table ) was then added slowly into the mixture and stirred at 100 rpm and room temperature for 20–30 min until the liquid turned into a highly viscous gel. The viscous gel was transferred into a heated bath at a temperature of 50 °C and allowed to perform the cross-linking reaction for 4–5 h. Thereafter, the cross-linked gel was diced into cubes (1 × 1 cm), immersed into excess d-water, and thoroughly washed to remove excess ECH. The d-water was changed several times until the water conductivity reached 700 S/cm. Finally, the gel was dried in an oven at 50–60 °C and then ground in a lab-scale, high-speed grinder to a particle size of 400–600 μm.

Characterization of Hydrogels

The carboxyl content of the CMC fibers and the cross-linked gels was determined using a conductometric titration method[31,41] with a METER pH/conductivity S470-KIT (Mettler Toledo GmbH, Greifensee, Switzerland) titrator. A certain amount of CMC fibers and cross-linked gels (with a solid content of ∼20 mg dry basis) and 2.5 mL of 0.02 M sodium chloride solution were added, and the mixture was sufficiently stirred to prepare a well-dispersed solution. Then, 0.1 M HCl was slowly added to the mixture to set the pH to 3. Then, the suspension was titrated with a 0.01 M NaOH solution at a rate of 0.1 mL/min until the mixture reached pH 11. The carboxyl group content of CMC fibers and the cross-linked gels was determined from the conductivity curves using the following equation (eq )[31]where [COOH]g is the carboxyl content of the CMC fibers or the cross-linked gels in mmol/g, VNaOH is the volume of NaOH (mL) required for deprotonation of the carboxylic groups, MNaOH is the molarity of NaOH (mol/L), and DWg is the weight (g) of the dry CMC fibers/gels initially dissolved in d-water.

The water retention value (WRV) of the cross-linked gels was measured by immersing the gels in d-water or 0.9 wt % NaCl aqueous solution (s-water). The swelling gels were withdrawn from the solution at each time interval and weighed out after removing the excess liquid from the surface of the gel. Excess d-water (or s-water) was removed by filtration using a 20 μm mesh nylon cloth. The free swell WRV, a measure of the dynamic water absorption properties of the gel, was calculated using eq (31,34)where W is the weight of the wet gel at time t and Wd is the weight of the dried gel.

To measure the water re-swelling kinetics, the hydrogel was first bone-dried in an oven at 50 °C for 12 h. Thereafter, 0.2 g of the dried gel was soaked in 200 mL of d-water at room temperature for up to 180 min. The WRV of the swollen gel was then measured according to eq. . The drying-soaking operation, described above, represented one re-swelling cycle. This re-swelling cycle was repeated four times using the same hydrogel.

The cross-linking density (CD) of the hydrogel is provided by the average molar mass (Mc) that is entrapped between any two cross-linking points contained in the gel. The CD value is inversely proportional to Mc, therefore, the higher the Mc, the lower the CD. The Mc value of any hydrogel can be calculated using a previously developed method[42] based on the Flory–Huggins theory[43]where Q is the equilibrium water absorption of the hydrogels, D2 is the density of the hydrogels, V1 is the molar volume of the solvent used for swelling, and X1 is the Flory–Huggins interaction parameter between solvents and hydrogels. Here, we assumed (as per the literature[31]) a density of 1.5 g/cm3 of the hydrogel (density of cellulose) and X1 = 0 because the volume fraction of methanol in water was 0 (the Flory–Huggins interaction parameter comes from the volume fractions of methanol in the methanol/water mixture).

The influence of ions (cations and anions obtained from NaCl, KCl, NH4Cl, CaCl2, and AlCl3 salts) on the swelling capability of hydrogels (for 40 h) was studied as described by Rodrigues et al.[44] The dimensionless salt sensitivity factor (f) was calculated[31,34] as follows (eq )where Ws and Ww is the equilibrium swelling capacity in s- and d-water, respectively. All salts were used at concentrations of 0.08, 0.11, and 0.15 mol/L.

The particle size was measured with an optical microscope (Olympus CKX41, Olympus Corp., Tokyo, Japan). Solid-state 13C NMR spectra of the samples were obtained on a Varian/Agilent VNMRS-400 instrument operating at 100.5 MHz. Samples were packed uniformly inside a 7.5 mm zirconium rotor and spun at 5500 Hz. Spinning sidebands were suppressed using the TOSS sequence.

Fourier transform infrared spectroscopy (FTIR) measurements of the samples were performed on an FTIR spectrometer using a Bruker Tensor 37 (Bruker, Ettlingen, Germany) with a PIKE MIRacle Diamond attenuated total reflectance (ATR) accessory. Solid samples were placed directly on the ATR crystal. The maximum pressure was applied by lowering the tip of the pressure clamp using a rachet-type clutch mechanism. The sample spectra of all 32 scans were averaged (from 550 to 4000 cm–1) with a resolution of 4 cm–1.

The crystalline properties of the cross-linked gels were examined by X-ray diffraction (XRD). The measurements were performed on a X′Pert Pro diffractometer (PANalytical B.V., Netherlands) with a PIXcel detector and Kα radiation (λ = 1.54 Å). The X-ray diffractograms were acquired with a 2θ (Bragg angle) range of 10°–30° at a scan rate of 0.005° s–1.

The crystallinity index (C.I.) of cellulose was calculated from the XRD spectra as follows[41]where I200 is the intensity of the (200) plane reflection, typically located around 2θ = 22.6°, and IAM is the intensity at 2θ = 18°, corresponding to the minimum in a diffractogram.[44]

Morphological changes in cellulose fibers were studied using Hoffman modulation contrast optical microscopy (HMC, Nikon Eclipse TE2000-U, Tokyo, Japan) and Hitachi Su-70 field emission scanning electron microscopy (SEM, Hitachi, Chiyoda, Tokyo, Japan). A few drops of 0.01% (w/v) toluidine blue solution were added to the fiber sample suspension at least 30 min prior to imaging for an optical microscopy measurement. For the SEM measurement, the gels were swollen to equilibrium in d- or s-water at 23 °C and neutral pH for 24 h and then freeze-dried using a LABCONCO FreeZone 2.5 instrument (LABCONCO, Kansas City, USA). The freeze-dried samples were placed on double-sided carbon adhesive discs attached to aluminum specimen stubs and then sputter-coated with gold to improve specimen conductivity. The images were taken at an accelerating voltage of 5 kV.