Chemical and Enzymatic Fiber Modification to Enhance the Mechanical Properties of CMC Composite Films

2.2. Plant Fiber Separation

Fibers of Yunnan pine wood, industrial hemp hurd, and bamboo culms were cut into pieces 3–5 by 3–5 by 5–10 mm (width by thickness by length) prior to chemical and enzymatic fiber extraction.

For chemical fiber separation: five grams of Yunnan pine wood, Bamboo culms, or industrial hemp hurd were placed in beakers and immersed in 100 mL of a 50:50 mixture of hydrogen peroxide (H₂O₂): glacial acetic acid (CH₃COOH) at 70 °C until the sample turned white. The samples were washed with distilled water until the pH was 7, then the samples were shaken slightly to separate the fibers. These procedures were performed in triplicate.

Enzymatic fiber separation: three enzymatic methods were used to obtain fibers. In the first method, five grams of a given material was treated with 100 mL of 5.00% lipase solution at 50 °C, stirred (900–1000 r/min) for 3 h, then the solution was filtered off and the residual materials were treated with GB2677.10-1995 (Determination of the content of holo-cellulose from paper raw materials) to remove most of the lignin. The residual material was immersed in 40 mL of 50 °C distilled water and stirred (900–1000 r/min) for more than 3 h until most of the fibers were separated, then washed with distilled water to obtain residues. The second method used the same procedure, but then immersed the fibers in 40 mL of 5% pectinase solution instead of distilled water. The final method used the same procedure, but then immersed the materials in 40 mL of 5% a 20:20 mixture of 5% pectinase solution and 5% hemicellulase solution instead of distilled water. Lipase (CAS:9001-62-1), pectinase (CAS:9032-75-1), and hemicellulose (CAS:9025-56-3) were obtained from Aladdin Biotechnology Co., Ltd., (Shanghai, China) and had enzyme activities of 100,000; 30,000; and 5000 U/g, respectively. Each fiber material/enzymatic treatment combination was prepared in triplicate.

2.3. Preparation of Cellulose, CMC, and CMC Composite Films

Four grams of cellulose extracted from eucalyptus bark according to Chinese Standard GB/T744-1989 (Determination of α-cellulose from pulp) was mixed with 80 mL of 100% ethanol(CH₃CH₂OH) and 20 mL of 30% NaOH solution and then stirred(900–1000 r/min) for 60 min at 30 °C. Five grams of sodium chloroacetate (C₂H₂ClNaO₂) were added and heated at 65 °C for 3 h. The sample was washed with 90% glacial acetic acid to a pH of 7, then washed with 80% ethanol 3 times and 95% ethanol once, before being oven-dried at 65 °C for 3 h to obtain CMC.

One gram of CMC was mixed with 0.40 g sodium alginate (C₆H₇NaO₆), 0.15 g glycerol (C₃H₈O₃), and 49.00 g distilled water and stirred (900–1000 r/min) at 70 °C for 15 min. The mixture was treated in an ultrasonic bath for 10 min and vacuumed for 45 min to remove air bubbles. The solution was poured into a polytetrafluoroethylene (PTFE) mold and cured at 30 °C for 48 h to obtain CMC composite films. The fiber-modified CMC composite film process was similar except that 0.1, 0.3, and 0.5 g of a given plant fiber was added during mixing (shown in Figure 1).

2.4. Fiber and Composite Film Characterization

Fiber dimensions: The length and width of 100 fibers, as well as the cell wall thickness and cell cavity width of each of the chemically or enzymatically prepared fibers were measured under a light microscope by ImageJ software.

Sample microstructure: The microstructure of eucalyptus bark powder, cellulose, CMC, and CMC composite films were observed by placing a sample on an aluminium stub and coating with gold/palladium before observation with the Czech TESCAN MIRA LMS field emission scanning electron microscope at an accelerating voltage of 200 eV to 30 KeV. A minimum of five fields were examined per material.

Fourier-transform infrared spectroscopy (FTIR): Eucalyptus bark powder, cellulose, and CMC were mixed with KBr and formed into a pellet while the CMC composite films were directly analyzed on a Nicolet i50 FTIR analyzer (Thermo Nicolet Corporation, Madison, WI, USA) with a scanning range of 500 to 4000 cm⁻¹ and 64 scans. Baseline correction was performed to analyze the spectral differences between plant fibers obtained by different treatments.

X-ray diffractometer analysis (XRD): The crystal structures of eucalyptus bark, cellulose, and the CMC films were studied by X-ray diffraction (XRD) on an Ultima IV X-ray diffractometer (Rigaku Corporation, Tokyo, Japan) using a scanning angle from 5 to 60°, a step size of 0.026° (accelerating current = 30 mA and voltage = 40 kV), and Cu-Kα radiation of λ = 0.154 nm. The degree of crystallinity (DOC, %) was calculated according to the formula:

 is the maximum intensity of the main peak (about 22°), and  is the diffraction intensity of amorphous cellulose (about 15°).

Thermogravimetric (TG) analysis: Approximately 5 to 6 mg of sample powder ground to pass through an 80 to 120 mesh screen and placed into sample holders for analysis on a TGA92 thermo gravimetric analyzer (KEP Technologies EMEA, Caluire, France). N₂ was used as the shielding gas and Al₂O₃ as the reference compound. The temperature was increased from 35 to 800 °C at a rate of 20 °C/min to generate a thermogravimetric curve.

Degree substitution (DS) of CMC: The degree of substitution of hydroxyl groups has an important influence on resulting CMC properties. The degree of substitution was determined by the acidimeter method by weighing 0.2 g (accuracy 0.1 mg) of the sample, dissolving it in 80 mL of water, stirring for 10 min, and adjusting the pH to 8.0. The sample was titrated using sulphuric acid (H₂SO₄) with continuous stirring to pH 3.74. The volume (mL) of sulphuric acid titration solution used was recorded (to the nearest 0.05 mL). The degree of substitution (DS) was then calculated using the amount required to reach the end point according to Equations (1) and (2), as follows (Table 1).

where B = amount of carboxymethyl substance contained in the sample, mmol/g;

m = quality of the sample, g;

c = concentration of sulphuric acid standard titration solution, mol/L;

V= volume value of standard titration solution of sulphuric acid, mL.

Physical Properties: Tensile strength (MPa) and elongation at break (%) were measured on ten 0.089 to 0.098 mm by 150 mm dog-bone samples of each material on a universal testing machine according to procedures described in GB/T 1040.1-2006 (Plastics—Determination of tensile properties). The load was applied to failure at a rate of 1 mm/min.

Film Opacity and Water Vapor Transmission: Opacity of the CMC composite films was tested by cutting 10 by 40 mm samples and placing them on the inner surface on one side of a cuvette and measuring absorbance at 600 nm on an XP Spectrum 752^# ultraviolet spectrophotometer (XP-Spectrum Company, Shanghai, China). Five measurements were made for each sample.

Water vapor transmission rate was assessed under controlled temperature and relative humidity conditions using unit time, unit water vapor pressure difference, and thickness through the unit, and expressed as the unit area of the water vapor volume of the specimen.

The water vapor transmission coefficient of the specimen was calculated according to Equation (3).

where P is the water vapor transmission coefficient of the sample in grams/square centimeter per second Pascal [g cm/(cm²·s·Pa)].

m is the mass change of the sample in grams (g) during the period t.

A is the sample area through the water vapor in square meters (m²).

t is the difference in time between two intervals after the mass change has stabilized in hours (h).

d is the thickness of the specimen in centimeters (cm).

p is the difference in water vapor pressure between the two sides of the specimen in Pascals (Pa).

3.2. Effects of Different Pretreatment Methods on Fiber Yield and Dimensions

Chemical treatment resulted in 100% fiber yield after 10 h of treatment at 70 °C (Table 2). Enzyme treatments produced lower yields. Distilled water treatment resulted in less than 5% fiber yields from bamboo culms and industrial hemp hurd and only 10–20% yield from Yunnan pine wood, despite the 12 h total treatment time. The subsequent use of pectinase alone or in combination with hemicellulase resulted in 90 to 95% fiber yield. Pectin plays important roles in cell wall interactions, especially in primary cell wall formation, and its disruption may facilitate fiber separation. Treatment times for the pectinase treatments were only 6 to 8 h whereas they were 5.5 to 8.5 h for the pectinase and hemicellulase treatments. These results illustrate the potential for producing high fiber yields using enzymatic treatments [27].

The distilled water treatment resulted in little fiber recovery and will not be further discussed. The other treatments had varying effects on the properties of the resulting fibers (Table 3). Pectinase treatment resulted in the shortest Yunnan pine wood fibers whereas the chemical treatment resulted in fibers that were nearly 50% longer. Similarly, pectinase and hemicellulase treated bamboo culms and industrial hemp hurd fibers were only half as long as those from the chemical treatment. Fiber length tended to be greater in all of the chemical treatments compared with the enzymatic treatments, although the pectinase treatment was sometimes similar to the chemical treatment (Table 3). The largest difference in fiber length was found between chemically and enzymatically treated industrial hemp hurd fibers. Decreased fiber lengths may reflect a tendency for enzymatically treated fibers to break more easily as they are separated, which would be detrimental to increasing the tensile strength of any composite. Fiber widths and lumen diameters tended to be similar for the same material regardless of whether the samples were chemically or enzymatically treated. The treatments are less likely to affect fiber width or lumen size, given that their primary effects would be on the cell walls themselves. Cell walls were slightly thicker in pectinase-treated Yunnan pine wood fibers than chemically treated fibers, whereas cell wall thicknesses of pectinase and hemicellulase and chemically treated Yunnan pine wood fibers were similar. Pectin is an important component in primary wood cell wall formation but becomes less important with subsequent lignification. However, the specificity of pectinase for pectin could lead to more efficient separation with reduced breakage.

Fiber length to width ratios can be a useful indicator of potential effects of fiber addition on tensile properties. The addition of fibers with higher length to width ratios may have a greater effect on tensile strength. Length to width ratios tended to be smaller in enzymatically treated fibers than in chemically treated fibers of the same species. As noted earlier, this may reflect a tendency for enzymatically treated fibers to be more brittle and produce shorter fibers, which would reduce tensile properties.

The relative crystallinity of untreated Yunnan pine wood, bamboo culms, and industrial hemp hurd were 38.8, 49.1, and 47.4%, respectively (Table 4). Almost all of the lignin was removed from materials treated chemically or enzymatically and crystallinity was increased. The degree of crystallinity was greatest in Yunnan pine wood but crystallinity also increased in bamboo culms and industrial hemp hurd, although the differences were not significant. Increased crystallinity indicates that chemical and enzymatic treatments removed some amorphous cellulose, resulting in an increase in the proportion of crystalline cellulose. Increased crystallinity may result in stronger reinforcing fibers.

3.3. CMC Characterization

Degree of Substitution: The substitution degree (DS) on CMC from eucalyptus bark cellulose was 0.89, which is similar to the values obtained for corn stover, straw, and reed CMCs, which ranged from 0.6 to 1.0 [28,29].

SEM: SEM examination of eucalyptus bark revealed that it consisted of many substances tightly aggregated together in small granular form (Figure 2a-1). The cellulose recovered from this material was in the form of polymerized fibrous bundles (Figure 2a-2), which became more discrete when the materials were reacted to form CMC with differing degrees of fiber breakage (Figure 2a-3). These changes reflect the effects of alkaline treatment and subsequent esterification, coupled with water penetration into the cellulose bundles, with resulting chain separation.

FTIR Analysis: FTIR spectra of commercially available CMC and eucalyptus bark CMC both contained the stretching vibrations of the CMC carboxylate anion COO^– at about 1630 cm⁻¹, with two characteristic absorption peaks at 1410 cm⁻¹ corresponding to the asymmetric (C=O) and symmetric (C-O) stretching vibrations caused by the carboxylic acid group, and an absorption peak at 1030 cm⁻¹ corresponding to the stretching vibration of the cellulose C-O-C group. The absorption peak at 898 cm⁻¹ is characteristic of the β-glycosidic bond in cellulose [30,31,32,33,34,35,36,37]. No absorption peaks were observed at 1518 and 1320 cm⁻¹, which are attributed to the aromatic vibration of the lignin ring and the C-O stretching vibration of the syringyl group, respectively [38], nor was there a peak at 1730 cm⁻¹, which is attributed to hemicellulose. These results indicate that cellulose extraction was nearly complete with little evidence of residual lignin or hemicelluloses, and the cellulose was successfully transformed into CMC (Figure 3).

X-ray Diffraction: Eucalyptus bark and holo-cellulose showed diffraction peaks at 2θ = 15.65, 16.45, 22.15, and 34.35°, whereas the cellulose diffraction peaks were at 14.85, 15.4, 21.75, and 34.35°, respectively (Figure 4). All three of the latter peaks correspond to the crystal planes of (101), (101⎯⎯), (200), and (004), which are typical reflections of cellulose Type I [39,40,41,42,43]. The relative crystallinity of eucalyptus bark and cellulose were 46.39 and 55.07%, respectively, again indicating that the cellulose was successfully extracted. The diffraction peaks of bark-derived CMC were at 2θ = 19.95 and 31.8°, which were similar to the diffraction peaks obtained from commercially available CMC, indicating successful transformation of cellulose to CMC (Figure 4) [4,43].