3.1. Mechanical-enzymatic pretreatment
Cellulolytic enzymes are glucoside hydrolases that break the β-(1à4)-glucosidic bonds of carbohydrates by inversion or retention of the anomeric carbon configuration (Ek et al. 2009).
Table 1 shows the percentage distribution of the solid residue and the fraction of solubilized carbohydrates from the eucalyptus pulp after the enzymatic pretreatments. As expected, the enzyme complex solubilized part of the hemicelluloses and cellulose of the raw material, whose initial chemical composition was: 77.7 ± 0.5% cellulose, 21.1 ± 0.5% hemicellulose and lignin <1%, which coincides with what reported by other authors for this type of pulp (Hu et al., 2018; Carrillo-Varela et al., 2019; Andrade et al., 2021). The first enzymatic treatment (2º- 0.000%), which was identical for the 5 CNFs, solubilized a lower amount of cellulose and xylan than the pulps with double enzymatic treatment, so the carbohydrate yield in the solid fraction was higher. This is due to the fact that in the first enzyme treatment the accessibility of the enzyme to the substrate was lower than the second. Both the mechanical refining process, as well as the first enzymatic treatment caused greater internal and external fibrillation of the fibers, increasing the exposed surface area of the fiber and the absorption of water, which facilitated the accessibility of the enzyme for second hydrolysis enzymatic.
The percentage of carbohydrates in the solid fraction decreases as the enzyme charge of the second enzyme treatment increases from 0% to 0.1%, which is mainly due to the hydrolysis of amorphous cellulose (Pääkko et al., 2007). Furthermore, in the second enzymatic treatment, the degradation of xylans was statistically the same for all samples, so the difference in the decrease in solid yields is clearly due to the hydrolysis of cellulose.
Table 1. Enzymatic hydrolysis and solubilization of carbohydrates.
Pulp
|
Enzyme charge a
|
Solubilized compounds
|
Solid carbohydrate yield (%)
|
Cellulose
(% initial cellulose)
|
Xylan
(% initial xylan)
|
Eucalyptus
|
2º - 0.000%
|
1.9±0.1
|
1.5%±0.3
|
96.6±0.4
|
2º - 0.025%
|
3.1±0.1
|
3.4%±0.2
|
93.5±0.2
|
2º - 0.050%
|
3.6±0.2
|
3.2%±0.2
|
93.2±0.3
|
2º - 0.075%
|
4.3±0.1
|
3.2%±0.2
|
92.5±0.1
|
2º - 0.100%
|
4.6±0.1
|
3.1%±0.1
|
92.3±0.2
|
a Enzyme charge of the second enzyme treatment.
3.2. Cellulose nanofibrils production
Determining the morphological characteristics of CNFs is relevant to understand the final properties of this nanomaterial. Table 2 shows the morphological characteristics of the CNFs produced from pulps with different enzyme charges and the same mechanical treatment. Regarding the width, length, and degree of polymerization of the CNFs, a decrease in these characteristics is observed as the enzyme charge increases, which can be attributed to a greater hydrolysis of the cellulose chains due to an increase enzyme concentration. Endoglucanases decrease drastically the degree of polymerization, which would increase the frequency of rupture or breakpoints within the fibers, facilitating the access of water to the interior of the cell wall and consequently the process of mechanical fibrillation.
Table 2. Morphological characteristics of CNFs.
|
Length (μm)
|
Width (nm)
|
Aspect ratio
|
Transmittance (%)
|
Degree of polymerization
|
CNF0
|
8.5
|
32.7
|
260.0
|
73.6
|
447
|
CNF0,025
|
7.4
|
26.9
|
275.0
|
74.1
|
400
|
CNF0,050
|
7.1
|
22.9
|
310.9
|
77.1
|
362
|
CNF0,075
|
6.1
|
21.8
|
278.9
|
78.5
|
334
|
CNF0,100
|
5.7
|
21.1
|
271.5
|
85.9
|
278
|
The transmittance of light at a specific wavelength through a CNF suspension indicates the presence of smaller and/or more homogeneous nano-objects and is often used as an indirect method to estimate the degree of fibrillation of CNF dispersions (Delgado-Aguilar, 2015; Tarrés et al., 2016). According to the above, the results of Table 2 show that the increase in the enzyme charge effectively allows greater fibrillation of the BHKP, which is reflected in the decrease in the lengths and widths of the CNFs. From a technological point of view, it is often interesting to produce fibrils with morphological characteristics that favor certain applications. For example, it is expected that a higher aspect ratio will favor the reinforcing properties of composite materials.
When observing the aspect ratio of the nanofibrils (Table 2) it can be clearly seen that this parameter does not have the same tendency as the transmittance of light and that its variation is affected by the enzyme charge during treatment, increasing with the enzyme charge a maximum value and then decreases. With the aim of getting a better understanding of how enzyme charge affects the morphological characteristics of CNFs, the relationships are shown in Figure 1.
For the length of the CNFs (Figure 1a) a negative linear relationship with the enzyme charge is observed, whose differences are statistically significant between the different samples (LSD method, 95% confidence). These results prove that a higher enzyme charge can generate significant changes in the lengths of the CNFs for the same mechanical treatment. In this case, a charge of 0.1% of enzyme (in the second enzymatic treatment) generates a 33% decrease in CNFs lengths.
As mentioned by Taheri and Samyn (2016), the minimum width of the fibrils produced during fibrillation depends on the operating conditions and the equipment used for mechanical fibrillation. Analyzing the widths of the CNFs (Figures 1b), it is observed that there is a drastic and significant decrease (p value <0.05) at low enzyme charges (<0.0005 ml/g). For enzyme charges greater than 0.0005 ml/g, the decrease in width is much less pronounced and there are no statistically significant differences (LSD method, 95% confidence). These results suggest that, for the same mechanical treatment, an increase in the enzyme charge facilitates fibrillation up to the minimum width given by the equipment. In this sense, there is evidence of the need to define a control parameter that encompasses the morphological properties of CNFs and optimize the enzymatic pretreatments for each mechanical process.
The morphological parameter that best reflects the concept of fibrillation corresponds to the aspect ratio of the CNFs, since it has been shown that an increase of the enzyme charge favors fibrillation as a result of the transversal break of the fibrils, reducing the length of these, and the longitudinal break that decrease the width until a minimum value. As a consequence of the above, the aspect ratio of the CNFs presents a maximum (p = 311) (Figure 1c) for an enzyme charge, in the second enzyme treatment, of 0.0005 ml/g.
An efficient fibrillation process seeks to produce a homogeneous material, with small widths (<50 nm), and attempting to decrease transversal break of the fibrils (length). Therefore, the aspect ratio is the parameter that best reflects these characteristics and corresponds to a good indicator of the effectiveness of the fibrillation process.
On one hand, Andrade et al. (2021) determined that for CNFs produced from the same raw material and with the same enzymatic-mechanical treatment, the aspect ratio was 330. On the other hand, Albornoz-Palma et al. (2020a) in their study produced CNFs with an aspect ratio of 303, from the same raw material and mechanical treatment, but with a different type and charge of enzyme.
3.3. Effect of mechanical-enzymatic treatment on the degree of polymerization of cellulose
The degree of polymerization is an important parameter that evaluates the length of cellulose chains and is frequently used to evaluate CNFs (Qing et al., 2013). Table 3 shows the variation in the degree of polymerization as a function of the applied treatment. As mentioned above, endoglucanase enzymes are characterized by a rapid decrease in the degree of polymerization (Ek et al. 2009).
The "Variation 1" in Table 3 indicates the decrease in DP with respect to the initial raw material (P), which decreases as the intensity of the mechanical and/or enzymatic pretreatment applied increases. The DP of cellulose decreases between 72 and 81% for the CNF produced, with DP values between 278 and 447, which coincides with that reported by various authors who produced CNF with similar enzymatic and mechanical pretreatments. Albornoz-Palma et al. (2020a) in their study used cellulase enzymes (1.2 wt%) to produce CNF with a degree of polymerization of 228; Tarres et al. (2016) used endoglucanase enzyme (0.032%) to produce CNF with a DP of 309; and Andrade et al. (2021) used the same raw material and enzyme cocktail, charge 0.1% of enzyme in two treatments (0.05% each) producing CNFs with a DP of 303, which means a decrease with respect to the initial raw material of 75%. Additionally, Qing et al. (2013) used a different mechanical treatment (SuperMassCollider- Microfluidization 15 passes at 1500 bar) and produced CNFs with a DP of 280 when dosing 3 FPU of enzyme/g fiber. From the above, it seems that the lower limit of the degree of polymerization is 220 for CNFs with enzymatic pretreatment with a high degree of fibrillation.
Table 3. Variation in degree of polymerization depending on the treatment applied.
Sample
|
Degree of polymerization
|
Variation 1
|
Variation 2
|
P
|
1438
|
---
|
|
PR
|
1231
|
-14%
|
|
PE
|
916
|
-36%
|
|
P0
|
760
|
-47%
|
|
P0,25
|
649
|
-55%
|
|
P0,05
|
601
|
-58%
|
|
P0,075
|
529
|
-63%
|
|
P0,1
|
497
|
-65%
|
|
CNF0
|
447
|
-69%
|
-41%
|
CNF0,025
|
400
|
-72%
|
-38%
|
CNF0,050
|
362
|
-75%
|
-40%
|
CNF0,075
|
334
|
-77%
|
-37%
|
CNF0,100
|
278
|
-81%
|
-44%
|
The "Variation 2" in Table 3 represents the decrease in the DP of the CNF after the homogenization process, with respect to the pulps with mechanical-enzymatic pretreatment. The results show that the decrease in DP due to the homogenization process is independent of the enzyme charge, since for the same mechanical treatment (15 passes through the homogenizer) the decrease in DP is close to 40% for all cases.
The length of the CNFs (Figure 2a) is linearly related to the degree of polymerization, with a coefficient of determination of 0.95. This line relationship: L (µm) = 0.02 DP coincides with that reported by Albornoz-Palma et al. (2020a), which produced CNFs with enzymatic pretreatment, but used another enzymes cocktail of cellulase enzymes and a higher enzyme charge (0.012 ml/g). The authors related the degree of polymerization of their samples to different number of passes through the homogenizer (0, 1, 2, 4, 7, 10, 15), showing the same linear regression. In view of the above, the length of the CNFs and the DP are linearly related, independent of the type of cellulase enzyme, enzymatic charge and intensity of the mechanical treatment applied.
As the relationship between length and degree of polymerization is positive linear (Figure 2a) and the relationship between length and enzyme charge is negative linear (Figure 1a), width and aspect ratio show the same trends as a function of the degree of polymerization than with the enzymatic charge, but in a specular way. Because of the above, the width of the CNFs (Figure 2b) at DP less than 362 did not show statistically significant differences (LSD method, 95% confidence). On the other hand, at DP greater than 362, the differences are significant, and the DP of the samples varies by up to 38%. The relationship between the aspect ratio and the DP has a maximum at DP = 362 (Figure 2c). This maximum is due to the fact that DP less than 362 there is a change in the lengths of CNFs but not in the widths, so the aspect ratio decreases.
In the literature, it has been observed that a higher degree of polymerization is strongly related to the improvement in the mechanical properties of nanofibrils (Zimmermann et al., 2010; Tarrés et al., 2016). Regarding the maximum load supported by the CNFs films (Figure 2d), it is observed that for DP values greater than 362, there are no statistically significant differences (Bonferroni method, 95% confidence). For DP less than 362, the maximum load decreases, and the values are statistically different (Bonferroni method, 95% confidence). When relating these results to the enzymatic charge and the aspect ratio, it can be seen that for charges less than or equal to 0.0005 ml/g (DP 362), where the aspect ratio is increasing to its maximum, the resistance of the CNF films remains constant. For enzyme charges greater than or equal to 0.0005 ml/g (DP 362), where the aspect ratio is decreasing, the resistance of the CNF films decreases. The above shows that the resistance of CNFs films is positively related to the degree of polymerization up to a maximum value, which corresponds to the maximum value of the aspect ratio. This is why the aspect ratio is the parameter that best predicts the final mechanical properties of the CNFs.