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Journal of Korea Technical Association of the Pulp and Paper Industry - Vol. 54 , No. 3

[ Article ]
Journal of Korea Technical Association of the Pulp and Paper Industry - Vol. 53, No. 5, pp. 5-15
Abbreviation: J. Korea TAPPI
ISSN: 0253-3200 (Print)
Print publication date 30 Oct 2021
Received 17 Aug 2021 Revised 16 Sep 2021 Accepted 23 Sep 2021

Combined Enzymatic Pretreatment of Pulp for Production of CNF
Shin Young Park1 ; Seokho Lee1, 2 ; Wanhee Im1, 3 ; Hak Lae Lee4, 5 ; Hye Jung Youn4, 5,
1Department of Forest Sciences, College of Agriculture and Life Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Graduate Student, Republic of Korea
2Current Address: R&D Center, Kleannara, Cheongju-Si, Chungcheongbuk-do 28174, Researcher, Republic of Korea
3Current Address: Moorim P&P Co. Ltd., Ulsan 45011, Senior Researcher, Republic of Korea
4Department of Agriculture, Forestry and Bioresources, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Professor, Republic of Korea
5Research Institute of Agriculture and Life Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Professor, Republic of Korea

Correspondence to : E-mail: (Address: Department of Agriculture, Forestry and Bioresources, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea)

Funding Information ▼


In this study, the effects of mixing treatment of endoglucanase and xylanase on characteristics of pulp and cellulose nanofibrils (CNF) were investigated. The degree of polymerization of the pulp was reduced and its crystallinity was increased after enzymatic pretreatment. The suspension viscosity of CNF prepared from enzymatic pretreated pulps was lower than that of untreated CNF. Compared with untreated CNF, more uniform and smaller fibrils could be obtained through combined pretreatment of endoglucanase and xylanase, but higher endoglucanase dosage led to wider fibrils width distribution. The aspect ratio of enzyme-treated CNF was lower than that of untreated CNF. The use of both endoglucanase and xylanase increased the tensile strength and elastic modulus of the sheet, whereas just endoglucanase-treated CNF sheet exhibited lower strength. The light transmittances of each CNF sheet were comparable; however, the casted sheet made using 1.0% endoglucanase-treated CNF was less transparent because of its morphological features. The combined use of endoglucanase and xylanase could facilitate the nanofibrillation and produce the sheet without any deterioration of strength.

Keywords: Cellulose nanofibrils, enzyme, pretreatment, tensile strength, transparent film

1. Introduction

Since petroleum-based materials have caused serious environmental problems, interests in sustainable biomaterials have been increased to replace them. Recently, cellulose nanomaterials of which a width is less than 100 nm have received a great attention owing to its abundance, sustainability, and biodegradability. Among them, cellulose nanofibril (CNF) is produced through mechanical treatment of pulp fibers, e.g. grinding, homogenization, and microfluidization. [1-5] In addition to the eco-friendliness of cellulose, CNF has advantages such as high mechanical properties and flexibility due to its high aspect ratio. Owing to these merits, CNF has a potential to be applied in various fields, including papermaking, [6,7] packaging, [8,9] composites, [10-12] electronics, [13,14] medicines, [15] and so on.

Although CNF has been noticed as an eco-friendly material, there are some obstacles for commercial application of CNF. One of the problems is its high price because of large energy consumption for CNF production. As CNF is produced by severe mechanical treatment, high amount of energy is consumed during the process, which leads to increase in the production cost. [16,17] Several pretreatment methods of pulp have been introduced to reduce the CNF production cost, including mechanical, [18,19] chemical, [20-23] and biological pretreatment. [24-26] Among them, biological pretreatment using enzymes does not need any harmful chemicals, and it is performed in a mild condition, thus it recognized as eco-friendly pretreatment method.

Enzymes have been studied for pulping and refining process for a long time. Several kinds of enzymes including endoglucanase, cellobiohydrolase, and hemicellulase have been used for these processes. Recently, these enzymes have been studied for CNF production. Endoglucanase and cellobiohydrolase which can degrade the structure of cellulose chain have mainly used for pretreatment for CNF production. [27,28] It revealed that pulp treated with enzymes showed lower clogging trouble during homogenization. [24,29] In addition, enzymatic pretreatment could reduce the energy cost for CNF production. It was also shown that enzymatic pretreatment affects the degree of polymerization, crystallinity, and mechanical properties of CNF. [24,27,30,31]

Although several studies of enzymatic pretreatment for CNF production have been performed, most of those studies used endoglucanase or cellobiohydrolase for pretreatment. Since bleached pulp fibers contain some amount of hemicelluloses in their structure, the role of hemicellulose in CNF production process should also be considered. Nonetheless, there have been only few studies which investigated the effect of hemicellulase on CNF production. Therefore, in this study, hardwood bleached kraft pulp was treated with endoglucanase and xylanase with various mixing ratios as a pretreatment for CNF production. Then, properties of CNF and CNF sheet including morphology, mechanical strength, and optical properties were measured. From this study, it was aimed to reveal the combined effect of endoglucanase and xylanase on the properties of CNF and the CNF sheet.

2. Materials and Methods
2.1 Materials

Never-dried hardwood bleached kraft pulp supplied by Moorim P&P Co. (Korea) was used as a raw material which contains about 79% of cellulose, 20% of xylose and 1% of lignin and ash. For enzymatic pretreatment, endoglucanase (Fibercare R, 4500 CNU-CA/g, Novozymes, Denmark) and endoxylanase (Pulpzyme HC 2500, 2500 AXU/g, Novozymes, Denmark) were used.

2.2 Enzymatic pretreatment of pulp

100 g pulp was disintegrated to 2.5% consistency with deionized (DI) water. Enzymatic pretreatment condition was determined by a preliminary test in the recommended conditions suggested by the enzyme supplier. Enzymes and pH 7 buffer solution (10 mL) were added to the pulp suspension. Mixing ratio and total dosage of endoglucanase (G) and endoxylanase (X) were adjusted to four different conditions as shown in Table 1. Enzymatic treatment was conducted at 55°C for 1h, and then the suspension was boiled to terminate the enzyme reaction. After termination of reaction, the suspension was washed sufficiently with DI water to remove the enzymes.

Table 1. 
Enzymatic pretreatment conditions
Condition Endoglucanasea Endoxylanasea Total enzyme dosagea
G 0.25%+X 0.25% 0.25% 0.25% 0.5%
G 0.5% 0.5% 0
G 0.5%+X 0.5% 0.5% 0.5% 1.0%
G 1.0% 1.0% 0
aEnzyme dosage was based on the oven dried weight of the pulp

To investigate the effect of enzymatic treatment of pulp, cupriethylenediamine (CED) viscosity and crystallinity of each treated pulp were measured. The CED viscosity measurement was performed in accordance with TAPPI method (T 230 om-99). A thin pulp sheet was prepared via vacuum filtration and drying, which was used for the determination of crystallinity using X-ray diffractometer (XRD, D8 ADVANCE with DAVINCI, Bruker, Germany) with Cu Kα radiation at 40 kV and 40 mA. The diffraction data (2θ) was collected from 3° to 40° and the crystallinity index was calculated by Segal method. [32]

2.3 Preparation and characterization of cellulose nanofibrils

CNFs were produced from untreated and enzyme-treated pulps, respectively. The pulp suspension of 1.5% consistency was mechanically ground using a Supermasscolloider (Masuko Sangyo, Japan) at 1500 rpm. The gap of grinding stones was -80 μm and each pulp suspension passed between grinder stones by 24 times. During CNF production, low shear viscosity of fiber suspensions was measured using Brookfield viscometer to evaluate the extent of fibrillation and viscosity of the CNF suspension. The measurement was conducted at 25℃ for 1 min using spindle #64.

To observe the morphology of nanofibrils, SEM images of fibers were taken at the different passing number using Field-Emission Scanning Electron Microscope (SUPRA 55VP, Carl Zeiss, Germany). In addition, the fibril width of CNFs was measured using Image J program from the SEM images at magnification of ×50000. More than 300 fibrils were measured and the average value and distribution of the fibril width were obtained.

Crystallinity of CNF was evaluated by XRD with the same manner in the pulp sample. As like the analysis of pulp, a thin sheet of CNF was used for the analysis.

To estimate the aspect ratio of CNF, sedimentation behavior of the CNF suspension was evaluated as described method by Varanasi et al. [33] CNF suspensions with different concentrations from 0.02 wt% to 0.08 wt% were prepared and left for sedimentation for 48 h. After the sedimentation, a sediment height of each suspension was measured. The quadratic regression equations were obtained from a plot of suspension consistency versus the ratio of sediment height (hs) to suspension height (ho). From the regression equations, a gel point of CNF suspension and average aspect ratio were obtained.

2.4 Preparation of CNF sheet

CNF sheet was prepared by two different methods, vacuum filtration and casting. In the case of vacuum filtration, 0.5 wt% CNF suspension was vacuum-filtrated on the cellulose ester membrane filter (0.2 μm pore Advantec, US) to form 40 g/m2 CNF sheet. Then it was wet pressed at 30 bar for 5 min. After wet pressing, the sheet was separated from membrane filter and then hot pressed at 100°C for 15 min. For casting, 0.5 wt% CNF suspension was poured into a petri dish in a grammage of 40 g/m2, and it was dried at 50℃ oven for 24 h.

2.5 Characterization of CNF sheet

Tensile properties of the CNF sheet were investigated using Universal Testing Machine (Instron, USA) with a 500 N load cell. CNF sheet was cut into rectangular specimen with 6 cm length×1.5 cm width for the tensile test. Test was performed at 3 cm span length and the cross-head speed of 10 mm/min. For each CNF sheet sample, the test was conducted more than four times. Tensile strength and strain at break of the sheet were measured, and the elastic modulus was calculated from the strain-stress curve.

In addition, light transmittance of the CNF sheet was measured at the wavelength range from 200 nm to 800 nm using Cary 100 UV-vis spectrophotometer (Agilent, USA). Each sample was analyzed in duplicate.

3. Results and Discussion
3.1 Effect of enzymatic pretreatment on pulp properties

To examine the effect of enzymatic pretreatment on the pulp properties, CED viscosity and crystallinity of the untreated and treated pulps were evaluated (Fig. 1). CED viscosity of pulp decreased by enzymatic pretreatment. This indicated that enzyme-treated pulps had lower degree of polymerization (DP) than untreated pulp due to the cleavage of cellulose chain by enzymes. [24,26,30] Among the pulps pretreated with different enzyme mixing ratio, pulp treated with 0.5% endoglucanase had quite lower viscosity than that treated with 0.25% endoglucanase and 0.25% xylanase, which means DP of cellulose decreased more at higher dosage of endoglucanase. However, there was no more decrease in the CED viscosity when the endoglucanase dosage was higher than 0.5%. In the case of crystallinity, because endoglucanase attack the amorphous region of the cellulose and hemicellulose, the crystallinity of enzymatic pretreated pulps was higher than that of untreated pulp. [26,30,34] On the contrary, the treatment of xylanase did not have significant influence on the DP and crystallinity of the pulp as described in the previous work. [35] From these results, it was confirmed that the enzymatic pretreatment of pulp with endoglucanase decreased the DP and increased the crystallinity of pulp, which may affect the process of CNF production and properties of CNF.

Fig. 1. 
CED viscosity (a) and crystallinity (b) of untreated and enzymatic pretreated pulps with different enzyme dosage.

3.2 Effect of enzymatic pretreatment on cellulose nanofibrils

Cellulose nanofibrils were prepared from untreated and enzyme-treated pulps, resepctively. Fig. 2 shows a change in the low shear viscosity of the fiber suspension during CNF production. As the pass number through a grinder increased, the viscosity of the suspension increased regardless of enzymatic treatment. It means that fibers were deconstructed into smaller fibrils, which led to an increase in surface area. However, an increment in the suspension viscosity was different depending on the enzymatic treatment conditions. The enzymatic treated fiber suspensions exhibited lower viscosity than the untreated fiber suspension. It means that the enzymatic pretreatment can relieve some problems related to the high viscosity of CNF suspension which may occur in mill production and application processes. Especially the suspension treated with 1.0% endoglucanase exhibited much lower viscosity than other enzyme-treated suspensions. The rheological properties of the pulp and CNF suspension are mainly affected by the consistency of the suspension and the morphological properties of the fibers. [36] Since the consistency of each suspension was same, differences in the morphological properties of CNF are likely to influence the viscosity of the suspension.

Fig. 2. 
Change in low shear viscosity of the suspension during CNF production.

Morphologies of cellulose nanofibrils with different enzymatic treatment conditions were examined using SEM (Fig. 3). The average fibril width and the fibril width distribution were obtained from these SEM images (Fig. 4 and Table 2). SEM images showed that most fibrils were nanofibrillated under 100 nm width for all conditions. However, the average fibril width and the width distribution were affected by the pretreatment conditions. As shown in Table 2, CNFs treated with total enzyme dosage of 0.5% showed narrower fibril width (27.7 nm for G 0.25%+X 0.25%, and 29.1 nm for G 0.5%) and more uniform distribution compared to untreated CNF (average width of 34.4 nm). This showed that enzymatic pretreatment could facilitate the nanofibrillation of fibers and make fibrils more uniform. [37] However, as total enzyme dosage increased up to 1.0%, the average width and the width distribution of CNFs got wider. CNF treated with 1.0% endoglucanase had relatively larger fibril width of approximately 40 nm and wider distribution than untreated CNF. It indicated that the appropriate dosage of endoglucanase and xylanase resulted in relatively homogeneous nanofibrils, whereas the higher amount of enzyme could decrease the efficiency of mechanical treatment and led to wider and heterogeneous distribution of fibril widths. [25]

Fig. 3. 
SEM images of untreated and enzymatic pretreated cellulose nanofibrils (24 times passes): Untreated CNF (a), CNF treated with G 0.25%+X 0.25% (b), G 0.5% (c), G 0.5%+X 0.5% (d), and G 1.0% (e).

Fig. 4. 
Fibril width distribution of untreated and enzyme-treated cellulose nanofibrils.

Table 2. 
Average fibril widths of untreated and enzyme-treated CNFs calculated from SEM images (24 times passes)
Enzyme condition Average fibril width (nm) Standard deviation of fibril width (nm)
Untreated 34.4 20.8
G 0.25%+X 0.25% 27.7 13.9
G 0.5% 29.1 15.3
G 0.5%+X 0.5% 31.1 19.7
G 1.0% 39.2 20.9

To evaluate the aspect ratio of CNF, the sedimentation behavior of CNF suspension was examined. The aspect ratio of each CNF was calculated by plotting the sediment height of the suspension versus the consistency of the suspension. [33] As shown in the Fig. 5, the aspect ratio of the CNF decreased when the dosage of endoglucanase increased. Considering with the result of the CNF width, it indicates that higher endoglucanase dosage led to production of fibrils with relatively short and wide shape. [30,37] The decrease in DP of fibers seemed to result in shorter fibrils by grinding. These morphological properties would also influence the viscosity of CNF suspension. CNF suspensions treated with enzymes had lower viscosity due to less entangled network between the fibrils because of the lower aspect ratio of enzyme-treated fibrils than untreated fibrils. On the contrary, compared to endoglucanase, xylanase did not affect the aspect ratio of CNF significantly.

Fig. 5. 
Estimated aspect ratio of untreated and enzyme-treated cellulose nanofibrils.

Although the crystallinity of pulp increased by enzymatic pretreatment, it might be further changed during CNF production by the mechanical treatment. Grinding treatment of pulp decreased the crystallinity of cellulose fibers. After grinding, CNF had the crystallinity of 60-70%, which was 12-14% lower than that of pulp. Enzyme treated CNF still exhibited higher crystallinity compared to untreated CNF, while the difference between them was similar (Fig. 6). Among enzymatic treated CNFs, CNF treated with 0.5% endoglucanase showed the highest crystallinity. As the total enzyme dosage increased, the crystallinity of CNF decreased due to the more severe cleavage of cellulose chain. [24,30]

Fig. 6. 
Crystallinity of CNFs depending on the enzymatic treatment conditions.

3.3 Effect of enzymatic pretreatment on properties of CNF sheet

CNF sheets were prepared using untreated and enzymatic pretreated CNFs by two different methods, and their tensile properties were evaluated. CNF sheets had different tensile properties depending on the enzymatic treatment condition and sheet prepartion method. The tensile stregth, elastic modulus, and strain at break of each sheet are presented in Fig. 7. Compared to untreated CNF sheet, pretreatment of only endoglucanase decreased the tensile strength of both vacuum filtrated and casted sheets (Fig. 7a). It seemed because endoglucanase-pretreated CNF formed a weaker network owing to shorter length of fibrils than other CNFs, which was expected from the aspect ratio as shown in Fig. 5. In addition, a decrease in the DP by enzymatic treatment might also led to the lower tensile strength of the sheet, which agreed with the results in the previous studies. [26,30,37,38] On the contrary, the addition of xylanase in enzymatic pretreatment led to the CNF sheet with higher mechanical properties than the sole glucanase addtion. It is known that prescence of hemicellulose is beneficial to the strength of paper or sheet. [39-41] However, the xylanase-treated CNF sheets in this study did not show such a decrease in strength. It appeared that some xylan still remained on the fibrils and more hyrdogen bonding between the fibrils were created by the combined effect of endoglucanase and xylanase. [31] In the case of strain at break, as total dosage of endoglucanase increased, enzyme treated CNF sheets exhibited lower strain values (Fig. 7b). Since enzyme treated fibers had lower aspect ratio and higher crystallinity than untreated fibers, their elongation ability was poor. [26,30,38] The elastic modulus of the CNF sheets showed similar tendency with tensile strength (Fig. 7c). The sheets treated with both endoglucanase and xylanase had higher modulus than untreated CNF sheets, whereas the treatment of only endoglucanase resulted in lower modulus vaule. Based on these results, it is expected that treatment of xylanase can prevent the loss of the mechanical strength of CNF sheet caused by endoglucanase.

Fig. 7. 
Tensile properties of vacuum filtrated and casted CNF sheets: tensile strength (a), strain at break (b), and elastic modulus (c).

The light transmittance of each CNF sheet is shown in Fig. 8. Transmittance of the filtrated CNF sheet was around 90%, while those of casted sheets ranged from 80% to 85%. It was due to a denser network of the filtrated sheets by pressing during sheet preparation. Between untreated and enzymatic pretreated CNF sheets, there was no noticable difference for filtrated sheets because the compaction by pressing process reduced the effect of CNF morphology on the optical property of the sheet. For casted sheets, sheet treated with 1.0% endoglucanase showed the lowest transmittances. Due to low aspect ratio and heterogeneous fibril width distribution of 1.0% endoglucanse-treated CNF, less packed structure between nanofibrils was formed during drying, which resulted in the low transmittance. [30,42]

Fig. 8. 
Light transmittances of vacuum-filtrated and casted CNF sheets at 600 nm wavelength.

4. Conclusions

In this study, the combined effect of endoglucanase and xylanase was investigated for the production of CNF in terms of the properties of CNF and CNF sheets. After enzymatic pretreatment, pulp had lower CED viscosity and higher crystallinity than untreated pulp due to the cleavage action of β-1,4-glucosidic chain by endoglucanase. CNF treated with both endoglucanase and xylanase showed narrower and more unifrom morphology than that of untreated or only endoglucanase-treated CNF. High dosage of endoglucanase led to the wider fibril width. Enzymatic pretreated CNF had smaller aspect ratio compared to untreated CNF. CNF sheets treated with only endoglucanse exhibited lower tensile strength and elastic modulus, whereas CNF sheets treated with mixture of endoglucanase and xylanase did not show noticable decresase in their mechanical properties. Consequently, compared to untreated and only endoglucanase treatment, the combined use of endoglucanse and xylanase led to the production of homogeneous CNFs and the prevented a redcution in the mechanical strength of the CNF sheet.


This work was supported by the Technological Innovation Program funded by the Ministry of Trade, Industry & Energy (project No. 10062717). We appreciate Buckman Laboratories, Korea for providing enzymes.

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