Enzyme-Assisted Preparation of Fibrillated Cellulose Fibers and Its

Ind. Eng. Chem. Res. 2010, 49, 2161–2168

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Enzyme-Assisted Preparation of Fibrillated Cellulose Fibers and Its Effect on Physical and Mechanical Properties of Paper Sheet Composites Sukjoon Yoo and Jeffery S. Hsieh* School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, 311 Ferst DriVe, Atlanta, Georgia 30332-0100

The fabrication of fibrillated cellulose fibers and their utilization have been of more interest recently because of their biodegradability, low cost, and mechanical and thermal properties comparable to those of glass fibers or carbon fibers. In this study, we applied the combination of a mechanical shearing process with a cooling system and an enzymatic pretreatment to produce fibrillated cellulose fibers from softwood pulps. The effects of the mechanical shearing process, the cooling system, and the enzymatic treatment on the formation of the fibrillated cellulose fibers as well as physical and mechanical properties of paper were investigated. It was indicated that the cooling system was necessary in the grinding process to prevent the adverse effect of heat generation on mechanical properties of paper sheets. It was observed that the enzyme-assisted mechanical shearing process improves the production of fibrillated cellulose fibers and results in a substantial improvement of tensile strength and elongation of paper while producing thinner and less bright paper with low water absorptiveness. 1. Introduction Polymeric or synthetic fillers such as glass fibers and carbon fibers have been widely used in many industrial applications due to their excellent mechanical and thermal properties. However, deposition through incineration of these materials causes severe environmental problems. The demand for environmentally friendly materials has been increased to replace the nonrenewable resources in the next generation of products and processes.1 Recently, the utilization of natural fibers obtained from renewable resources such as wood has been of more interest as an alternative to synthetic fibers because of their biodegradability, low cost, and comparable mechanical and thermal properties.2,3 Fibrillated cellulose fibers are the natural fibers which have been studied most extensively as renewable materials due to their abundance and promising performance. The fibrillated cellulose fibers form a highly crystalline structure by laterally packing long cellulose molecules with hydrogen bonding. This stable structure brings high mechanical properties such as a Young’s modulus close to 138 GPa and a low coefficient of thermal expansion around 10-7 K-1.4,5 A mechanical treatment was employed mainly to disintegrate the cellulose fibrils, tightly hooked to one another by multiple hydrogen bonds, from the complex plant cell wall.6 Turbak et al. and Herrick et al. reported the first production of microfibrillated cellulose fibers by using a high pressure homogenizer. However, this mechanical homogenization usually requires a higher number of passes, which obviously increases the energy usage.7-9 In order to achieve the proper degree of fibrillation as well as to improve the efficiency of process, several mechanical methods have been introduced including a supergrinding method,10 a cryocrushing method,11,12 and an ultrasonic method.13 Chemical treatments such as acid hydrolysis prior to mechanical treatments was also attempted to facilitate the fibrillation and reduce the number of passes.14 However, it is noted that acid hydrolysis results in short, fibrous crystallites which decrease the reinforcement effect.15 TEMPO-mediated oxidation16 was reported as another * To whom correspondence should be addressed. Tel.: (404) 8943556. Fax: (404) 385-6317. E-mail: [email protected].

chemical treatment method to obtain nanofibers. Recently, high pressure mechanical treatment combined with mild enzymatic hydrolysis was introduced as an efficient and environmentally friendly method to prepare fibrillated cellulose fibers in contrast to acid hydrolysis.17,18 Fibrillated cellulose fibers have been used mostly for the reinforcement of composite materials.11,14,15,19-21 The application of fibrillated cellulose fibers has been extended into several areas including transparent materials,22-25 biomedical applications,26 and gas barrier films.25 Fibrillated cellulose fibers, which have smaller particle size and higher surface area than regular pulp fibers, have an enormous advantage in the production of paper with better paper sheet properties such as tensile strength. Recently, Henriksson et al. reported the synthesis of cellulose nanopaper structure with high toughness via a tensile test.27 In the present work, we synthesized fibrillated cellulose fibers by applying the strong mechanical shearing process combined with a cooling system and an enzymatic pretreatment, and explored the effect of mechanical grinding and enzyme treatment on the formation of fibrillated cellulose fibers and the improvement of several important paper properties including thickness, brightness, opacity, water absorptiveness, tensile strength, and elongation. 2. Experimental Section Materials. Southern softwood bleached pulp with about 90% brightness was obtained from a five-stage bleaching process of a commercial manufacturing facility. Cellulase from Aspergillus species (Product No. C2605) was obtained from Sigma-Aldrich and used without further purification. Tris(hydroxymethyl)aminomethane (Product No. 93358) was obtained from SigmaAldrich and used without further purification. Enzymatic Pretreatment. A 3.00 wt % sample of softwood pulp and 0.02 wt % cellulase enzyme were dispersed in deionized water with 0.79 wt % tris(hydroxymethyl)aminomethane. Tris(hydroxymethyl)aminomethane is one of the most common buffers used to simulate the typical physiological pH of living organisms between pH 7 and pH 9. The prepared pulp solution was incubated in 50 °C for 2 h to activate the

10.1021/ie901621n  2010 American Chemical Society Published on Web 01/26/2010

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Ind. Eng. Chem. Res., Vol. 49, No. 5, 2010

Figure 1. Schematic drawing of grinder system.

enzyme, followed by washing with water twice on a Bu¨chner funnel to remove cellulase enzyme and tris(hydroxymethyl)aminomethane. The cleaned pulp was mixed with about 1 L of deionized water and incubated again at 80 °C for 30 min to stop the activity of the remnant enzymes. Afterward, the pulp was washed again with water twice. Fibrillation. For fibrillation, the laboratory-scale grinder system was designed and manufactured by our laboratory. Figure 1 shows the schematic of the grinder system. It consists of a grinding reactor with a motor, a continuous feed pumping system, and a circulating cooling water system. The grinding mechanism consists of two slit and hole drilled stainless disks fit inside the reactor with a 20 hp motor to provide intensive grinding energy. The pulp material is fed onto the top disk and passed through the openings. The pulp is ground between the top and bottom disks and then forced through the opening of the bottom disk. The refined material is collected from the bottom of the reactor and fed back into the reactor again in a continuous manner through a gear pump (Liquiflo Model CF8M). The time used for energy input is reflected in the degree of pulp downsizing. The equipment is a traditional grinding and refining device but upgraded with a 20 hp motor instead of a typical 1 hp motor. The intensified energy input to the material is the key to making the pulp size smaller. Thirteen grams of the pulp was dispersed in water, and the concentration of the pulp suspension was adjusted to 0.5 wt % by adding water. The consistency of less than 1.0 wt % was found to be optimum for the smooth circulation of pulp solution through the grinder system. The suspension was continuously passed through the grinder system until the designated reaction time between 0 and 90 min. The flow rate of the circulated pulp suspension is around 1 L/min. Thus, it takes around 3 min to finish one pass of grinding for 3 L of pulp solution. Initially, the pulp suspension was at room temperature, but the heat generated by the grinder system increased the temperature of the pulp suspension by 5 °C every 15 min. The grinder system was cooled by the circulating cooling water outside the batch to prevent the effect of the generated heat. The grinder speed was set to 7.00 Hz. The ground pulp was recovered by filtration and further air-drying. Sheet Formation. The handsheets were formed using TAPPI Standard T205 om-88 titled “Forming Handsheets for Physical Tests of Pulp”. A 0.3 wt % sample of air-dried pulp was dispersed in 2.8 L of water. A 420 mL volume of solution was used for the formation of one handsheet, so the handsheet will be around 1.2 g. The handsheet was pressed at 50 psi two times

for 7 and 2 min. Then the handsheets were conditioned prior to testing based on TAPPI Standard T402 om-93, “Standard Conditioning and Testing Atmospheres for Paper, Board, Pulp Handsheets, and Related Products”. The handsheets were dried at 22-28 °C for at least 24 h and kept in a conditioning chamber of 50.0% relative humidity for at least 4 h. The Sanpia Dry Keeper (Sanpiatec Corp., Japan) was used as a conditioning chamber. Scanning Electron Microscopy. Scanning electron microscope (SEM) images of the fibrillated pulps were taken on a LEO 1530 thermally assisted field emission (TFE) scanning electron microscope (SEM) operating at 10 kV. Measurement of Paper Sheet Properties. Thickness measurements were made with a Model 89-100 thickness tester (Thwing-Albert Instrument Co., Philadelphia, PA) according to TAPPI Standard T411 om-89. Brightness and opacity were measured with a Model Color Touch 2 (Technidyne Corp., New Albany, IN) according to ISO Standard 2469 and ISO Standard 2471, respectively. Tensile and elongation were measured with a Model QC-1000 tensile tester (Thwing-Albert Instrument Co., Philadelphia, PA) according to TAPPI Standard T494 om-88. The water absorptiveness was measured according to TAPPI Standard T441 om-90. Zero span tensile was measured with a Pulmac TS-B1 (Pulmac Instruments International, Montpelier, VT) according to TAPPI Standard T231 cm-85. Canadian Standard Freeness (CSF) was measured with a CSF tester from Testing Machines, Inc. (Mineola, NY) according to TAPPI Standard T227 om-94. The air resistance of a paper sheet was measured with a Gurley densometer (Gurley Precision Instruments, Troy, NY) according to TAPPI Standard T460 om-88 and reported as seconds per 100 mL per 6.4 cm2, which are commonly referred to as Gurley seconds. The viscosity of pulp was measured according to TAPPI Standard T230 om-89. All data were normalized to basis weight (60 g/m2) for proper comparison. 3. Results and Discussion Scanning Electron Microscopy. The morphological change of pulps through mechanical grinding with a cooling system and an enzymatic treatment was observed via scanning electron microscopy (SEM) analysis. Figure 2 shows the SEM images of pulps before and after mechanical grinding with or without a cooling system and an enzymatic treatment. When a cooling system was not applied to the mechanical grinding, the morphology of pulps (Figure 2C,D) was not changed much compared with the original pulps (Figure 2A,B). However, after the mechanical grinding process with a cooling system, pulps were stretched and squeezed by the mechanical friction between two disks in the grinder (Figure 2E,F). When enzyme pretreatment was applied with the temperature controlled mechanical grinding process, the pulps were fibrillated to produce cellulose fibers from the cell wall (Figure 2G,H). This result indicates that the enzymatic pretreatment facilitates disintegration of cellulose fibers from the pulp surface by breaking or loosening the bonding between fibers through enzymatic hydrolysis. Effect of Heat Generation in Mechanical Grinding Process on Paper Sheet Properties. The pulp suspension was at room temperature initially. However, because of heat generated by the grinding system, the temperature of pulp suspension increases as it passes through the grinder process. After running 1 h, the temperature of the pulp suspension rises from 20 to 40 °C, with an average rise of 5 °C every 15 min. The temperature condition in the grinding process can influence the formation of cellulose fibers and the quality of products made of cellulose


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Figure 2. Scanning electron micrographs of softwood pulp raw materials (A and B), after mechanical grinding without a cooling system (C and D), after mechanical grinding with a cooling system (E and F), and after temperature controlled mechanical grinding with an enzymatic pretreatment (G and H).

fibers. The effect of the heat generation in the mechanical grinding process on paper sheet properties was explored by testing paper sheet properties after the grinding processes with or without the cooling system. The grinding system was cooled by the circulating cooling water outside the batch to prevent the effect of the heat generation. When there is no cooling water circulating around the bath reactor, the temperature rises by about 20 °C for 60 min (20 passes) of grinding. Figure 3 shows the change of paper sheet properties after 20 passes of grinding with or without a cooling water system. Table 1 shows the absolute values of each paper sheet property after 20 passes of the mechanical grinding with or without a cooling water system. Regardless of the existence of a cooling system, the grinding process itself increases tensile strength and elongation of paper sheets and reduces the thickness and water absorptiveness of paper sheets, compared with the paper sheets which are made of the original pulps. The addition of the cooling system into the grinding process enables the stabilization of the temperature around 20 °C and results in 44% more increase of tensile strength and 15% more decrease of thickness than the case of grinding without cooling. It is possible that the cell wall may be more brittle in the cold water and that it can make the disintegration of fibers from cell walls easier than the high

Figure 3. Effect of the cooling system in the mechanical grinding process on paper sheet properties.

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Table 1. Paper Sheet Properties after 20 Passes of the Mechanical Grinding with or without a Cooling System raw pulp thickness (mm) brightness (%) opacity (%) tensile (N m/g) elongation (%) water absorptiveness (g/m2)

0.112 ((0.001) 83.04 ((0.02) 72.53 ((0.41) 23.36 ((1.37) 0.65 ((0.12) 105.84 ((2.35)

grinding without a grinding with a cooling system cooling system 0.106 ((0.005) 87.75 ((3.48) 78.42 ((0.62) 43.74 ((2.70) 1.01 ((0.05) 76.30 ((7.51)

0.090 ((0.003) 79.70 ((2.79) 73.95 ((0.50) 54.17 ((0.93) 0.98 ((0.03) 81.26 ((6.12)

temperature condition. Another possible reason for the reduction of mechanical properties in the high temperature condition is the thermal degradation of cellulose fibers at high temperature.28 The high temperature condition activates the cleavage of the glycosidic linkage of cellulose and initiates the depolymerization of cellulose into glucose units, which brings the significant reduction of mechanical properties such as tensile strength. This result indicates the adverse effect of heat generation on the mechanical properties of paper sheet clearly. Therefore, the mechanical grinding with a cooling system is preferable since better mechanical properties are expected. After this point, all results presented in this paper were produced by mechanical grinding with a cooling system. Effect of Grinding Period on Paper Sheet Properties. The pulp suspension, treated by mechanical grinding with a cooling system, was used to synthesize the paper sheet. Different grinding periods were applied to see the effect of the mechanical grinding on paper sheet properties such as thickness, brightness, opacity, tensile, elongation, and water absorptiveness. Figures 4-9 show the change of paper sheet properties as a function of the number of grinding passes. As many grinding passes were applied with the longer treatment time, the tensile strength and elongation of paper sheet were improved while the thickness, brightness, opacity, and water absorptiveness decreased. The paper sheets obtained from pulp suspension after 30 passes of grinding showed a 2-fold increase in tensile strength (Figure 4) and a 1.5-fold increase in the elongation (Figure 5), as compared to those from original pulps. It is expected that the intense mechanical grinding by applying a long grinding period enlarges the fibrillated portion of cell walls and increases the amount of fibrillated cellulose fibers. The production of more fibrillated fibers results in a larger surface area per mass unit, which increases the accessible hydroxyl groups in the fiber cell wall and creates the stronger cross-linkage among fibers caused by hydrogen bonds. This positive effect of the increase of the

Figure 4. Tensile strength of paper sheets vs number of grinding passes.

Figure 5. Elongation of paper sheets vs number of grinding passes.

Figure 6. Thickness of paper sheets vs number of grinding passes.

bonded area and the strong network on the tensile strength can be explained based on the Page equation29 shown below. 9 12gC 1 ) + T 8Z Plb(RBA) RBA )

S0 - S S0

(1)

(2)

where T is the tensile breaking length (length), l is the fiber length (length), b is the fiber-fiber bond strength (N/m2), RBA is the relative bonded area (unitless); S is the light scattering coefficient of paper sheet (m2/kg), S0 is the light scattering coefficient of the unbonded sheet (m2/kg), g is the gravitational constant (9.8 m/s2), Z is the zero span tensile (length), C is the fiber coarseness (weight/length), and P is the fiber perimeter (length). Results indicate that the probable strong network formation by fibrillated fibers could enhance the tensile strength of the paper sheet and bring the synergistic improvement of the elongation. The thickness (Figure 6) of paper sheets was also affected by the extent of mechanical grinding. The fibrillated fibers are expected to have finer structure and larger surface area with high density of hydroxyl groups than original pulps. The finer structure of fibrillated fibers enables the compact packing of fibers by efficient space filling. The strong hydrogen bond also induces the closer cross-linking between fibers. The combined effect of the strong network and the finer structure of fibrillated fibers can reduce the total volume of fibrils, which


Figure 7. Brightness of paper sheets vs number of grinding passes.

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Figure 9. Water absorptiveness of paper sheets vs number of grinding passes.

Figure 8. Opacity of paper sheets vs number of grinding passes.

was observed as the reduction of paper sheet thickness. The brightness of paper is the measure of a paper’s ability to reflect light. It is presented as the volume of light reflected off the paper sheet, which is rated on a scale of 0-100. Figure 7 shows that the brightness of paper sheet decreased as the pulp suspension was exposed to longer mechanical shearing. Fibrillated cellulose fibers were known to have high transmittance of light.22-24 It is possible that addition of fibrillated fibers increases the absorption or transmittance of light while decreasing the light reflection of a paper sheet. The opacity is another measurement of the paper’s ability to reflect and scatter visible light. Figure 8 shows that the opacity also decreases as the number of grinding passes increases and presents that intensive grinding increases the content of fibrillated fibers in the paper sheets. Figure 9 shows the change of water absorptiveness with the increase of the number of grinding passes. All samples were conditioned in a chamber with 50.0% relative humidity. The water absorptiveness of paper sheet decreases from 105 g of H2O/m2 to 87 g of H2O/m2 with 30 times of grinding passes. Water absorptiveness is a physical characteristic related to the expected hydroxyl groups inside the fiber cell wall. It is believed that the increase of hydrogen bonds by the formation of strong network structure among fibrillated fibers decreases the accessible hydroxyl groups in the paper sheets and that it ultimately reduces the ability of paper sheet to absorb the water.

Figure 10. Change of paper sheet properties by mechanical grinding with or without enzymatic pretreatment.

Effect of Enzymatic Pretreatment on Paper Sheet Properties. Enzymatic pretreatment was applied to the pulp suspension prior to the mechanical grinding process. Its effect on disintegration of fibers and the properties of paper sheet which is made of fibrillated fiber suspension was investigated. Figure 10 shows how paper sheet properties change with combination of the mechanical shearing process and the enzymatic pretreatment. Table 2 shows the absolute values of paper sheet properties after 20 passes of grinding with or without the enzymatic pretreatment. Even without the enzymatic pretreatment, the mechanical shearing treatment itself was proved to affect the paper sheet properties through the production of more fibrillated cellulose fibers with high surface area per unit mass. The air resistance of paper (Gurley seconds) in Table 2 can be used as an indirect indicator of surface area per unit mass of fibrillated fibers. Gurley seconds increase by 540% after applying 20 passes of mechanical grinding only and increase by 3200% after 20 passes of grinding with the enzymatic pretreatment. The more production of fibrillated fibers with high surface area per unit mass enables the compacter packing of fibers, which increases the air resistance of paper sheets. The mechanical grinding combined with the enzymatic treatment also brings the increase of zero span tensile strength (Table 2), indicating the possible increase

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Table 2. Paper Sheet Properties after 20 Passes of the Temperature Controlled Mechanical Grinding with or without an Enzymatic Pretreatment

thickness (mm) brightness (%) opacity (%) tensile (N m/g) elongation (%) zero span tensile (N m/g) Gurley seconds (s) Canadian Standard Freeness (mL) viscosity (mPa · s)

raw pulp

grinding without an enzymatic pretreatment

grinding with 0.2 g of enzymatic pretreatment

grinding with 0.4 g of enzymatic pretreatment

0.112 ((0.001) 83.04 ((0.02) 72.53 ((0.41) 23.36 ((1.37) 0.65 ((0.12) 3.45 ((1.05) 2.7 ((0.0) 698.5 ((1.7) 20.8 ((0.7)

0.101 ((0.001) 75.38 ((2.91) 70.77 ((1.41) 48.81 ((0.79) 0.86 ((0.03) 14.59 ((3.96) 17.3 ((1.3) 612.1 ((0.9) 18.2 ((2.7)

0.085 ((0.003) 64.93 ((2.65) 64.48 ((0.86) 58.47 ((0.62) 1.49 ((0.14) 409.68 ((177.68) 89.3 ((4.5) 611.1 ((11.2) 14.1 ((0.1)

0.083 ((0.002) 67.35 ((1.43) 60.73 ((2.25) 55.13 ((0.94) 1.31 ((0.21) N/A N/A N/A N/A

of fiber strength through treatment. This increase of fiber strength can be another reason for the increase of tensile strength based on the Page equation (eqs 1 and 2). Canadian Standard Freeness (CSF), shown in Table 2, decreases by 13% after 20 passes of mechanical grinding to present that the mechanical grinding produces finer fibers. The tensile strength and the elongation increase by 100% and 30%, respectively, while the thickness and the brightness of paper sheet decrease by around 10% after 20 passes of mechanical grinding only. The addition of an enzymatic pretreatment prior to the mechanical shearing process enlarges the extent of these changes of paper sheet properties with the same tendency. Compared to paper sheet made of untreated original pulp, the mechanical shearing treatment after an enzymatic pretreatment brings a 250% increase in tensile strength and a 225% increase in elongation, which are 25% more and 77% more than the case of the mechanical grinding only. On the other hand, the enzymatic pretreatment furthers a 10% more decrease in thickness, brightness, and opacity of paper sheet than the case of the mechanical grinding only. These additional changes of paper sheet properties by the enzymatic pretreatment with the same tendency as the case of mechanical grinding only are reasonable because the enzymatic hydrolysis is expected to break the linkage of the crystalline structure of cellulose and to facilitate the disintegration of fibrillated fibers. The viscosity of pulps decreases by 12% after 20 passes of the mechanical grinding

Figure 11. Enzymatic processes by three types of cellulases.

only and decreases by 32% after 20 passes of grinding with the enzymatic pretreatment (Table 2). These decreases of the viscosity of pulps with the mechanical grinding and the enzymatic pretreatment indicate that the average degree of polymerization of the cellulose is decreased and presents relatively the reduction of molecular weight. The enzymatic processes were done generally by three types of enzymes (Figure 11). Endocellulase breaks internal bonds to disrupt the crystalline structure of cellulose and expose individual cellulose polysaccharide chains. Exocellulase cleaves 2-4 units such as cellobiose from the ends of the exposed chains produced by endocellulase. Cellobiase or β-glucosidase hydrolyzes the exocellulase product into individual monosaccharides. The cell walls become softened by these enzymatic breakages of networks among fibers prior to the mechanical grinding. This facilitates the cell wall disintegration and increases the content of fibrillated fibers in cellulosic fiber suspensions for pulp sheet synthesis. Based on the same reasons we discussed in the results section on the effect of mechanical grinding, the amplification of fibril production by the combined effect of mechanical shearing and enzymatic treatment raises the tensile strength and elongation while decreasing the thickness, brightness, and opacity of paper sheet. Combined with SEM analysis (Figure 2G,H), this result presents that the enzymatic pretreatment facilitates the disintegration of fibrillated fibers from the cell wall effectively and improves the efficiency of the fibril production in the mechanical


shearing process. It suggests the possible economic benefit of the enzymatic treatment to reduce the operating cost by lowering the processing time or the number of passes in the mechanical shearing process. For example, in the case of the tensile strength, the grinding process with the enzymatic pretreatment requires 25% less number of grinding passes than the grinding process only to achieve the same improvement in tensile strength, which reduces the energy requirements and therefore makes it more environmentally friendly also. The effect of cellulase enzyme dosage was also investigated. A 2 times higher dosage of cellulase enzyme, 0.4 g, was used in the enzymatic pretreatment and the other conditions of the whole procedure were same as the previous trials. Table 2 shows the paper sheet properties after enzyme pretreatment with two different dosages. It indicates that a 2-fold higher dosage of cellulase enzyme does not bring an additional effect on the paper sheet quality. Thickness and brightness of paper sheet do not change even with a high dosage of enzyme. Tensile strength and elongation were reduced by 6% and 10%, respectively. The enzyme amount in the initial trials was 0.2 g per 30 g of pulp, which was estimated based on the activity of cellulase enzyme we used. One cellulase unit (CU), the unit of enzyme activity, is defined as the amount of enzyme that liberates reducing sugar at the rate of 1 µmol/min. The cellulase enzyme we used in this paper has an activity of more than 1000 CU/g, indicating that addition of 1 g of cellulase can liberate 1000 µmol of sugars/1 min. The simple arithmetic calculation shows that 0.2 g of cellulase enzyme is the amount that is enough to treat 30 g of pulp. Therefore, this result indicates that fibril production cannot be improved by just adding more enzymes and also points out that an excessive dosage of enzyme can cause the deterioration of mechanical properties of cellulose fibers due to too much hydrolysis of cellulose fibers. 4. Conclusion In the present study, we investigated the effect of the mechanical shearing process with the cooling system and the enzymatic pretreatment on the fabrication of fibrillated cellulose fibers from softwood pulps and explored the utilization of the obtained fibril suspension to improve the physical and mechanical properties of paper. Application of the intensive mechanical shearing treatment through the longer grinding period results in the substantial improvement of tensile strength and elongation of paper while producing thinner and less bright paper with low water absorptiveness. It is expected that strong mechanical treatment enables the production of more fibrillated fibers which have finer structure, large surface area, and high density of accessible hydroxyl groups. The increase of these characteristic fibrils in a fiber suspension induces the formation of strong fibril networks and compact packing, which brings the changes in paper sheet properties mentioned here. The influence of heat generation in the mechanical shearing process was investigated by controlling the temperature inside the grinding vessel through the circulating cooling system. Results indicate the adverse effect of heat generation on the mechanical properties of paper and suggest the necessity of a cooling system to prevent the temperature elevation of the grinding process. Finally, it was explored how the enzyme pretreatment prior to the mechanical shearing process affects the fibrillation and the paper sheet properties. It is shown that the enzymatic pretreatment improves the efficiency of fibril production in mechanical shearing by facilitating the disintegration of fibril-

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lated fibers from the cell wall. The combination of mechanical shearing and enzyme pretreatment improves the tensile strength by 250% and the elongation by 200% compared to paper made of the original pulp. It was also proved that an excessive dosage of enzyme does not provide the improvement of fibrillation performance. Overall, these results suggest the possible economic benefit and environmental advantage of the enzymatic treatment to reduce the operating cost and provide environmentally friendly operating conditions. In conclusion, the combination of the mechanical shearing process and the enzymatic treatment successfully fabricated the fibrillated fibers from softwood pulp. The facilitation of fibril disintegration by the enzyme-assisted mechanical shearing process enables substantial improvement in the tensile strength and the elongation of paper and the production of thinner and less bright paper. Acknowledgment The authors thank Tae-Hyun Bae for his expertise and help with the scanning electron microscopy analysis. The authors also thank John Melnyczuk, Guozhou Mo, Peter Zou, and Cosmas Bayuadri for their help for the construction and operation of the mechanical shearing process. Literature Cited (1) Mohanty, A. K.; Misra, M.; Drzal, L. T. Sustainable bio-composites from renewable resources: Opportunities and challenges in the green materials world. J. Polym. EnViron. 2002, 10 (1-2), 19–26. (2) Samir, M.; Alloin, F.; Dufresne, A. Review of recent research into cellulosic whiskers, their properties and their application in nanocomposite field. Biomacromolecules 2005, 6 (2), 612–626. (3) Berglund, L. A. Cellulose-based nanocomposites. In Natural Fibers, Biopolymers, and their Biocomposites; Mohanty, A. K., Misra, M., Drzal, L. T., Eds.; CRC Press: Boca Raton, FL, 2005; pp 807-832. (4) Sakurada, I.; Nukushina, Y.; Ito, T. Experimental determination of elastic modulus of crystalline regions in oriented polymers. J. Polym. Sci. 1962, 57 (165), 651–660. (5) Nishino, T.; Matsuda, I.; Hirao, K. All-cellulose composite. Macromolecules 2004, 37 (20), 7683–7687. (6) Lepoutre, P.; Hui, S. H.; Robertson, A. A. Some properties of polyelectrolyte-grafted cellulose. J. Macromol. Sci.: Chem. 1976, A10 (4), 681–693. (7) Herrick, F. W.; Casebier, R. L.; Hamilton, J. K.; Sandberg, K. R. Microfibrillated cellulose: morphology and acessibility. J. Appl. Polym. Sci.: Appl. Polym. Symp. 1983, 37, 797–813. (8) Nakagaito, A. N.; Yano, H. The effect of morphological changes from pulp fiber towards nano-scale fibrillated cellulose on the mechanical properties of high-strength plant fiber based composites. Appl. Phys. A: Mater. Sci. Process. 2004, 78 (4), 547–552. (9) Turbak, A. F.; Snyder, F. W.; Sandberg, K. R. Microfibrillated cellulose, a new cellulose product: properties, uses, and commercial potential. J. Appl. Polym. Sci.: Appl. Polym. Symp. 1983, 37, 815–827. (10) Taniguchi, T.; Okamura, K. New films produced from microfibrillated natural fibres. Polym. Int. 1998, 47 (3), 291–294. (11) Bhatnagar, A.; Sain, M. Processing of cellulose nanofiber-reinforced composites. J. Reinf. Plast. Compos. 2005, 24 (12), 1259–1268. (12) Chakraborty, A.; Sain, M.; Kortschot, M. Cellulose microfibrils: A novel method of preparation using high shear refining and cryocrushing. Holzforschung 2005, 59 (1), 102–107. (13) Zhao, H. P.; Feng, X. Q.; Gao, H. J. Ultrasonic technique for extracting nanofibers from nature materials. Appl. Phys. Lett. 2007, 90, 073112. (14) Boldizar, A.; Klason, C.; Kubat, J.; Naslund, P.; Saha, P. Prehydrolyzed cellulose as reinforcing filler for thermoplastics. Int. J. Polym. Mater. 1987, 11 (4), 229–262. (15) Zimmermann, T.; Pohler, E.; Geiger, T. Cellulose fibrils for polymer reinforcement. AdV. Eng. Mater. 2004, 6 (9), 754–761. (16) Saito, T.; Kimura, S.; Nishiyama, Y.; Isogai, A. Cellulose nanofibers prepared by TEMPO-mediated oxidation of native cellulose. Biomacromolecules 2007, 8 (8), 2485–2491.

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ReceiVed for reView March 31, 2009 ReVised manuscript receiVed January 4, 2010 Accepted January 12, 2010 IE901621N

Enzyme-Assisted Preparation of Fibrillated Cellulose Fibers and Its

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