An environmentally friendly method for enzyme-assisted preparation of microfibrillated cellulose (MFC) nanofibers
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1 European Polymer Journal 43 (2007) EUROPEAN POLYMER JOURNAL An environmentally friendly method for enzyme-assisted preparation of microfibrillated cellulose (MFC) nanofibers M. Henriksson a, G. Henriksson a, L.A. Berglund a, T. Lindström b, * a Department of Fibre and Polymer Technology, Royal Institute of Technology, SE Stockholm, Sweden b STFI-Packforsk AB, Box 5604, SE Stockholm, Sweden Received 20 February 2007; received in revised form 4 May 2007; accepted 20 May 2007 Available online 8 June 2007 Abstract Microfibrillated cellulose nanofibers (MFC) provide strong reinforcement in polymer nanocomposites. In the present study, cellulosic wood fiber pulps are treated by endoglucanases or acid hydrolysis in combination with mechanical shearing in order to disintegrate MFC from the wood fiber cell wall. After successful disintegration, the MFC nanofibers were studied by atomic force microscopy (AFM). Enzyme-treatment was found to facilitate disintegration, and the MFC nanofibers produced also showed higher average molar mass and larger aspect ratio than nanofibers resulting from acidic pretreatment. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Microfibrillated cellulose; Nanofibers; Endoglucanase; Hydrolysis; Degree of polymerization 1. Introduction Microfibrillated cellulose nanofibers (MFC) disintegrated from wood pulp can be used in polymer nanocomposites of high mechanical performance [1]. These nanocomposites show a Young s modulus approaching 20 GPa. The reported strength is also high, although the data should be interpreted with care since flexural strength of brittle materials depends strongly on specimen geometry. Still, nanocomposites based on MFC nanofibers from wood pulp is a new class of materials with potential for exceptionally high mechanical performance. * Corresponding author. Fax: address: tom.lindstrom@stfi.se (T. Lindström). MFC nanofibers are expected to show high stiffness since the Young s modulus of the cellulose crystal is as high as 134 GPa [2]. Its polymer molecules crystallize in extended-chain conformation during biosynthesis and form microfibrils with a lateral dimension of around 4 nm [3]. In wood, cellulose microfibrils show preferred orientation and often in a direction close to the axial fiber direction. Wood microfibrils form aggregates of nm thickness in cellulosic wood pulp fibers [3]. Cellulosic pulp fibers can be disintegrated into fine fragments by mechanical refining methods. However, such methods tend either to damage the microfibril structure by reducing molar mass and degree of crystallinity or fail to sufficiently disintegrate the pulp fiber. Herrick et al. [4] and Turbak et al. [5] reported on a method for producing /$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi: /j.eurpolymj
2 M. Henriksson et al. / European Polymer Journal 43 (2007) microfibrillated cellulose. The MFC typically consist of disintegrated microfibril aggregates with a lateral dimension in the scale of tens of nanometers. The method was to pass a dilute cellulosic wood pulp fiber/water suspension through a mechanical homogenizer, where a large pressure drop facilitates microfibrillation. The function of a homogenizer is explained in detail by Rees [6]. When a cellulosic pulp fiber suspension is homogenized, the procedure is often repeated several passes in order to increase the degree of fibrillation [1,4]. A higher number of passes obviously increases the energy required for disintegration. In order to facilitate disintegration, one may reduce fiber length by mechanical cutting [4] or the fiber cell wall can be treated and embrittled by acid hydrolysis [7] prior to homogenization. However, acid hydrolysis can significantly lower the molecular weight of cellulose and result in short, fibrous crystallites in the form of microcrystalline cellulose (MCC) [8]. As a consequence, acid hydrolysis may reduce the reinforcement effect [9]. Nakagaito and Yano demonstrated improved disintegration as the number of passes through the homogenizer was increased to 30 times [1]. However, MFC nanofibers were recently disintegrated in a more efficient process where 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidation was utilized as a pretreatment step prior to mechanical treatment [10]. The use of enzymatic pre-treatment may also lower processing cost by lowering the number of passes through the homogenizer, and has advantages from the environmental point of view as compared with chemical methods. In nature, cellulose does not degrade by single enzymes. Instead aerobic fungi like Trichoderma-, Phanerochaete- and Aspergillus-species degrade cellulose by excreting a mixture of independently acting, but cooperating hydrolytic enzymes [11]. Such a set of cellulases can contain seven or more different enzymes belonging to different protein families [12], but at least four functional types of cellulases can be recognized: A- and B-type cellulases are termed cellobiohydrolases, and are able to attack also highly crystalline cellulose, whereas C- and especially D-type cellulases, endoglucanases with a common name, generally require some disorder in the structure in order to attack the cellulose [13,14]. Cellobiohydrolases and endoglucanases show strong synergetic effects [11,15]. In the present study, environmentally friendly pretreatment of high cellulose content wood pulp fibers with a pure C-type endoglucanase was investigated in order to facilitate disintegration of MFC nanofibers. 2. Experimental 2.1. Materials Two different commercial bleached wood sulphite pulps based on Norway Spruce (Picea abies) from Domsjö Fabriker AB, Sweden, were used. One softwood sulphite pulp intended for special paper qualities, termed special paper pulp (SPP), delivered both as never dried and dried pulp, and one softwood dissolving pulp (DSP), delivered dry. The pulp fibers are about 40 lm wide and more than 1 mm long. The pulps are close to lignin- and extractives-free and carbohydrate analyses showed that SPP contained 85% cellulose while the cellulose content of DSP was 93%. The rest is mainly hemicelluloses. The enzyme used is Novozym 476, an endoglucanase manufactured by Novozymes A/S, Denmark. The poly(diallyldimethylammonium chloride) (poly-dadmac) used in atomic force microscopy, was Alcofix-109 purchased from CDM, Sweden. Prior to use the sample was fractionated and the fraction above 500 k was used. This fraction had M w 870, Pretreatment procedures The fibers were treated by four different methods. Three of the methods included beating in a PFI-mill, manufactured by HAM-JERN, Norway, according to EN :1994 [16] with one modification: 40 g fibers diluted with water to a total weight of 300 g (13.3% dry content) was used. The enzymatically treated fibers were first mechanically beaten 1000 revolutions in a PFImill in order to increase the swelling in water and make the cellulose more accessible for the enzyme. Enzymatic treatment was performed as follows: 3% pulp was dispersed in 50 mm tris(hydroxymethyl)aminomethane/hcl buffer with ph 7 and 3%, 1.5%, 0.5% and 0.02% enzyme, related to the weight of the pulp (SPP- 3%, DSP-3%, DSP-1.5%, DSP-0.5%, and DSP- 0.02%). The fibers were incubated at 50 C for 2 h, washed on a Büchner funnel, thereafter incubated again at 80 C for 30 min, to stop the
3 3436 M. Henriksson et al. / European Polymer Journal 43 (2007) activity of the enzymes, and then washed again. The fibers were finally beaten 4000 revolutions in a PFI-mill. No-enzyme references termed SPP-ref, and DSPref were also prepared, and treated in the same way as the enzymatically treated pulp, but without exposure to enzymes. DSP samples were also treated with the enzymatic step replaced by mild hydrolysis and termed DSP-MH. A 3% fiber suspension was adjusted to ph 1 with HCl and incubated at 50 C for 1 h. After incubation, the fibers were washed on a Büchner funnel. DSP-MH was finally beaten 4000 revolutions in a PFI mill. A much stronger hydrolysis was also performed to form DSP-SH. This method did not include any beating. A 7% pulp suspension was first swollen in 3% NaOH at 50 C for 10 min and then washed. Thereafter the hydrolysis was performed with 7% pulp concentration in 2.5 M HCl at 90 C for 2 h. After incubation, the fibers were again washed, but not beaten Homogenization After treatments, 2% fiber suspensions in water were subjected to the homogenizing action of a high-pressure slit homogenizer, Laboratory Homogenizer 15 M, Gaulin Corp., USA. The fibers were passed 20 times through the slit. Processing was performed in a similar way as described in Ref. [4]. Efforts were made to keep the pressure constant for the different test-runs. During the process, the suspension viscosity increased with increasing number of passes. Initially the suspension was at room temperature but the temperature increased with increasing number of passes to 70 C. Herrick et al. [4] found that increasing the temperature from 20 C to C facilitates homogenization Estimation of cellulose molecular weight The molecular weight of cellulosic fibers can be estimated from an average intrinsic viscosity value. The measurements were performed on dissolved fibers, prior to homogenization, according to SCAN-CM 15:99 [17] with cupriethylendiamine as solvent. Fibers were weighed in the wet state. The water content of the sample was taken into account when adding water to the samples. Two samples were made for each set of fibers. The intrinsic viscosity, g (ml/g), is related to the degree of polymerization (DP) and an empirical relationship for this polymer-solvent system is suggested as g = 2.28 DP 0.76 [18]. The presence of any hemicelluloses is not considered when calculating DP. The DP data after homogenization for DSP-0.5% and DSP-0.02% reported in Table 1, were obtained from samples homogenized 12 passes in a different type of homogenizer, a Microfluidizer M 110-EH, Microfluidics Inc., USA. Molecular weight distribution was estimated for three dissolving pulp samples (DSP, DSP- 1.5%, DSP-SH) by size exclusion chromatography (SEC). The solvent system used was dimethylacetamide (DMAc)/LiCl. Pullulan was used for calibration and a refractive index detector was used. Water was removed from the MFC suspensions by solvent exchange with methanol and DMAc before being dissolved in DMAc/LiCl containing ethylisocyanate. Sample preparation and measurements were carried out at MoRe Research AB, Sweden. Table 1 DP estimated at different stages in the process and statement of whether the pulp was possible to homogenize into nanofibers or not (yes/no) Pulp Method DP Possible to homogenize? DP after homogenization SPP special paper pulp SPP 2930 SPP-ref, no-enzyme reference 2620 No SPP-3%, 3% enzyme 910 Yes 640 DSP dissolving pulp DSP 1280 DSP-ref, no-enzyme reference 1200 No DSP-3%, 3% enzyme 540 Yes 340 DSP-1.5%, 1.5% enzyme 640 Yes 480 DSP-0.5%, 0.5% enzyme 600 Yes 470 DSP-0.02%, 0.02% enzyme 910 Yes 740 DSP-MH, mild hydrolysis 1100 No DSP-SH, strong hydrolysis 170 Yes Not measured Please note that with the exception of DSP-SH, all treated pulps were beaten in a PFI-mill prior to homogenization (see Section 2).
4 M. Henriksson et al. / European Polymer Journal 43 (2007) Microscopy Optical micrographs were obtained by placing a drop of fiber suspension of low concentration on a glass slide. Scanning electron microscopy (SEM) images were captured using a Hitachi s-4300 field emission SEM operating at 2 kv. The sample was mounted onto a substrate with carbon tape and coated with a thin layer of gold. The image in Fig. 3 was obtained from a sample of DSP-0.5% homogenized 12 passes in a different type of homogenizer, a Microfluidizer M 110-EH, Microfluidics Inc., USA. Atomic force microscope (AFM) images were measured using a Nanoscope IIIa, from Vecco Inc., USA. The scan was performed in tapping mode in air with a standard silica cantilever. Amplitude images are presented. Silica plates were used as substrate for the samples. The surfaces of the silica plates were first oxidized at 1000 C for 3 h and then hydroxylized in a plasma oven to ensure that the surfaces were hydrophilic. Thereafter the surface charge of the plates was modified by placing the plates into a 100 mg/l poly-dadmac/1 mm NaCl solution for 10 min. The plates were rinsed with 1 mm NaCl to remove excessive poly-dadmac. The plates were placed in 0.01% fiber suspension for 5 s and rinsed with 0.5 ml, 1 mm NaCl solution. The samples were dried at room temperature prior to AFM analysis. 3. Results and discussion 3.1. Physical appearance of pretreated pulp Two major grades of cellulosic fibers (SPP and DSP) were divided into three different groups, the first pretreated with a commercial endoglucanase at different concentrations (SPP-3%, DSP-3%, DSP-1.5%, DSP-0.5%, and DSP-0.02%), the second pretreated with mild or strong acid hydrolysis (DSP-MH, DSP-SH), and the third was not chemically treated (SPP-ref, DSP-ref). The fibers were then subjected to mechanical beating, except from DSP-SH, and homogenization in order to disintegrate MFC nanofibers. Beating is used in industry in order to increase the flexibility of the fiber as well as its surface roughness. The physical appearance of the fibers after the different treatments, was studied by optical microscopy, see Fig. 1. Initially the fibers are about 40 lm wide and more than 1 mm long. Estimates of DP for the materials are presented in Table 1. Fig. 1a presents the DSP-ref after mechanical beating in a PFI-mill. The length is unchanged although surface fibrillation and presence of fine material (cell wall fragments) is observed. The effect of enzymatic treatment is apparent by comparison of Fig. 1a and b. The fiber length is somewhat reduced for DSP-3% and the extent of fine material is increased. However, corresponding observations of DSP-0.02% show only limited fiber shortening compared with DSP-3%. For the case of DSP-3%, there is also a reduction in DP from 1280 to 540 with the pretreatment, see Table 1. However, lower enzyme concentration (1.5%, 0.5%, and 0.02%) results in an estimated DP of 640, 600, and 910, respectively. The high DP for DSP-0.02% is interesting. As high DP as possible is desirable for MFC nanofibers, since this is expected to increase the inherent tensile strength of the cellulose [19]. Procedures with low enzyme concentration are therefore preferable from this point of view, since the reduction in molecular weight is smaller. The strongly hydrolyzed fibers in Fig. 1c show substantially reduced fiber length and DP (see Table 1). This material is closer to microcrystalline cellulose [8] and the resulting microfibrils are expected to have low aspect ratio Ease of homogenization Prior to homogenization, the two-phase nature of the water fiber suspension is apparent since sedimentation occurs rapidly for all of the suspensions. After successful homogenization, suspensions are stable. The viscosity also increases substantially with the number of passes through the homogenizer. This is related to the increasing degree of MFC nanofiber disintegration from the cellulosic fiber cell wall. The purpose of acid or enzymatic pretreatment is to facilitate microfibrillation during the homogenization procedure. From Table 1, it is apparent that not all types of cellulosic fibers were possible to homogenize. However, all fibers subjected to enzyme pretreatment were successfully homogenized. In several other cases, the fibers blocked the slit in the homogenizer. As a consequence, the flow of the suspension was stopped and microfibrillation was prevented. When fibers agglomerate and block the slit, the equipment must be emptied, demounted and cleaned before the processing may continue. This occurred repeatedly with some cellulosic fibers and they were impossible to homogenize. There was
5 3438 M. Henriksson et al. / European Polymer Journal 43 (2007) Fig. 1. Optical microscopy images of (a) DSP-ref, no-enzyme reference of dissolving pulp after beating, (b) DSP-3%, enzyme-treated dissolving pulp (3%) after beating, and (c) DSP-SH, strongly acid hydrolyzed dissolving pulp. Scale bar is 100 lm. no difference in ease of homogenization between the special paper pulp SPP (85% cellulose) and dissolving pulp DSP (93% cellulose). In addition, both never dried and dried SPP pulps were tried since never dried pulp has been reported to facilitate homogenization [4], but no such effect was observed. Previous work showed that MFC nanofibers were easier to disintegrate from mechanically cut cellulosic fibers [4]. However, the specific procedure used is impractical, whereas the present PFI-milling and other mechanical beating methods can be used industrially. The observed fiber blockage problems are caused by fiber agglomeration. The tendency for agglomeration is reduced with reduced fiber length. Although the present cellulosic fibers of lowest molar mass are easy to disintegrate, molar mass is not the major parameter influencing ease of homogenization. This is demonstrated by the following case. The DSP-3% could not be homogenized directly after enzyme treatment. However, when this pulp was subjected to PFI-milling, with little change in DP but significant fiber shortening and cell wall disintegration, homogenization was possible. Ease of disintegration in the homogenizer therefore is influenced by fiber length, although other factors are also important. The combination of enzyme treatment and mechanical beating certainly provides an advantageous starting state for fiber disintegration. It is possible that the endoglucanase treatment increases cell wall swelling in the water suspension and, as a consequence, the cell wall is more easily disintegrated into MFC nanofibers as it is subjected to mechanical shearing during the homogenization process. It was observed that cellulosic fiber suspensions possible to homogenize, showed slower agglomeration and sedimentation in the inlet reservoir. The presence of smaller fiber fragments in the enzymatically treated and strongly hydrolyzed pulps seemed to act as a suspending agent. This is in agreement with previous experience [4] where pulp dispersion in a 2% suspension of uncut pulp fibers was improved by the addition of MFC. The stability of the cellulosic fiber suspension therefore appears to be another factor facilitating homogenization.
6 3.3. After homogenization M. Henriksson et al. / European Polymer Journal 43 (2007) There is a significant decrease in DP during homogenization, see Table 1. For instance, DP of DSP-3% decreases from 540 to 340 after homogenization (20 passes through the homogenizer). The cellulosic fiber with the higher original DP (SPP) shows higher final DP. Herrick et al. [4] and Turbak et al. [5] observed a 27% reduction in cellulose DP due to homogenization, similar to the present case. Boldizar et al. [7] found effects of DP on the reinforcement efficiency of MFC nanofibers in polyvinylacetate. Higher DP gave better mechanical properties, although DP in the range gave significant reinforcement effects. The MFC nanofibers produced using the present enzymatic methods were therefore expected to function well as composite material reinforcement. This was confirmed in a recent study [20]. Fig. 2 shows the molecular weight distribution for DSP, DSP-1.5%, and DSP-SH. DSP-SH is severely degraded. A shift in molecular weight distribution towards lower values is also apparent for the enzymatically treated molecules, although the higher molecular weight fractions are well preserved. Weight average molecular weights are presented in the text of Fig. 2. The MFC nanofiber samples were studied by SEM and AFM. In Fig. 3 there is a SEM image of DSP-0.5%. The original pulp fiber structure is Fig. 3. SEM image of freeze-dried DSP-0.5% after homogenization. Scale bar is 30 lm. destroyed and the resulting nanofibers are of large aspect ratio. AFM images of the DSP-3% nanofibers show highly microfibrillated nanofibers of large aspect ratio (length/diameter), see Fig. 4a. The nanofibers are about nm wide and several micrometer long. There is also a fraction of shorter nanofibers with thickness of about 5 10 nm. The acid hydrolyzed DSP-SH nanofibers show much smaller aspect ratio, see Fig. 4b. These nanofibers have thickness of about 5 15 nm. Battista demonstrated strong correlation between DP and length of cellulose nanofibers [8]. The DSP-SH also contains larger fragments of cellulosic fiber cell walls. At higher magnification, the DSP-SH nanofibers varied significantly in thickness, whereas MFC from DSP-3% showed more uniform nanofiber thickness Mechanisms facilitating disintegration of MFC nanofibers Fig. 2. Molecular weight distributions for MFC nanofibers based on dissolving pulp. Samples are termed DSP, dissolving pulp, DSP-1.5%, enzyme treated dissolving pulp (1.5%) after homogenization, and DSP-SH, strongly acid hydrolyzed dissolving pulp after homogenization. The weight average molecular weights are DSP: 285,700, DSP-1.5%: 280,000, and DSP-SH: 63,600. Acid hydrolysis facilitates disintegration of MFC nanofibers from cellulosic pulp fibers. Boldizar et al. suggested that the reduced molar mass associated with hydrolysis embrittles the cell wall and facilitates disintegration [7]. However, the reduction in molecular weight appears difficult to control. In the present study, the resulting MFC also shows a wide distribution in aspect ratio and diameters. Endoglucanase pretreatment in combination with mechanical beating greatly facilitates disintegration of MFC nanofibers from cellulosic wood fibers by mechanical shearing of a 2% fiber suspension (homogenization). Although fiber cutting during the mechanical beating stage contributes to
7 3440 M. Henriksson et al. / European Polymer Journal 43 (2007) Fig. 4. AFM images (5 5 lm 2 ) of MFC nanofibers, (a) DSP-3%, enzyme treated dissolving pulp (3%), (b) DSP-SH, strongly acid hydrolyzed dissolving pulp. ease of disintegration for the higher enzyme concentrations, the DSP-0.02% showed only limited fiber shortening and yet was easily disintegrated. A recent study [21] suggests an additional mechanism facilitating MFC disintegration. Endoglucanase treatment increased the reactivity of cellulosic pulp fibers, even after mild treatment where the molecular weight distribution of cellulose was virtually unchanged. It was suggested that endoglucanase treatment increases swelling of cellulosic pulp fibers in water suspension. A hypothesis is therefore that this effect also contributes to MFC nanofiber disintegration. Indirect support for this explanation is provided by the limited fiber shortening and small change in DP for the sample subjected to 0.02% enzyme concentration. Despite little reduction in DP due to enzyme treatment (from 1280 to 910), the DSP-0.02% pulp was easily disintegrated. 4. Conclusions Endoglucanase pretreatment facilitates disintegration of cellulosic wood fiber pulp into MFC nanofibers by mechanical shearing in an environmentally friendly process without solvents or chemical reactants. The MFC successfully produced from enzymatically pretreated cellulosic wood fibers showed a more favorable structure than nanofibers resulting from fibers subjected to hydrolysis by strong acid. AFM images showed high aspect ratio of the MFC nanofibers prepared after enzymatic pretreatment. These high aspect ratio nanofibers are of great interest as reinforcement in nanocomposite materials. The acid hydrolyzed MFC showed inhomogeneous distribution of nanofiber geometry and a large extent of thick cell wall fragments of low aspect ratio. One effect of high concentration enzymatic pretreatment and mechanical beating is to reduce fiber length and increase the extent of fine material. However, pretreated fibers subjected to the lowest enzyme concentration, 0.02%, were also successfully disintegrated although molecular weight and fiber length were well preserved. The estimated DP was only reduced from 1280 to 910. This indicates that additional mechanisms are in operation than lowered molecular weight, associated embrittlement, and fiber shortening. It is suggested that increased cellulosic fiber swelling in water due to enzyme-treatment facilitates preparation of MFC nanofibers. Acknowledgements Biofibre Materials Centre (BiMaC) at KTH (GH) and the Swedish Research Council (MH) are gratefully acknowledged for financial support. Dr S. Notley and Prof L. Wågberg at KTH are gratefully acknowledged for help with the AFM work. Novozyme is acknowledged for the gift of the enzyme.
8 M. Henriksson et al. / European Polymer Journal 43 (2007) References [1] Nakagaito AN, 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 2004;78: [2] Sakurada I, Nukushina Y, Ito T. Experimental determination of the elastic modulus of crystalline regions in oriented polymers. J Polym Sci 1962;57: [3] Hult EL, Iversen T, Sugiyama J. Characterization of the supermolecular structure of cellulose in wood pulp fibres. Cellulose 2003;10: [4] Herrick FW, Casebier RL, Hamilton JK, Sandberg KR. Microfibrillated cellulose: morphology and accessibility. J Appl Polym Sci, Appl Polym Symp 1983;37: [5] Turbak AF, Snyder FW, Sandberg KR. Microfibrillated cellulose, a new cellulose product: properties, uses, and commercial potential. J Appl Polym Sci, Appl Polym Symp 1983;37: [6] Rees LH. Evaluating homogenizers for chemical processing. Chem Eng 1974;13: [7] Boldizar A, Klason C, Kubát J, Näslund P, Sáha P. Prehydrolyzed cellulose as reinforcing filler for thermoplastics. Int J Polym Mater 1987;11: [8] Battista OA. Microcrystal polymer science. New York: McGraw Hill Inc.; [9] Zimmerman T, Pöhler E, Geiger T. Cellulose fibrils for polymer reinforcement. Adv Eng Mat 2004;6: [10] Saito T, Nishiyama T, Putaux JL, Vignon M, Isogai A. Homogeneous suspensions of individualized microfibrils from TEMPO-catalyzed oxidation of native cellulose. Biomacromolecule 2006;7: [11] Rabinovich ML, Melnick MS, Bolbova AV. The structure and mechanism of action of cellulolytic enzymes. Biochem Moscow 2002;67: [12] Henrissat B. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 1991;280: [13] Johansson G, Ståhlberg J, Lindeberg G, Engström Å, Pettersson G. Isolated fungal cellulose terminal domains and a synthetic minimum analogue bind to cellulose. FEBS Lett 1989;243: [14] Henriksson G, Nutt A, Henriksson H, Pettersson B, Ståhlberg J, Johansson G, et al. Endoglucanase 28 (Cel12A), a new Phanerochaete chrysosporium cellulase. Eur J Biochem 1999;259: [15] Berghem LER, Pettersson G. The mechanism of enzymatic cellulose degradation. Purification of a cellulolytic enzyme of Trichoderma viridae active on highly ordered cellulose. Eur J Biochem 1973;37: [16] EN :1994. European Standard. European Standards are published by European Committee for Sandardization (CEN), rue de Strassart 36, B-1050 Brussels, Belgium. [17] SCAN-CM 15:99. SCAN Standard. SCAN-test methods are published and recommended by central laboratories for the pulp and paper industry in Denmark, Finland, Norway and Sweden. They can be obtained from Scandinavian Pulp, Paper and Board Testing Committee, Box 5604, SE Stockholm, Sweden. [18] Gruber E, Gruber R. Viskosimetriche bestimmung des polymerisationsgrades von cellulose. Das Papier 1981;35: [19] Mark RE. Cell wall mechanics of tracheids. New Haven and London: Yale University press; p [20] Henriksson M, Berglund LA. Structure and properties of cellulose nanocomposite films containing melamine formaldehyde. J Appl Poly Sci, 2007, in press. [21] Engström AC, Ek M, Henriksson G. Improved accessibility and reactivity of dissolving pulp for the viscose process: pretreatment with monocomponent endoglucanase. Biomacromolecules 2006;7:
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