The change in the morphology of the fiber mat during the enzymatic treatment showed the development of smoothened surface possibly due to the accumulation of the enzyme in the fiber matrix and subsequent removal of the unwanted microfibrils (Fig.
16). The SEM clearly showed that refined fiber would be more ragged with treated endoglucanase. The changes in the fiber properties were caused; however, not only by the quality of modified spruce fibers, but also by the bonding process following the web formation stage. To investigate the effect of water jets on the spruce fibers, and the images revealed that the samples made of endoglucanase-treated fibers had more cracks present on the fiber surface than those containing untreated fibers. The appearance of cracks was not widespread for the spruce fibers pictured right after enzymatic process.
It was believed that the impact forced of the water jets triggered the cracks caused by the enzymes to propagate and visibly increase in size. This also resulted in the fibrillation of some PFI fibers. The changes in fiber characteristics, that is, cracks of different sizes and fibrillation, might have a significant impact on the strength properties of the fibers.
Fig. 16. Scanning electron microscopic images of PFI revolutions 0, 2000 and 4000 surface image of control and endoglucanase treated (5 IU) fibers.
Control 0 5 IU 0
Control 4000 5 IU 4000
Control 2000 5 IU 2000
V Conclusions
The study showed that irrelevant proteins preferentially adsorbed on pulp surface with higher lignin contents than endoglucanase, and the addition of yeast extract strengthened the above trend. Complicating factors impacted the endoglucanase hydrolysis rates of this system: the presence of irrelevant proteins, pulp lignin, and cellulose contents (or lignin-associated microfibril structures). Addition of yeast extract further promoted preferential adsorption of irrelevant proteins, and increased the hydrolysis rates by endoglucanase and altered the mode of hydrolysis.
On the other hand, the pretreatment of endoglucanase from recombinant Paenibacillus through refined would raise the pulp properties. According to the study, the cellulase modified the interfacial properties of fibers, increasing the affinity for water, such as pulp drainability and fiber hydration. Moreover, the enzymatic pretreatment also changed the distribution of microfiber (Morfi), causing wider fiber, less kink angle and small fines, more broken ends and rate in length of microfiber. The strength properties of the pulp had an impact by the studied endoglucanase. The SEM also showed that refined fiber would increase the coarseness with endoglucanase treated.
VI References
Akaracharanya, A., Lorliam,W., Tanasupawat, S., Lee, K. C., and Lee, J. S. (2009).
“Paenibacillus cellulositrophicus sp. nov., a cellulolytic bacterium from Thai soil.,”
International Journal of Systematic and Evolutionary Microbiology 59, 2680-2684.
Andrić, P., Meyer, A. S., Jensen, P. A., and Johansen, K. D. (2010). “Reactor design for minimizing product inhibition during enzymatic lignocellulose hydrolysis: I.
Significance and mechanism of cellobiose and glucose inhibition on cellulolytic enzymes,” Biotechnology Advances 28, 308-324.
Bajpai, P. (1999). “Application of enzymes in the pulp and paper industry,”
Biotechnology Progress 15, 147-157.
Bajpai, P., Mishra, S. R., Mishra, O. P., Kumar, S., and Bajpal, P. K. (2006). “Use of enzymes for reduction in refining energy-laboratory studies,” Tappi Journal 5, 25-32.
Bansal, P, Hall, M., Realff, M. J., Lee, J. H., and Bommarius, A. S. (2009). “Modeling cellulase kinetics on lignocellulosic substrates,” Biotechnology Advances 27(6), 833-848.
Berlin, A., Balakshin, M., Gilkes, N., Kadla, J., Maximenko, V., Kubo, S., and Saddler, J. (2006). “Inhibition of cellulase, xylanase and β-glucosidase activities by softwood lignin preparations,” Journal of Biotechnology 125, 198-209.
Bhat, M. K. (2000). “Cellulases and related enzymes in biotechnology,” Biotechnology Advances 18, 355-383.
Boussaid, A. and Saddler, J. N. (1999). “Adsorption and activity profiles of cellulases during the hydrolysis of two Douglas fir pulps,” Enzyme and Microbial Technology 24, 138-143.
Börjesson, J., Peterson, R., and Tjerneld, F. (2007). “Enhanced enzymatic conversion of
softwood lignocellulose by poly (ethylene glycol) addition,” Enzyme and Microbial Technology 40, 754-762.
Cadena, E. M., Chriac, A. I., Pastor, F. I. J., Diaz, P., Vidal, T., and Torres, A. L. (2010).
“Use of cellulases and recombinant cellulose binding domains for refining TCF kraft pulp,” Biotechnology Progress 26(4), 960-967.
Calvini, P. (2005). “The influence of levelling-off degree of polymerization on the kinetics of cellulose degradation,” Cellulose 12, 445-447.
Chernoglazov, V. M., Ermolova, A. A., and Klyosov, A. A. (1988). “Adsorption of high-purity endo-1,4-β-glucanases from Trichoderma reesei on components of lignocellulosic materials: Cellulose, lignin, and xylan,” Enzyme and Microbial Technology 10, 503-507.
Dienes, D., Borjesson, J., Stalbrand, H., and Reczey, K. (2006). “Production of Trichoderma reesei Cel7B and its catalytic core on glucose medium and its application for the treatment of secondary fibers,” Process Biochemistry 41, 2092-2096.
Fan, L. T., Lee, Y. H., and Beardmore, D. H. (1980). “Mechanism of the enzymatic hydrolysis of cellulose: Effects of major structural features of cellulose on enzymatic hydrolysis,” Biotechnology and Bioengineering 22, 177-199.
Galbe, M. and Zacchi, G. (2002), “A review of the production of ethanol from softwood,” Applied Microbiology and Biotechnology 59, 618-628.
García, O., Torres, A. L., Colom, J. F., Pastor, F. I. J., Díaz, P., and Vidal, T. (2002).
“Effect of cellulase-assisted refining on the properties of dried and never-dried eucalyptus pulp,” Cellulose 9, 115-125.
Garmaroody, E. R., Resalati, H., Fardim, P., Hosseini, S. Z., Rahnama, K. A., Saraeeyan, R., and Mirshokraee, S. A. (2011). “The effects of fungi pre-treatment of poplar chips on the kraft fiber properties,” Bioresource Technology 102, 4165-4170.
Gil, N., Gil, C., Amaral, M. E., Costa, A. P., and Duarte, A. P. (2009). “Use of enzymes to improve the refining of a bleached Eucalyptus globulus kraft pulp,” Biochemical Engineering Journal 46, 89-95.
Henrissat, B. (1994). “Cellulases and their interaction with cellulose,” Cellulose 1, 169-196.
Himmel, M. E., Ding, S. Y., Johnson, D. K., Dney, W. S., Nimlos, M. R., Brady, J. W., and Foust, T. D. (2007). “Biomass recalcitrance: engineering plants and enzymes for biofuels production,” Science 315, 804-807.
Hubbell, C. A., and Ragauskas, A. J. (2010). “Effect of acid-chlorite delignification on cellulose degree of polymerization,” Bioresource Technology 101, 7410-7415.
Ishizawa, C. I., Jeoh, T., Adney, W. S., Himmel, M. E., Johnson, D. K., and Davis, M. F.
(2009). “Can delignification decrease cellulose digestibility in acid pretreated corn stover?” Cellulose 16, 677-686.
Jarvis, M. (2003). “Cellulose stacks up,” Science 426, 611-612.
Jeffries, T. W. (2008). “Introduction of a special issue on biotechnology for the pulp and paper industry,” Enzyme and Microbial Technology 43(2), 77.
Jeoh, T., Ishizawa, C. I., Davis, M. F., Himmel, M. E., Adney, W. S., and Johnson, D. K.
(2007). “Cellulase digestibility of pretreated biomass is limited by cellulose accessibility,” Biotechnology and Bioengineering 98, 112-122.
König, J., Grasser, R., Pikor, H., and Vogel, K. (2002). “Determination of xylanase, beta-glucanase, and cellulase activity,” Analytical and Bioanalytical Chemistry 374, 80-87.
Kamaya, Y. (1996). “Role of endoglucanase in enzymatic modification of bleached kraft pulp,” Journal of Fermentation and Bioengineering 82, 549-553.
Klyosov, A. A. (1990). “Trends in biochemistry and enzymology of cellulose
degradation,” Biochemistry 29, 10577-10585.
Ko, C. H., Chen, W. L., Tsai, C. H., Jane, W. N., Liu, C. C., and Tu, J. (2007).
“Paenibacillus campinasensis BL11: A wood material-utilizing bacterial strain isolated from black liquor,” Bioresource Technology 98, 2727-2733.
Ko, C. H., Lin, Z. P., Tu, J., Tsai, C. H., Liu, C. C., Chen, H. T., and Wang, T. P. (2010a).
“Xylanase production by Paenibacillus campinasensis BL11 and its pretreatment of hardwood kraft pulp bleaching,” International Biodeterioration and Biodegradation 64(1), 13-19.
Ko, C. H., Tsai, C. H., Lin, P. H., Chang, K. C., Tu, J., Wang, Y. N., and Yang, C. Y.
(2010b). “Characterization and pulp refining activity of a Paenibacillus campinasensis cellulase expressed in Escherichia coli,” Bioresource Technology 101(20), 7882-7888.
Ko, C. H., Tsai, C. H., Tu, J. S., Tang, H., and Liu, C. C. (2011). “Expression and thermostability of Paenibacillus campinasensis BL11 pectate lyase and its applications in bast fibre processing,” Annals of Applied Biology 158(2), 218-225.
Kristensen, J. B., Thygesen, L. G., Felby, C., Jorgensen, H., and Elder, T. (2008).
“Cell-wall structural changes in wheat straw pretreated for bioethanol production,”
Biotechnology for Biofuels 1(5).
Kumar, R., and Wyman, C. E. (2009). “Cellulase adsorption and relationship to features of corn stover solids produced by leading pretreatments,” Biotechnology and Bioengineering 103, 252-267.
Lawoko, M., Henriksson, G., and Gellerstedt, G. (2006). “Characterisation of lignin-carbohydrate complexes (LCCs) of spruce wood (Picea abies L.) isolated with two methods,” Holzforschung 60, 156-161.
Lecourt, M., Meyer, V., Sigoillot, J. C., and Petit-Conil, M. (2010). “Energy reduction of refining by cellulases,” Holzforschung 64, 441-446.
Lynd, L. R., Weimer, P. J., van Zyl, W. H., and Pretorius, I. S. (2002). “Microbial cellulose utilization: fundamentals and biotechnology,” Microbiology and Molecular Biology Reviews 66, 506-577.
Maki, M., Leung, K. T., and Qin, W. (2009). “The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass,” International Journal of Biological Sciences 5(5), 500-516.
Mamma, D., Hatzinikolaou, D., Stamatis, H., Kalogeris, D., and Kekos, E. (2009).
“Adsorption of major endoglucanase from Thermoascus aurantiacus on cellulosic substrates,” World Journal of Microbiology and Biotechnology 25, 781-788.
Miller, G. L. (1959). “Use of dinitrosalicylic acid reagent for determination of reducing sugar,” Analytical Biochemistry 31, 426-428.
Mizutani, C., Sethumadhavan, K., Howley, P., and Bertoniere, N. (2002). “Effect of a nonionic surfactant on Trichoderma cellulase treatments of regenerated cellulose and cotton yarns,” Cellulose 9, 83-89.
Mutjé, P., Pèlach, M. A., Vilaseca, F., García, J. C., and Jiménez, L. (2005), “A comparative study of the effect of refining on organosolv pulp olive trimmings and kraft pulp from eucalyptus wood,” Bioresource Technology 96, 1125-1129.
Nidetzky, B., Steiner, W., and Claeyssens, M. (1994), “Cellulose hydrolysis by the cellulases from Trichoderma reesei: adsorptions of two cellobiohydrolases, two endocellulases and their core proteins on filter paper and their relation to hydrolysis,”
Biochemical Journal 303, 817-823.
Oksanen, T, Pere, J., Paavilainen, L., Buchert, J., and Viikari, L. (2000). “Treatment of recycled kraft pulps with Trichoderma reesei hemicellulases and cellulases,” Journal of Biotechnology 78, 39-48.
Ooshima, H., Burns, D. S., and Converse, A. O. (1990). “Adsorption of cellulase from
Trichoderma reesei on cellulose and lignacious residue in wood pretreated by dilute sulfuric-acid with explosive decompression,” Biotechnology and Bioengineering 36, 446-452.
Ouyang, J., Dong, Z., Song, X., Lee, X., Chen, M., and Yong, Q. (2010). “Improved enzymatic hydrolysis of microcrystalline cellulose (Avicel PH101) by polyethylene glycol addition,” Bioresource Technology 101, 6685-6691.
Pala, H, Mota, M., and Gama, F. M. (2002). “Enzymatic modification of paper fibres,”
Biocatalysis and Biotransformation 20, 353-361.
Palonen, H., Tjerneld, F., Zacchi, G., and Tenkanen, M. (2004). “Adsorption of Trichoderma reesei CBH I and EG II and their catalytic domains on steam pretreated softwood and isolated lignin,” Journal of Biotechnology 107, 65-72.
Pason, P., Kosugi, A., Waeonukul, R., Tachaapaikoon, C., Ratanakhanokchai, K., Arai, T., Murata, Y., Nakajima, J., and Mori, Y. (2010). “Purification and characterization of a multienzyme complex produced by Paenibacillus curdlanolyticus B-6,” Applied Microbiology and Biotechnology 85, 573-580.
Pason, P., Kyu, K. L., and Ratanakhanokchai, K. (2006). “Paenibacillus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzyme system that degrades insoluble polysaccharides,” Applied and Environmental Microbiology 72, 2483-2490.
Ramírez, F., Puls, J., Zúñiga, V., and Saake, B. (2008). “Sorption of corn cob and oat spelt arabinoxylan onto softwood kraft pulp,” Holzforschung 62, 329-337.
Reeve, D. W. (1996). “Chlorine dioxide in delignification, in Pulp Bleaching-Principles and Practice,” ed by Dence CW and Reeve DW. Tappi Press, Atlanta, pp. 263-287.
Shi, J., Shi, S. Q., Barnes, H. M., and Pittman, C. U. (2011). “A chemical process for preparing cellulosic fibers hierarchically from kenaf bast fibers,” BioResources 6(1), 879-890.
Sihtola, H., Kyrklund, B., Laamanen, L., and Palenius, I. (1963). “Comparison and conversion of viscosity and DP-values determined by different methods,” Paperi ja Puu 45(4), 225-323.
Silvy, J., Romatier, G., and Chiodi, R. (1968). “Méthodes pratiques de contrôle du raffinage,” Revue ATIP 22, 31-53.
Siqueira, G., Milagres, A. M. F., Carvalho, W., Koch, G., and Ferraz, A. (2011).
“Topochemical distribution of lignin and hydroxycinnamic acids in sugar-cane cell walls and its correlation with the enzymatic hydrolysis of polysaccharides,”
Biotechnology for Biofuels 4(7).
Suchy, M., Hakala, T., Kangas, H., Kontturi, E., Tammelin, T., Pursula, T., and Vuorinen, T. (2009). “Effects of commercial cellobiohydrolase treatment on fiber strength and morphology of bleached hardwood pulp,” Holzforschung 63, 731-736.
Tomme, P., van Tilbeurgh, H., Pettersson, G., van Damme, J., Vandekerckhove, J., Knowles, J., Teeri, T., and Claeyssens, M. (1988). “Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis,” European Journal of Biochemistry 170, 575-581.
Várnai, A., Siika-aho, M., and Viikari, L. (2010). “Restriction of the enzymatic hydrolysis of steam-pretreated spruce by lignin and hemicellulose,” Enzyme and Microbial Technology 46, 185-193.
Várnai, A., Viikari, L., Marjamaa, K., and Siika-aho, M. (2011). “Adsorption of monocomponent enzymes in enzyme mixture analyzed quantitatively during hydrolysis of lignocellulose substrates,” Bioresource Technology 102, 1220-1227.
Verenich, S., Arumugam, K., Shim, E., and Pourdeyhimi, B. (2007). “Effect of cellulase pretreatment of raw and bleached cotton fibers on properties of hydroentangled
nonwoven fabrics,” Journal of Applied Polymer Science 105, 492-499.
Waeonukul, R., Kyu, K. L., Sakka, K., and Ratanakhanokchai, K. (2009). “Isolation and characterization of a multienzyme complex (cellulosome) of the Paenibacillus curdlanolyticus B-6 grown on Avicel under aerobic conditions,” Biotechnology and Bioengineering 107(6), 610-614.
Wang, C. M., Shyu, C. L., Ho, S. P., and Chiou, S. H. (2008). “Characterization of a novel thermophilic, cellulose-degrading bacterium Paenibacillus sp strain B39,”
Letters in Applied Microbiology 47, 46-53.
Wong, K. K. Y., Alison, R. W., and Spehr, S. (2001). “Effects of alkali and oxygen extractions of kraft pulp on xylanase-aided bleaching,” Journal of Pulp and Paper Science 27, 229-234.
Yang, B., and Wyman, C. E. (2004). “Effect of xylan and lignin removal by batch and flow through pretreatment on the enzymatic digestibility of corn stover cellulose,”
Biotechnology and Bioengineering 86, 88-95.
Yang, B., and Wyman, C. E., (2006). “BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates,” Biotechnology and Bioengineering 94, 611-617.
Zhang, Y. H. P., Himmel, M. E., and Mielenz, J. R. (2006). “Outlook for cellulase improvement: screening and selection strategies,” Biotechnology Advances 24, 452-481.
Zhang, Y. H. P., and Lynd, L. R. (2004). “Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems,” Biotechnology and Bioengineering 88, 797-824.
Zheng, Y., Pan, Z. L., Zhang, R. H., Wang, D. H., and Jenkins, B. (2008). “Non-ionic surfactants and non-catalytic protein treatment on enzymatic hydrolysis of pretreated
creeping wild ryegrass,” Applied Biochemistry and Biotechnology 146, 231-248.
Zhang, Y., Zhang, Y., and Tang, L. (2010). “Effect of PEG4000 on cellulase catalysis in the lignocellulose saccharification processes,” Journal of Chemical Technology and Biotechnology 86, 115-120.