The present work investigated the use of sorbitol as a soluble carbon source, in association with cellulose, to produce cellulases and xylanases in submerged cultures of Penicillium echinulatum 9A02S1. Because cellulose is an insoluble carbon source, in cellulase production, there are some problems with rheology and oxygen transfer. The submerged fermentations containing media composed of 0, 0.25, 0.5, 0.75, and 1% (w/v) sorbitol and cellulose that were added at different times during the cultivation; 0.2% (w/v) soy bran; 0.1% (w/v) wheat bran; and a solution of salts. The highest filter paper activity (FPA) ( ？IU·mL？1) was obtained on the seventh day in the medium containing 0.5% (w/v) sorbitol and 0.5% (w/v) cellulose added 24 h after the start of cultivation. However, the CMCases showed an activity peak on the sixth day ( ？IU·mL？1) in the medium containing 0.75% (w/v) sorbitol and 0.75% (w/v) cellulose added after 12 h of cultivation. The xylanases showed the highest activity in the medium with 0.75% (w/v) sorbitol and 0.25% (w/v) cellulose added 36 h after the start of cultivation. This strategy enables the reduction of the cellulose concentration, which in high concentrations can cause rheological and oxygen transfer problems. 1. Introduction Lignocellulosic biomass has been projected to be one of the main resources for economically attractive bioethanol production, and enzymatic hydrolysis is the most potent alternative process for the saccharification of its polymers. Cellulase is an enzyme complex capable of hydrolyzing cellulose into glucose molecules , and xylanases degrade xylan, the main carbohydrate present in some hemicelluloses, into xylose . Although cellulases and xylanases have several industrial uses, the greatest potential use of these enzymes is in the enzymatic hydrolysis of lignocellulosic materials to produce second-generation ethanol . The cellulase complex has three major hydrolases: the endo- -1,4-glucanases (EG I, EG II, EG III, EG IV, and EG V; EC 22.214.171.124), which hydrolyze the glucosidic bonds randomly in cellulose fiber; the exo- -1,4-glucanases or cellobiohydrolases (CBH I and CBH II; EC 126.96.36.199), which act on the reducing and nonreducing ends of polymers, releasing cellobiose; and the -1,4-glucosidases (BG I and BG II; EC 188.8.131.52), which hydrolyze oligosaccharides and cellobiose into glucose . The xylanolytic complex capable of hydrolyzing xylan is usually composed of several enzymes, such as -1,4-endoxylanase, -xylosidase, -L-arabinofuranosidase, -glucuronidase, acetyl xylan esterase, and the phenolic,
J. Pérez, J. Mu？oz-Dorado, T. de la Rubia, and J. Martínez, “Biodegradation and biological treatments of cellulose, hemicellulose and lignin: an overview,” International Microbiology, vol. 5, no. 2, pp. 53–63, 2002.
H. Xiong, N. von Weymarn, O. Turunen, M. Leisola, and O. Pastinen, “Xylanase production by Trichoderma reesei Rut C-30 grown on L-arabinose-rich plant hydrolysates,” Bioresource Technology, vol. 96, no. 7, pp. 753–759, 2005.
M. Chandra, A. Kalra, P. K. Sharma, H. Kumar, and R. S. Sangwan, “Optimization of cellulases production by Trichoderma citrinoviride on marc of Artemisia annua and its application for bioconversion process,” Biomass and Bioenergy, vol. 34, no. 5, pp. 805–811, 2010.
M. Hrmová, E. Petráková, and P. Biely, “Induction of cellulose- and xylan-degrading enzyme systems in Aspergillus terreus by homo- and heterodisaccharides composed of glucose and xylose,” Journal of General Microbiology, vol. 137, no. 3, pp. 541–547, 1991.
E. Margolles-Clark, M. Ilmén, and M. Penttil？, “Expression patterns of ten hemicellulase genes of the filamentous fungus Trichoderma reesei on various carbon sources,” Journal of Biotechnology, vol. 57, no. 1–3, pp. 167–179, 1997.
N. Aro, A. Saloheimo, M. Ilmén, and M. Penttil？, “ACEII, a novel transcriptional activator involved in regulation of cellulase and xvlanase genes of Trichoderma reesei,” Journal of Biological Chemistry, vol. 276, no. 26, pp. 24309–24314, 2001.
R. K. Sukumaran, R. R. Singhania, G. M. Mathew, and A. Pandey, “Cellulase production using biomass feed stock and its application in lignocellulose saccharification for bio-ethanol production,” Renewable Energy, vol. 34, no. 2, pp. 421–424, 2009.
P. Vermette and A. Ahamed, “Culture-based strategies to enhance cellulase enzyme production from Trichoderma reesei RUT-C30 in bioreactor culture conditions,” Biochemical Engineering Journal, vol. 40, no. 3, pp. 399–407, 2008.
A. J. P. Dillon, S. O. Paesi-Toresan, and L. P. Barp, “Isolation of cellulase-producing mutants from a Penicillium sp strain denominated 3MUV24,” Revista Brasileira de Genetica, vol. 15, no. 3, pp. 491–498, 1992.
A. J. P. Dillon, C. Zorgi, M. Camassola, and J. A. P. Henriques, “Use of 2-deoxyglucose in liquid media for the selection of mutant strains of Penicillium echinulatum producing increased cellulase and β-glucosidase activities,” Applied Microbiology and Biotechnology, vol. 70, no. 6, pp. 740–746, 2006.
M. Camassola and A. J. P. Dillon, “Production of cellulases and hemicellulases by Penicillium echinulatum grown on pretreated sugar cane bagasse and wheat bran in solid-state fermentation,” Journal of Applied Microbiology, vol. 103, no. 6, pp. 2196–2204, 2007.
M. Camassola, L. Ramos de Bittencourt, N. T. Shenem, J. Andreaus, and A. J. Pinheiro Dillon, “Characterization of the cellulase complex of Penicillium echinulatum,” Biocatalysis and Biotransformation, vol. 22, no. 5-6, pp. 391–396, 2004.
L. F. Martins, D. Kolling, M. Camassola, A. J. P. Dillon, and L. P. Ramos, “Comparison of Penicillium echinulatum and Trichoderma reesei cellulases in relation to their activity against various cellulosic substrates,” Bioresource Technology, vol. 99, no. 5, pp. 1417–1424, 2008.
M. Ilmén, A. Saloheimo, M.-L. Onnela, and M. E. Penttil？, “Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei,” Applied and Environmental Microbiology, vol. 63, no. 4, pp. 1298–1306, 1997.
X. Sun, Z. Liu, K. Zheng, X. Song, and Y. Qu, “The composition of basal and induced cellulase systems in Penicillium decumbens under induction or repression conditions,” Enzyme and Microbial Technology, vol. 42, no. 7, pp. 560–567, 2008.
H. J？rgensen, A. M？rkeberg, K. B. R. Krogh, and L. Olsson, “Production of cellulases and hemicellulases by three Penicillium species: effect of substrate and evaluation of cellulase adsorption by capillary electrophoresis,” Enzyme and Microbial Technology, vol. 36, no. 1, pp. 42–48, 2005.
J. C. Carle-Urioste, J. Escobar-Vera, S. El-Gogary et al., “Cellulase induction in Trichoderma reesei by cellulose requires its own basal expression,” Journal of Biological Chemistry, vol. 272, no. 15, pp. 10169–10174, 1997.
A. Illanes, J. C. Gentina, and M. P. Marchese, “Production and stabilization of cellulases from Trichoderma reesei,” MIRCEN Journal of Applied Microbiology and Biotechnology, vol. 4, no. 4, pp. 407–414, 1988.
C. P. Kubicek and M. E. Penttil？, “Regulation of production of plant polysaccharide degrading enzymes by Trichoderma,” in Trichoderma and Gliocladium, Enzymes, Biological Control and Commercial Applications, G. E. Harman and C. P. Kubicek, Eds., pp. 49–67, Taylor & Francis, Bristol, UK, 1999.
S. J. B. Duff, D. G. Cooper, and O. M. Fuller, “Effect of colloidal materials on cellulase production by Trichoderma reesei Rut-C30,” Applied and Environmental Microbiology, vol. 49, no. 4, pp. 934–938, 1985.
X. Fang, S. Yano, H. Inoue, and S. Sawayama, “Strain improvement of Acremonium cellulolyticus for cellulase production by mutation,” Journal of Bioscience and Bioengineering, vol. 107, no. 3, pp. 256–261, 2009.
T. K. Ghose and V. Sahai, “Production of cellulases by Trichoderma reesei QM 9414 in fed-batch and continuous-flow culture with cell recycle,” Biotechnology and Bioengineering, vol. 21, no. 2, pp. 283–296, 1979.
E. A. Ximenes, B. S. Dien, M. R. Ladisch, N. Mosier, M. A. Cotta, and X.-L. Li, “Enzyme production by industrially relevant fungi cultured on coproduct from corn dry grind ethanol plants,” Applied Biochemistry and Biotechnology, vol. 137–140, no. 1–12, pp. 171–183, 2007.
P. Richard, M. Putkonen, R. V？？n？nen, J. Londesborough, and M. Penttil？, “The missing link in the fungal L-arabinose catabolic pathway, identification of the L-xylulose reductase gene,” Biochemistry, vol. 41, no. 20, pp. 6432–6437, 2002.