Effect of physical parameters such as initial pH, agitation (rpm), and temperature (°C) for cellulase production from Bacillus subtilis AS3 was investigated. Central composite design of experiments followed by multiple desirability function was applied for the optimization of cellulase activity and cell growth. The effect of the temperature and agitation was found to be significant among the three independent variables. The optimum levels of initial pH, temperature, and agitation for alkaline carboxymethylcellulase (CMCase) production predicted by the model were 7.2, 39°C, and 121？rpm, respectively. The CMCase activity with unoptimized physical parameters and previously optimized medium composition was 0.43？U/mL. The maximum activity (0.56？U/mL) and cell growth (2.01？mg/mL) predicted by the model were in consensus with values (0.57？U/mL, 2.1？mg/mL) obtained using optimized medium and optimal values of physical parameters. After optimization, 33% enhancement in CMCase activity (0.57？U/mL) was recorded. On scale-up of cellulase production process in bioreactor with all the optimized conditions, an activity of 0.75？U/mL was achieved. Consequently, the bacterial cellulase employed for bioethanol production expending (5%, w/v) NaOH-pretreated wild grass with Zymomonas mobilis yielded an utmost ethanol titre of 7.56？g/L and 11.65？g/L at shake flask and bioreactor level, respectively. 1. Introduction Cellulases have versatile applications in textile, laundry, pulp and paper, fruit juice extraction, and animal feed additives [1]. In addition, they find use in saccharification of lignocellulosic agroresidues to fermentable sugars which can be used for production of bioethanol, lactic acid, and single-cell protein [2]. Bacteria have been widely explored for cellulase production owing to their high growth rate, expression of multienzyme complexes, stability at extreme temperature and pH, lesser feedback inhibition, and ability to withstand variety of environmental stress [1]. Among them, Bacillus sp. continues to be dominant bacterial workhorse due to the capacity to produce and secrete large quantities of extracellular enzymes [3, 4]. However, physical process parameters such as temperature, pH, and agitation speed play a vital role for the cellulase production efficiency of the microorganisms. Agitation speed is an important factor which governs the dissolved oxygen level in the culture broth that affects cell growth of cellulase producing microorganism [5]. However, higher agitation speed has been shown to inhibit cellulase activity [5, 6]. Analogous profile in
References
[1]
M. K. Bhat, “Cellulases and related enzymes in biotechnology,” Biotechnology Advances, vol. 18, no. 5, pp. 355–383, 2000.
[2]
O. J. Sánchez and C. A. Cardona, “Trends in biotechnological production of fuel ethanol from different feedstocks,” Bioresource Technology, vol. 99, no. 13, pp. 5270–5295, 2008.
[3]
C. Mawadza, F. C. Boogerd, R. Zvauya, and H. W. Van Verseveld, “Influence of environmental factors on endo-β-1,4-glucanase production by Bacillus HR68, isolated from a Zimbabwean hot spring,” Antonie van Leeuwenhoek, vol. 69, no. 4, pp. 363–369, 1996.
[4]
G. Rastogi, G. L. Muppidi, R. N. Gurram et al., “Isolation and characterization of cellulose-degrading bacteria from the deep subsurface of the Homestake gold mine, Lead, South Dakota, USA,” Journal of Industrial Microbiology and Biotechnology, vol. 36, no. 4, pp. 585–598, 2009.
[5]
K. I. Jo, Y. J. Lee, B. K. Kim et al., “Pilot-scale production of carboxymethylcellulase from rice hull by Bacillus amyloliquefaciens DL-3,” Biotechnology and Bioprocess Engineering, vol. 13, no. 2, pp. 182–188, 2008.
[6]
R. Lejeune and G. V. Baron, “Effect of agitation on growth and enzyme production of Trichoderma reesei in batch fermentation,” Applied Microbiology and Biotechnology, vol. 43, no. 2, pp. 249–258, 1995.
[7]
Z. A. Chipeta, J. C. Du Preez, and L. Christopher, “Effect of cultivation pH and agitation rate on growth and xylanase production by Aspergillus oryzae in spent sulphite liquor,” Journal of Industrial Microbiology and Biotechnology, vol. 35, no. 6, pp. 587–594, 2008.
[8]
B. H. Lee, B. K. Kim, Y. J. Lee, C. H. Chung, and J. W. Lee, “Industrial scale of optimization for the production of carboxymethylcellulase from rice bran by a marine bacterium, Bacillus subtilis subsp. subtilis A-53,” Enzyme and Microbial Technology, vol. 46, no. 1, pp. 38–42, 2010.
[9]
M. Latifian, Z. Hamidi-Esfahani, and M. Barzegar, “Evaluation of culture conditions for cellulase production by two Trichoderma reesei mutants under solid-state fermentation conditions,” Bioresource Technology, vol. 98, no. 18, pp. 3634–3637, 2007.
[10]
D. Deka, P. Bhargavi, A. Sharma, D. Goyal, M. Jawed, and A. Goyal, “Enhancement of cellulase activity from a new strain of Bacillus subtilis by medium optimization and analysis with various cellulosic substrates,” Enzyme Research, vol. 2011, Article ID 151656, 8 pages, 2011.
[11]
S. Nagar, V. K. Gupta, D. Kumar, L. Kumar, and R. C. Kuhad, “Production and optimization of cellulase-free, alkali-stable xylanase by Bacillus pumilus SV-85S in submerged fermentation,” Journal of Industrial Microbiology and Biotechnology, vol. 37, no. 1, pp. 71–83, 2010.
[12]
M. Tagaki, S. Abe, G. H. Emert, S. Suzuki, and N. Yata, “A method for the production of alcohol directly from cellulose using cellulase and yeast,” in Proceedings of Bioconversion of Cellulosic Substances Into Energy, Chemicals and Microbial Protein, pp. 551–571, 1977.
[13]
R. L. Howard, E. Abotsi, E. L. J. Van Rensburg, and S. Howard, “Lignocellulose biotechnology: issues of bioconversion and enzyme production,” African Journal of Biotechnology, vol. 2, no. 12, pp. 702–733, 2003.
[14]
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.
[15]
D. da Silveira dos Santos, A. C. Camelo, K. C. P. Rodrigues, L. C. Carlos, and N. Pereira Jr., “Ethanol production from sugarcane bagasse by Zymomonas mobilis using Simultaneous Saccharification and Fermentation (SSF) process,” Applied Biochemistry and Biotechnology, vol. 161, no. 1–8, pp. 93–105, 2010.
[16]
G. Derringer and R. Suich, “Simultaneous determination of several response variables,” Journal Quality Technology, vol. 12, no. 4, pp. 214–219, 1980.
[17]
E. C. Harrington, “The desirability function,” Industrial Quality Control, vol. 21, no. 10, pp. 494–498, 1965.
[18]
B. C. Okeke and S. K. C. Obi, “Saccharification of agro-waste materials by fungal cellulases and hemicellulases,” Bioresource Technology, vol. 51, no. 1, pp. 23–27, 1995.
[19]
Y. Sun and J. Cheng, “Hydrolysis of lignocellulosic materials for ethanol production: a review,” Bioresource Technology, vol. 83, no. 1, pp. 1–11, 2002.
[20]
N. Nelson, “A photometric adaptation of the Somogyi method for the determination of glucose,” Journal of Biological Chemistry, vol. 153, pp. 375–380, 1944.
[21]
M. Somogyi, “A new reagent for the determination of sugars,” Journal of Biological Chemistry, vol. 160, pp. 61–68, 1945.
[22]
H. B. Seo, H. J. Kim, O. K. Lee, J. H. Ha, H. Y. Lee, and K. H. Jung, “Measurement of ethanol concentration using solvent extraction and dichromate oxidation and its application to bioethanol production process,” Journal of Industrial Microbiology and Biotechnology, vol. 36, no. 2, pp. 285–292, 2009.
[23]
M. S. Tanyildizi, D. ？zer, and M. Elibol, “Optimization of α-amylase production by Bacillus sp. using response surface methodology,” Process Biochemistry, vol. 40, no. 7, pp. 2291–2296, 2005.
[24]
S. Singh, J. C. Du Preez, B. Pillay, and B. A. Prior, “The production of hemicellulases by Thermomyces lanuginosus strain SSBP: influence of agitation and dissolved oxygen tension,” Applied Microbiology and Biotechnology, vol. 54, no. 5, pp. 698–704, 2000.
[25]
G. Rastogi, A. Bhalla, A. Adhikari et al., “Characterization of thermostable cellulases produced by Bacillus and Geobacillus strains,” Bioresource Technology, vol. 101, no. 22, pp. 8798–8806, 2010.
[26]
H. Purkarthofer, M. Sinner, and W. Steiner, “Effect of shear rate and culture PH on the production of xylanase by Thermomyces lanuginosus,” Biotechnology Letters, vol. 15, no. 4, pp. 405–410, 1993.
[27]
A. K. Ray, A. Bairagi, K. Sarkar Ghosh, and S. K. Sen, “Optimization of fermentation conditions for cellulase production by Bacillus subtilis CY5 and Bacillus circulans TP3 isolated from fish gut,” Acta Ichthyologica et Piscatoria, vol. 37, no. 1, pp. 47–53, 2007.
[28]
H. Ariffin, M. A. Hassan, U. K. M. Shah, N. Abdullah, F. M. Ghazali, and Y. Shirai, “Production of bacterial endoglucanase from pretreated oil palm empty fruit bunch by Bacillus pumilus EB3,” Journal of Bioscience and Bioengineering, vol. 106, no. 3, pp. 231–236, 2008.
[29]
M. Sohail, R. Siddiqi, A. Ahmad, and S. A. Khan, “Cellulase production from Aspergillus niger MS82: effect of temperature and pH,” New Biotechnology, vol. 25, no. 6, pp. 437–441, 2009.
[30]
M. I. Rajoka, “Influence of various fermentation variables on exo-glucanase production in Cellulomonas flavigena,” Electronic Journal of Biotechnology, vol. 7, no. 3, pp. 256–263, 2004.
[31]
Y. Liang, Z. Feng, J. Yesuf, and J. W. Blackburn, “Optimization of growth medium and enzyme assay conditions for crude cellulases produced by a novel thermophilic and cellulolytic bacterium, Anoxybacillus sp. 527,” Applied Biochemistry and Biotechnology, vol. 160, no. 6, pp. 1841–1852, 2010.
[32]
N. Beukes and B. I. Pletschke, “Effect of sulfur-containing compounds on Bacillus cellulosome-associated CMCase and Avicelase activities,” FEMS Microbiology Letters, vol. 264, no. 2, pp. 226–231, 2006.
[33]
M. Jahani, M. Alizadeh, M. Pirozifard, and A. Qudsevali, “Optimization of enzymatic degumming process for rice bran oil using response surface methodology,” Food Science and Technology, vol. 41, no. 10, pp. 1892–1898, 2008.
[34]
X. H. Júlio, P. F. Hertz, and M. A. Z. Ayub, “Cellulase and xylanase production by isolated amazon Bacillus strains using soybean industrial residue based solid-state cultivation,” Brazilian Journal of Microbiology, vol. 33, no. 3, pp. 213–218, 2002.
[35]
Y. Yamashita, A. Kurosumi, C. Sasaki, and Y. Nakamura, “Ethanol production from paper sludge by immobilized Zymomonas mobilis,” Biochemical Engineering Journal, vol. 42, no. 3, pp. 314–319, 2008.
[36]
E. Sasikumar T and Viruthagiri, “Simultaneous Saccharification and Fermentation (SSF) of sugarcane bagasse-kinetics and modeling,” International Journal of Chemical and Biological Engineering, vol. 3, no. 2, pp. 57–64, 2010.
