Received: 26 January, 2011. Accepted: 2 April, 2011. Original Research Paper Dynamic Biochemistry, Process Biotechnology and Molecular Biology ©2011 Global Science Books Application of Response Surface Methodology (RSM) for Optimization of Physico-chemical Parameters for the Production of Endoglucanase by Trichoderma ressei Rut C-30 using Agro-residues Vishal Kapoor 1* • Renu Singh 1 • Rintu Banerjee 2 • Vijay Kumar 1 1 Laboratory of Microbiology, Christ Church College, Kanpur, 208001, India 2 Microbial Biotechnology and Downstream Processing Laboratory, Agricultural and Food Engineering Department, Indian Institute of Technology, Kharagpur, West Bengal, 721302, India Corresponding author: * vishal.262570@gmail.com ABSTRACT Response surface methodology (RSM) involving central composite design (CCD) was employed to optimize the physico-chemical parameters for the production of endoglucanase by Trichoderma ressei Rut C-30 under solid state fermentation using a novel mixture of waste paper and wheat bran. Most effective variables for the endoglucanase production in screening experiments were incubation day, substrate ratio, solid: liquid ratio and pH of the medium. A quadratic model was developed through RSM in terms of related independent variables to maximize the endoglucanase production as the response. Incubation day and solid: liquid ratio were found to be the most significant factors. The predicted optimal parameters were tested in the laboratory and the final endoglucanase activity obtained was very close to the predicted value (22.93 IU/g, predicted; 25.43 IU/g, tested). After optimization, endoglucanase activity increased by ~1.77-fold. Our result shows that optimization of enzyme production is the most useful way to obtain concentrated enzyme extracts from solid state cultivation and that T. ressei Rut C-30 using cheap agro-residues can be an attractive source for endoglucanase production. _____________________________________________________________________________________________________________ Keywords: agro-residues, endoglucanase, response surface methodology, solid-state fermentation, Trichoderma reesei Rut C-30 Abbreviations: CCD, central composite design; CMC, carboxymethyl cellulose; RRL, Regional Research Laboratory; RSM, response surface methodology; SSF, solid state fermentation; SmF, submerged fermentation INTRODUCTION Lignocellulose is a potentially valuable source of energy. It is composed of holocellulose bound to lignin. Different plant materials contain various ratios of cellulose, hemicel- lulose and lignin (Sánchez 2009). Lignocellulolytic micro- organisms produce cellulases and hemicellulases that hyd- rolyze plant lignocelluloses (Esterbauer et al. 1991; Nieves et al. 1998; Jørgensen et al. 2003). These microorganisms and their enzymes are being used for valorisation of plant residues together with many other industrial and environ- mental applications (Bhat 2000). Currently the most impor- tant applications of cellulases are the production of dis- solving pulp, treatment of wastewater, deinking of recycled waste paper in the paper and pulp industry and in the enzy- matic saccharification of lignocellulosics present in agricul- tural residues (Ko et al. 2009; Sukumaran et al. 2009). Cellulolytic enzyme systems can be produced by a number of different microorganisms, such as aerobic and anaerobic bacteria (Gilkes et al. 1991), white rot fungi (Uzcategui et al. 1991), soft rot fungi (Wood et al. 1988) and anaerobic fungi (Barichievic and Calza 1990). T. reesei Rut C-30 culture is the most widely utilized soft rot fungus that has been employed for the production of various cel- lulases using various agro-residues (Chahal et al. 1982; Singhania et al. 2006). High cost of production and low yield of the enzyme are the major impediments for their industrial application. Therefore, investigations on the ability of the cellulose hydrolyzing microbial strains to utilize inexpensive sub- strate have been under taken. A little work has been directed for improvement of the fermentation processes (Esterbauer et al. 1991; Haltrich et al. 1996). Solid state fermentation (SSF) offers advantages over submerged fermentation (SmF), such as in the nature of the substrates, low media cost, higher productivity, less effort in downstream pro- cessing, reduced energy and cost requirements. Aeration also tends to require lower pressure than that needed for liquid cultivation (Smits et al. 1996). Although there have been many studies regarding endo- glucanase (EC 3.2.1.4) production using Trichoderma strains, a complete optimization of operational conditions for the production of the enzyme has received little atten- tion in the literature (Reczey et al. 1996; Gerber et al. 1997). The effect of particle size, aeration rate and harvest time for the production of cellulase by T. reesei QM 9414 was eval- uated through RSM by Rocky-Salimi and Hamidi-Esfahani (2010). Optimization of such processes with RSM may pro- vide great benefits in the production of endoglucanase. RSM is a combination of mathematical and statistical techniques that is useful in analyzing the effects of several independent variables on the system response without the need of a predetermined relationship between the objective function and the variables (Draper and John 1988; Draper and Lin 1990; Myers and Montgomery 2002; Tanyildizi 2005). The present study was performed to investigate and optimize the conditions for endoglucanase production em- ploying agro-residues (waste paper and wheat bran) through RSM for batch runs designed with composite central design (CCD). Four factors, viz., incubation day, substrate ratio, solid: liquid ratio and medium pH were selected as process (independent) parameters while endoglucanase activity was ®