IJRRAS 6 (2) ● February 2011 www.arpapress.com/Volumes/Vol6Issue2/IJRRAS_6_2_02.pdf 132 OPTIMIZATION OF CULTURE CONDITIONS FOR THE PRODUCTION OF XYLANASE IN SUBMERGE FERMENTATION BY PENICILLIUM CITRINUM USING RESPONSE SURFACE METHODOLOGY Gargi Ghoshal 1 , Ashwini Kamble 2 , U.S. Shivhare 3* & U.C. Banerjee 4 1.3 University Institute of Chemical Engineering and Technology, Panjab University, Chandigarh - 160014, India 2,4 Department of Pharmaceutical Technology, National Institute of Pharmaceutical Education and Research, SAS Nagar-160062, Punajb, India. Tel: +91172 2534906; Fax +91 172 2779173, *Email:usshiv@yahoo.com ABSTRACT In the present study Response surface methodology (RSM) was used to investigate the combined effect of relevant process variables to maximize the production of xylanase in submerge fermentation by Penicillium citrinum MTCC 2553. The process variables include pH (6.5, 7.0, and 7.5); temperature (25, 30, and 35°C); agitation speed (190, 200, and 210 rpm); and, substrate (xylan) concentration (0.70%, 0.75%, and 0.80%). A 2 4 factorial central composite design (CCD) using response surface methodology (RSM) was employed to obtain interaction between the process variables and optimizing these variables. Total 22 experiments were carried out in shake flask and a three dimensional response surface was generated to determine the effect of process variables on xylanase production. The optimal calculated values of tested variables for maximal production of xylanase were: pH 7.49, temperature 29°C, agitation speed 209 rpm, and substrate concentration of 0.75%. This approach for process parameter optimization yielded enhance xylanase activity by three - folds compared to the unoptimized media. Key words: Xylanase; Penicillium citrinum MTCC 2553; RSM; CCD; Submerge fermentation; Process parameter optimization. 1. INTRODUCTION Xylanases are glycosidases (o-glycoside hydrolases, EC 3.2.1.8) which catalyse the endohydrolysis of 1,4-β-D- xylosidic linkages in xylan in a random manner and are extensively used in paper pulp, food, and animal feed industry. Xylan is a heterogeneous carbohydrate of β 1,4 linked D-xylopyranose units and short chain branches of O-acetyl, α L-arabinofuranosyl and α D-glucuronyl residues. Xylan is the major component of hemicelluloses which is the second most abundant renewable resources in nature. Monocotyls contain about 40% hemicelluloses whereas soft wood and hard wood contain 15-25% and 25-32% hemicelluloses respectively [1]. Conversions of hemicelluloses to valuable products by xylanases hold strong promise for the degradation of a variety of unutilized or underutilized agricultural residues for industrial applications including hydrolysis of lignocelluloses to fermentable sugars for fuel ethanol production, bread making, and clarification of beer and fruit juices [2]. Xylanases derived from microorganisms have immense potential applications in the food, feed, and paper pulp industries. Xylanases are produced by prokaryotes and eukaryotes. A large number of bacteria and fungi are known to produce xylanases [3-5]. Filamentous fungi are the most appropriate producer of xylanases and other xylan degrading enzymes as it gives higher yield compared to bacteria and yeast. As xylanases are extracellular enzymes, from industrial point of view, filamentous fungi are the most suitable one to produce xylanases and other xylan degrading enzymes. In the present investigation, we report the production of xylanases by P. citrinum MTCC 2553 in submerged fermentation (SmF). Preliminary selection of the suitable medium formulation for xylanases by P. citrinum MTCC 2553 was conducted in SmF. Initially nutrient broth (NB) media, Mandels & Sternburg’s basal (MS) media and Czapek yeast extract (CYE) media were used as growth media for P.citrinum MTCC 2553. The organism exhibited better activity in CYE media. Further experiments were conducted using the CYE media. Subsequently, the feasibility of using different levels of initial pH, temperature, agitation speed, and substrate concentration combinations were also investigated. Optimization of media and process conditions are the most important factors to reduce the production cost. In preliminary study, optimization of xylanase production was done using conventional method, which involved varying one variable at a time while keeping the other variables constant. This method is lengthy and often does not produce the effect of interaction of different variables. To overcome this difficulty, response surface methodology (RSM) was used to optimize the media composition and few process variables [6-8]. RSM is the most commonly used statistical practice for bioprocess optimization. RSM is a compilation of numerical and statistical techniques useful for analyzing the effect of several independent variables. The process consists of a