Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 16, No. (1) : 2014 : 63-70 © Global Science Publications ISSN-0972-3005 *Corresponding author’s email: btrbio@gmail.com OPTIMIZATION OF BIODIESEL PRODUCTION FROM WASTE COOKING OIL USING PURE LIPASE AND RHIZOPUS ORYZAE THROUGH RESPONSE SURFACE METHODOLOGY B. BHARATHIRAJA 1 *, A. SARAVANA RAJ 1 , J. JAYAMUTHUNAGAI 2 , M. JAYAKUMAR 3 , M. ARUL KIRUBAKARAN 1 , P. VIVEK 1 , R. PRAVEEN KUMAR 4 AND S. PALANI 4 1 Department of BioTechnology, Vel Tech High Tech Dr. Rangarajan Dr. Sakunthala Engineering College, Avadi, Chennai, India 2 Center for BioTechnology, Anna University, Chennai, India 3 Department of Basic Sciences, Sri Ramana Maharishi College of Engineering, Cheyar, Kancheepuram, India 4 Department of BioTechnology, Arunai Engineering College, Tiruvannamalai, 606 603, India (Received : 28 August, 2013; accepted : 30 September, 2013) Key words : Response surface methodology, Transesterification, Fatty acid alkyl esters, Acyl acceptor Abstract–Biodiesel is the Fatty Acid Alkyl Ester of vegetable oils produced through transesterification reaction. Immobilized pure lipase and Rhizopus oryzae was used as the catalyst for the conversion of Waste Cooking Oil (WCO) into biodiesel using methanol as an acyl acceptor. Response surface methodology (RSM) was adopted to optimize the parameters such as temperature, time, oil to solvent ratio, enzyme concentration for lipase and pH, olive oil concentration, rpm for Rhizopus oryzae. A high conversion of 94.01% was obtained through lipase catalyzed reaction at oil to solvent ratio of 1:3, enzyme concentration of 10% at 30 o C after 24 hrs. Similarly, a high conversion of 83.76% was obtained at an optimum pH of 5.5, olive oil concentration 25g/L and 150 rpm was obtained for Rhizopus oryzae. The lipase resulted in higher yield of biodiesel when compared to the Rhizopus oryzae. Thus the influential parameters were optimized using RSM. INTRODUCTION The use of fossil fuels and its depletion has become a major threat to today’s world. Most of the vehicles rely on diesel as a source of energy. But their continuous use and less availability has increased their demands and led to their price rise. Hence the need for an alternative fuel (biodiesel) would be necessary to meet the energy needs. The production of biodiesel by chemical methods using oil and acid base system is costly, as it will involve major downstream processing steps for the purification of biodiesel and glycerol. The best alternative is biological method using whole cell biocatalyst and purified enzymes. The production of biodiesel has been practiced by several countries like Belgium, France, Italy, Germany and United States (Fukuda et al., 2007).The synthesis of biodiesel has been done by a process called as transesterification. It is a three step reaction process catalyzed by chemical catalyst or biological /biocatalyst. Lipase will retain its catalytic activity for more than 100 days (Shimada et al., 2002). The common biological catalyst used is the enzyme lipase, which catalyses transesterification and esterification reaction resulting in the synthesis / production of biodiesel commonly known as Fatty Acid Alkyl Esters (FAAE).The various fungi that produces enzyme lipase are Candida Antarctica (Novozyme 435), C.rogosa , Psuedomonas cepacia , P.fluroscens , Rhizopus oryzae , R. Chinensis , Thermomyces lanuginosus , Penicillium roqueforti , P.camembertii, Mucor javanicus (Salis et al., 2008; Qin et al., 2008). In our study, both the purified lipase and the wild R.O. strain are immobilized through entrapment method in calcium alginate beads. This increases the specificity of the enzyme action and