Enzyme and Microbial Technology 45 (2009) 188–194 Contents lists available at ScienceDirect Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt Solvent-free methanolysis of canola oil in a packed-bed reactor with use of Novozym 435 plus loofa M. Hajar a , S. Shokrollahzadeh b , F. Vahabzadeh a,∗ , A. Monazzami a a Food Process Engineering and Biotechnology Research Center, Department of Chemical Engineering, Amirkabir University of Technology (Tehran Polytechnic), #424 Hafez Ave., Tehran, Iran b Department of Chemical Industries, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran article info Article history: Received 27 January 2009 Received in revised form 13 April 2009 Accepted 29 May 2009 Keywords: Methanolysis Biodiesel Packed-bed reactor Novozym 435 Loofa Canola oil Response surface methodology abstract Enzymatic methanolysis of canola oil in the solvent-free system was studied in a packed-bed reactor (PBR) using small pieces of loofa plus Novozym 435. Response surface methodology (RSM) was applied to determine the effect of the transesterification conditions, namely flow rate of substrate (x 1 ), temperature (x 2 ) and methanol to canola oil molar ratio (x 3 ) as the regressors, on the methyl ester production. A central composite design (CCD) was employed to optimize the reaction. A second-order polynomial multiple regression model was chosen and analysis of variance (ANOVA) showed a high coefficient of determination (R 2 ) value of 0.996, thus adjustment of the model with experimental data was ensured. The methyl ester yield increased as the flow rate of the reaction mixture in the PBR increased from its low to the middle level thereafter, increasing the flow rate corresponded to decreasing the yield. The same trends of changes were observed for the other two factors. The optimum process conditions for biodiesel production in the PBR were found to be: x 1 = 6.3 mL/min, x 2 = 38 ◦ C and x 3 = 4.3. The same batch was successfully used repeatedly in the PBR for six enzymatic cycles (432h), where the methyl ester yield was maintained above 97%. © 2009 Elsevier Inc. All rights reserved. 1. Introduction Products of alcoholysis of vegetable oils are esters of fatty acids among them methyl ester, which is characterized as a well-known biodiesel, are currently subject of interest because of biodegrad- ability, having non-toxic behavior, and low emission as compared to that of fossil diesel fuel [1]. Several difficulties exist in chemical transesterification, problems in: glycerol recovery from the reaction mixture, release of used catalyst and salts to the environment, and high energy consumption rate [2,3]. Extensive researches thus have been carried out on enzymatic production of biodiesel to present this process as an appropriate alternative to the chemical methods [4,5]. In general, packed-bed reactor permits repeated use of enzymes while this configuration of the bioreactor provides better atmo- sphere for biocatalysts damages since the shear stresses in lower compared to that of the agitated bioreactors [6]. High porosity, low density and high pore volume of loofa sponge have made this lignocellulosic material acts positively in cells and/or enzyme immobilization areas [7]. Considering characteristics listed above, small pieces of loofa sponges plus Novozym 435 were used to pack the PBR in the present study. ∗ Corresponding author. Tel.: +98 21 64543161; fax: +98 21 66405847. E-mail address: far@aut.ac.ir (F. Vahabzadeh). For modeling and optimizing of a particular process of inter- est, chemical or biochemical, use of response surface methodology (RSM) as a collection of statistical and mathematical techniques has been suggested. RSM uses a design of experiment such as the cen- tral composite design (CCD) to fit a mathematical model by least squares techniques in order to define more appropriately quanti- tative relationships between several different factors in a system under study [8]. In the present work, methanolysis of canola oil in the solvent- free system in a packed-bed reactor (PBR) with use of Novozym 435 (commercially immobilized form) plus loofa sponge (small pieces), has been monitored. Results of the study were indicative of loofa and enzyme together as the packing system of the reactor, performed well in the process of biodiesel production. Applica- tion of RSM facilitated development of the mathematical model to describe quantitative mutual relationships exist between three independent variables, namely molar ratio of methanol to canola oil, reaction temperature, and substrate flow rate while determina- tion of the methyl ester yield was used as the response variable. 2. Materials and methods 2.1. Materials Refined canola oil was purchased from Behshahr Industrial Co. (Tehran, Iran). Commercial immobilized lipase from Candida antarctica, namely Novozym 435, was provided as a gift from Novo Nordisk (A.S., Denmark-Tehran Office). Methanol and 0141-0229/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2009.05.013