Preparation and Kinetic Study of Rhizopus oryzae Whole-cell as Biocatalyst for Biodiesel Synthesis through Non-alcohol Route H. Hermansyah, M. B. Faiz, R. Arbianti, and T. S. Utami Department of Chemical Engineering, Universitas Indonesia, Depok 16424, Indonesia Email: heri@che.ui.ac.id, merisa.bestari21@ui.ac.id, arbianti, nana@che.ui.ac.id Yopi Research Center for Biotechnology, Indonesian Institute of Sciences, Cibinong 16911, Indonesia Email: yopi@lipi.go.id Abstract—Direct use of lipase producing whole-cell as biocatalyst for biodiesel synthesis gain extensively attention since it could reduce the catalyst cost. Whole-cell of Rhizopusoryzaewas cultivated by single-step and two- stepmethod. At reaction time of 48 h, the single-step cultivated whole-cell produced23% FAME yield. It is higher comparable with the two-step cultivated whole-cell with 19% FAME yield.Kinetic model based Michaelis- Mentenmechanism was found to fit fairly the substrate and product concentration profileof experimental result.K m and V max value for R. oryzaewhole-cell are 3mole L -1 , 0.09 mole L - 1 h -1 (one-step) and 3 mole L -1 , 0.065mol L -1 h -1 (two-step). Index Terms—biodiesel, michaelis-menten, whole-cell I. INTRODUCTION In recent years, fatty acid methyl esters (FAMEs), commonly referred to biodiesel fuel, have become viewed as promising renewable sources of fuel[1]. Current technology for biodiesel production, using an alkali or acid catalyst, has several important limitations such as requires feedstock free of water, produce salts, generate glycerin as a low grade by-product, etc.[2]. In the other side, utilizing lipase as catalyst for biodiesel production over several advantage, such as require lower heating, works even in the presence of water, produces no salts [2]. Lipases have up to 74 times higher productivity than alkali catalyst [3]. Lipase is easy to be deactivated by alcohol, which is a reactant in biodiesel synthesis reaction. Therefore, we have developed new method to maintain the activity and stability of the biocatalyst during reaction byusing methyl acetate as an acyl donor [4]. The price of lipase is expensive due to purification procedures [2]. Haas et. al. reported that catalyst cost in biodiesel production by using enzyme is 0.14US$ per kg per kg ester [5]. It is much higher than using NaOH0.006 US$ per kg ester [5]. In order to lower the catalyst cost, we used enzyme immobilization method in various support to make the lipase become reusable due to its easy recovery from the reaction mixture [6]–[8]. We have also developed kinetic model based on Michaelis- Menten and Ping Pong Bi Bi Mechanism describing the interesterification behavior of the triglyceride with methyl acetate to produce biodiesel using lipase [4], [6]. Although immobilization make enzyme become reusable, but the process is time and cost consuming [6], [7]. Utilizing whole-cell overproducing intracellular lipase, such as Rhizopusoryzae, as biocatalyst in biodiesel synthesis is a potential way to reduce the biocatalyst cost [9]. The preparation of a biocatalyst requires no complex purification process, and the prepared whole-cells could be directly used as lipase containers [10]. In this research, we have done preparation of R. oryzaewhole-cell using two different cultivation methods, i.e. single-step and two-step method. Works that deal with modeling of enzymatic interesterification for whole-cell catalyzed biodiesel production are not often found in the literature. This works also presents a kinetics study of enzymatic reaction between cooking oil and methyl acetate for biodiesel production using suspendedR. oryzaewhole-cell as biocatalyst. The kinetic was based on Michaelis- Menten(MM) mechanism. II. MATERIALS AND METHODS A. Materials All experiments were carried out using R. oryzaeobtainedfrom School of Life Science and Technology, Bandung Institute of Technology, Indonesia. Potato Dextrose Broth (PDB) was obtained from BD Difco TM (USA). Trypton, NaNO 3 , KH 2 PO 4 , MgSO 4 .7H 2 O, glutaraldehyde and methyl acetate were purchased from Merck (Darmstadt, Germany). Olive oil and cooking oil were obtained locally. B. Preparation of R. Oryzae Whole-cell There are two different methods used for fungi propagation, i.e. single-step and two-step method. One- step cultivation was carried out using 50ml of PDB 2013 Engineering and Technology Publishing Journal of Life Sciences and Technologies Vol. 1, No. 3, September 2013 168 doi: 10.12720/jolst.1.3.168-171 Manuscript received June 10, 2013; revised August 21, 2013.