Co-immobilization of Rhizomucor miehei lipase and Candida antarctica lipase B and optimization of biocatalytic biodiesel production from palm oil using response surface methodology Mansour Shahedi a , Maryam Yousefi b, *, 1 , Zohreh Habibi a, ** , Mehdi Mohammadi c , Mohammad Ali As'habi d a Department of Pure Chemistry, Faculty of Chemistry, Shahid Beheshti University, G.C., Tehran, Iran b Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran c Bioprocess Engineering Department, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran d Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin, Tehran, Iran article info Article history: Received 7 July 2018 Received in revised form 24 February 2019 Accepted 9 April 2019 Available online 12 April 2019 Keywords: Biodiesel Co-immobilization Lipase Response surface methodology abstract Lipases from Candida antarctica B (nonspecific lipase) and Rhizomucor miehei (1,3-specific lipase) were simultaneously immobilized on epoxy functionalized silica gel under mild conditions. The results showed rapid and simple immobilization of 4e15 mg of CALB:RML (different ratios 4:1, 2:1,1.5:1,1:1) on 1 g of support after 6 h. The thermal stability of derivatives and also their stability in methanol were greatly improved compared to the single immobilized enzyme. All the derivatives were also used to catalyze the transesterification of palm oil with methanol to produce fatty acid methyl esters (FAMEs). Response surface methodology (RSM) and a central composite rotatable design (CCRD) was used to study the effects of five factors, reaction temperature, methanol/oil ratio, reaction time, t-butanol concentra- tion and CALB:RML ratio on the fatty acid methyl esters (FAME) yield. A quadratic polynomial equation was obtained for methanolysis reaction by multiple regression analysis. The optimum combinations for the reaction were CALB:RML ratio (2.5:1), t-butanol to oil (39.9 wt%), temperature (35.6  C), methanol:oil ratio (5.9), reaction time 33.5 h. FAME yield of 78.3.5%, which was very close to the predicted value of 75.2%, was obtained. Verification experiment confirmed the validity of the predicted model. © 2019 Elsevier Ltd. All rights reserved. 1. Introduction Fossil fuels have been used for many years as the most dominant fuel for motor engines. However the serious crisis of declining fossil fuel resources and environmental pollution have led to a search for new renewable biofuels and finding novel alternative fuel sources. Biodiesel (monoalkyl esters of long-chain fatty acids) has a great potential as an alternative diesel fuel [1]. Biodiesel is produced by alcoholysis of renewable lipid sources, such as vegetable oil or animal fat. From an environmental point of view it shows several advantages over conventional fuel: biode- gradability, renewability, reduction of greenhouse gas emissions, reduced CO, hydrocarbons, NOx and particles in exhaust emission and therefore, significantly reduces pollution, also biodiesel can be pumped, stored and handled using the same infrastructure employed for compression ignition engines with little or no modifications. Conventionally the synthesis of alkyl esters is accomplished by chemical transesterification from which, short reaction times and high yields are obtained. Though the yield is high, the process has many disadvantages such as high energy requirements, difficulty in the transesterification of triglycerides with high free fatty acid content, pretreatment of the substrate when water is present and difficulties in the recovery of catalyst and glycerol [2]. Enzymatic approaches serve as a promising technology for biodiesel production due to the mild reaction conditions, easy re- covery of product, being environmentally friendly and low demanding on raw materials compared with chemical methods. In contrast, biocatalysts allow the synthesis of specific alkyl esters, * Corresponding author. ** Corresponding author. E-mail addresses: M.yousefi@ari.ir (M. Yousefi), Z_habibi@sbu.ac.ir (Z. Habibi). 1 These authors contributed equally to this work. Contents lists available at ScienceDirect Renewable Energy journal homepage: www.elsevier.com/locate/renene https://doi.org/10.1016/j.renene.2019.04.042 0960-1481/© 2019 Elsevier Ltd. All rights reserved. Renewable Energy 141 (2019) 847e857