Enzymatic synthesis of biodiesel from high free fatty acid feedstock using a recombinant rhizopus oryzae lipase

Resumen

The present thesis is focused, in general terms, on the enzymatic synthesis of biodiesel using a recombinant Rhizopus oryzae lipase (rROL), expressed in a methylotrophic yeast (Pichia pastoris) as a cell factory and immobilised onto a polymethacrylate support. The main feature of this enzyme is its regioespecificity, which allows to catalyse the alcoholysis of sn-1 and sn-3 ester bonds of the triglyceride into two fatty acid alkyl esters (biodiesel). The use of rROL becomes a key factor since glycerol is not formed as a by-product in favour of 2-monoglyceride. The first part of the thesis is focused on the evaluation of a novel feedstock as a substrate for biodiesel production. Alperujo oil is a vegetable oil, which can be representative of other high-FFA feedstocks, considered waste-oils. Preliminary studies are performed to find out the role of this FFA in terms of initial reaction rate and stability of the rROL. Moreover, the enzyme has been immobilised by covalent binding to ensure its stability and recovery. During the following parts, emphasis is put on the improvement of the enzymatic reaction itself using 10-mL vials. Temperature and initial water activity are set up in order to increase initial rate and enzyme stability. In addition, the two most used acyl-acceptors are compared. Methanol and ethanol are added using three stepwise strategies: one, five and ten pulses. Stability and productivity are also compared in order to find out the best one. Then, scale up to a 50-mL stirred-reactor is carried out by reproducing the previous experiments. Initial rates and stability are compared. Further analysis allowed to calculate enzyme’s half-life times and productivities in the different reactions. Semi-continuous addition of the acyl-acceptor which best results were obtained with, is attempted by using an automatised micro-burette. Last chapter is focused on the simulation of an industrial process of enzymatic production of biodiesel using all the previous obtained results. Specific software (SuperPro Designer®) is used to raise and develop a process to produce and purify the biodiesel as well as its by-product (2-monoglyceride). Then, viability studies are performed and some modifications are suggested in order to find out a profitable and feasible process.