**4. Conclusions**

In this research work, the immobilization of Lipozyme RM IM, a *Rhizomucor miehei* lipase, on different supports has been evaluated by its use as biocatalyst in the synthesis of Ecodiesel, a biodiesel-like biofuel obtained by selective transesterification of sunflower oil with ethanol. The best results have been obtained using a commercial silica gel 60 as support, achieving similar results to those obtained over the non-immobilized Lipozyme. Likewise, the optimization of the reaction parameters as well as the influence of these parameters in the enzymatic transesterification has been carried out by a statistical multifactorial design of experiments with three factors run by the Software StatGraphics. The analysis of variance showed that, in order to obtain an improvement in selectivity and kinematic viscosity, the reaction conditions should be an oil/ethanol molar ratio of 1/6 and a high amount of 10 N NaOH, 50 μL, whereas the amount of lipase employed depends on which parameter wanted to be optimized, i.e., 0.012 g of lipase to achieve the best selectivity value or 0.017 to achieve the best kinematic viscosity. Furthermore, the quadratic models obtained showed good results in terms of predicting the selectivity and viscosity of the investigated systems, as it was corroborated by experimental reactions.

The stability of the heterogeneous biocatalysts was studied by successive reactions in order to evaluate the feasibility and economic viability of its application in industrial scale. In this way, by means of simple physical immobilization of the Lipozyme on silica-gel, 15 reuses can be obtained without an evidence of activity loss. This fact allows us to assume that it is possible to perform a much greater number of reuses. These results are very remarkable, taking into account that the commercial Lipozyme RM IM, immobilized in an organic polymer is able to perform only 6 reuses before the total loss of activity.

Besides, an improvement in the operational separation of the biocatalyst from the reaction medium was obtained, since the commercial Lipozyme has to be separated by centrifugation, whereas the supported Lipozyme can be separated by decantation.

**Author Contributions:** This research article is part of the doctoral thesis of J.C., directed by D.L., and F.M.B., who in general conceived and designed the experiments. C.L., A.A.R., B.H. and R.E. made substantive intellectual contributions to this study, making substantial contributions to conception and design of it, as well as to the acquisition, analysis and interpretation of data. Furthermore, J.C., D.L. and R.E. wrote the paper. All the authors have been also involved in drafting and revising the manuscript, so that everyone has given final approval of the current version to be published in Energies Journal.

**Acknowledgments:** This research is supported by the MEIC funds (Project ENE 2016-81013-R), Junta de Andalucía and FEDER (P11-TEP-7723), that cover the costs to publish in open access.

**Conflicts of Interest:** The authors declare no conflict of interest.
