## 4 Biocatalytic Synthesis of Biodiesel in Supercritical Fluids
The usual advantages of continuous processes are even more evident in the case of the enzymatic synthesis of biodiesel because products are directly separated, and the methanol concentration has a low impact on the enzyme deactivation [13, 17]. [PERSON] _et al._[83] were pioneers in the applicability of scCO\({}_{2}\) in the continuous enzymatic synthesis of biodiesel from soybean and corn oils with reaction conversions above 98% at 1 mL/min CO\({}_{2}\) flow rate, followed by complete fractionation of the reaction mixture. In order to improve the efficiency of the continuous process for biodiesel synthesis in scCO\({}_{2}\) using immobilized enzymes as catalysts, several authors described how the appropriate design of the continuous reactor may provide operational advantages (e.g., efficient mixture of substrates, continuous separation of products, enhanced operational stability of enzymes, _etc._) [77]. [PERSON] and co-workers [84] implemented a continuous process for biodiesel production in supercritical carbon dioxide by using virgin sunflower oil and methanol as substrates, which were incorporated in the reactor using a static mixer with 27 elements (See Figure 7). The application of Lipozyme TL IM as biocatalyst led to FAMEs yields that exceeded 98% at 20.0 MPa and 40 \({}^{\circ}\)C, for a residence time of 20 s and an oil to methanol molar ratio of 1:24. Even for moderate reaction conversions, a fractionation stage based on two high pressure cyclone separators provided FAMEs of up to 96% purity. In the same context, [PERSON] _et al_[85] reported the continuous production of fatty acid ethyl esters from soybean oil in compressed carbon dioxide, propane and _n_-butane, using immobilized Novozym 435 as the catalyst. The experiments were performed in a packed-bed bioreactor. An evaluation of the effects of pressure and temperature in the range of 5.0 to 15.0 MPa and 30-70 \({}^{\circ}\)C, respectively, as well as the oil to ethanol molar ratio (from 1:6 to 1:18) and solvent to substrates mass ratio (from 4:1 to 10:1) was carried out. The best results (up to 98% yield) were obtained for lipase-catalyzed alcohols in a continuous tubular reactor using compressed propane (70 \({}^{\circ}\)C, 6.0 MPa), which it is suggested as another potential route to biodiesel production. The similar dielectric constants of compressed propane and carbon dioxide, together with the higher pressure phase transition values generally found in systems formed by carbon dioxide and high molecular weight compounds (e.g., triglycerides)lead weight to the belief that propane may also be suitable as reaction medium for enzyme-catalyzed bioconversions [29, 86]. Biphasic systems based on ILs and scCO\({}_{2}\) have recently been used for the enzymatic synthesis of biodiesel [87]. For example, a continuous enzymatic reactor, based on supported biocatalyst particles coated with hydrophobic ILs having imidazolium cations with C\({}_{12}\)-C\({}_{18}\) alkyl side chains was studied for the methanolysis of triolein in scCO\({}_{2}\) at 60 \({}^{\circ}\)C and 18.0 MPa. The operational stability of the immobilized lipase was improved by its coating with the ILs, _i.e._, 1-methyl-3-octadecylimidazolium hexafluorophosphate. A final two-phase system was obtained, revealing a good catalytic behaviour in continuous operation under supercritical conditions, with up to 82% biodiesel yield after 12 cycles of 4 h. For these IL/scCO\({}_{2}\) systems, the unique properties of long chain ILs, providing a very appropriate microenvironment for enzyme-catalyzed reactions, led to a clear improvement in the efficiency for the biotransformation of vegetable oils into biodiesel. However, the full miscibility of these hydrophobic ILs with both the triolein substrate and the methyl oleate product was related with the continuous activity decay observed for long term operation cycles.