## 4 Biocatalytic Synthesis of Biodiesel in Supercritical Fluids  
The use of dense gases, mainly scCO\({}_{2}\) or propane, as alternatives to organic solvents, to provide efficient reaction media for the enzymatic synthesis of biodiesel might be considered as the cleanest approach. As indicated before, the non-miscibility between triacylglycerides and methanol starting substrates, and glycerol and biodiesel final products, is rate-limiting because of the low efficiency in mass-transfer phenomena during the process, as well as the loss of activity of the biocatalyst through direct contact with methanol and glycerol. SFCs have shown to be suitable systems to overcome these drawbacks following the appropriate design of both reactor and reaction conditions. Investigation of the phase behaviour of the reaction system is important for better understanding the enzymatic synthesis of biodiesel in supercritical fluids. Pressure and temperature can affect the reaction rate by affecting the partitioning of the reaction components between the two phases, leading to poor conversions at the enzyme surface. Thus, [PERSON] _et al._ studied the phase behaviour of corn oil-methanol-CO\({}_{2}\) mixtures as a function of pressure by using a phase equilibrium cell equipped with a sapphire window [77]. Visual observation of the phase behaviour of the reaction mixture upon pressurization at 55 \({}^{\circ}\)C showed that the immiscible biphasic liquid phase, consisting of corn oil and methanol, approached a single liquid phase at pressures above the critical pressure of CO\({}_{2}\) and also expanded through the dissolution of CO\({}_{2}\). However, as a single homogeneous phase was not fully formed at the pressure levels investigated in this study (11.0, 20.0 and 35.0 MPa), authors consider reaction mixture as a two phase (lower single liquid phase and upper CO\({}_{2}\) phase) system. Other authors studied the solubility of olive husk oil in scCO\({}_{2}\) (40-80 \({}^{\circ}\)C, 15.0-35.0 MPa), and the influence of methanol and ethanol (1-5% v/v) as cosolvents, by using a dynamic flow method. It was observed how the solubility of the triglyceride increased moderately when increasing the CO\({}_{2}\) pressure at constant  
Figure 6: Biodiesel synthesis by the two-step supercritical approach. Reaction equations [76]. temperature, but doubled when 5% (v/v) of cosolvents was added [78]. The phase behaviour of the CO\({}_{2}\)-biodiesel system in the presence of cosolvents (e.g., methanol [79], ethanol [80], _etc._) has also been studied, leading to the observation that the addition of alcohol improves their miscibility. The design of the supercritical reactor used has been seen to be key element for the efficiency of the (bio)catalytic synthesis of biodiesel. Both continuous and discontinuous reactors have been assayed using several types of immobilized lipases as biocatalysts, and optimized reaction conditions (pressure, temperature, cosolvents, stirring, _etc._) and yields near to 100% biodiesel have been achieved. As an example of the use of discontinuous reactors, [PERSON] _et al._[81, 82] carried out biodiesel synthesis in scCO\({}_{2}\) using a mixture of vegetable oil and methanol (1:4 molar ratio) and a mixture of immobilized of _Candida rugosa_ and _Rhizopus oryzae_ lipases, as biocatalysts. In these studies, various factors, such as temperature, pressure, stirring speed, and the concentration of immobilized enzymes were investigated, resulting in optimal conditions for biodiesel production (100% yield at 2 h) under the following conditions: 13.0 MPa pressure, 45 \({}^{\circ}\)C temperature, 250 rpm stirring speed, and 20% (w/w) immobilized enzyme. Furthermore, an optimized stepwise addition of methanol allowed for the maintenance of immobilized lipase activity and the recycling of the immobilized biocatalysts. The yield of these stepwise reactions was still 85% after 20 reuses.