## 4 Biocatalytic Synthesis of Biodiesel in Supercritical Fluids
Thus, an excess in the triolein inlet flow or the continuous release of biodiesel product from the enzyme particle to the scCO\({}_{2}\) flow may dissolve the protective IL shell, enhancing continuous enzyme deactivation. Similarly, the low efficiency of the hydrophobic scCO\({}_{2}\) phase in transporting the hydrophilic by-product glycerol could led to its retention in the closest enzyme microenvironment and to the continuous biocatalyst poisoning, preventing the entry of new triolein substrate molecules. Different experimental approaches to efficiently desorb glycerol from the catalytic matrix in biodiesel synthetic processes under supercritical conditions have been proposed. Desorption was carried out using absolute ethanol under atmospheric conditions at different mass flows (10-30 g/min) or using ethanol-modified supercritical CO\({}_{2}\) (1:3 molar ratio of ethanol:CO\({}_{2}\)), under a pressure of 14.0 MPa, within a temperature range of 106-134 \({}^{\circ}\)C and with mass flow rates of 6-34 g/min. The results showed that ethanol is an efficient solvent for this process and that the supercritical desorption is much faster than conventional desorption processes [88]. Figure 7: Set-up of the continuous enzymatic reactor for biodiesel synthesis [84]. T: temperature indicator; P: pressure indicator; V: valve; BPR: Back-pressure regulator; CS: cyclone separator; F: Flow. The development of solid supports with a covalently attached IL phase is a further step towards reducing the amount of ILs used in catalytic processes in scCO\({}_{2}\), providing permanent protection of the enzymes against the adverse effect of this SCF. Through this approach, the properties of ILs are transferred to the solid phase, leading to either particle- or monolithic-Supported Ionic Liquid-Like Phases (SILLPs) [89]. This kind of material with tunable properties can be prepared on demand and they have been shown to efficiently immobilize a variety of catalysts. Indeed, the microenvironment provided by these \"solid ionic liquid phases\" can be modified by the modulation of the different design vectors of SILLPs [90]. Bioreactors based on _Candida antarctica_ lipase B (CALB) adsorbed onto SILLPs have been successfully applied as macroporous monolithic mini-flow systems for the continuous synthesis of citronellyl propionate in scCO\({}_{2}\)[91], as well as the KR and DKR of _rac_-1-phenylthethanol [92], showing in both cases good enzymatic activity and operational stability. In this context, the use of different nano-structured supports, based on different 1-decyl-2-methylimidazolium cations covalently attached onto a polystyrene-divinylbenzene (PS-DVB) porous matrix as carriers to immobilize _Candida antarctica_ lipase B (CALB), was recently reported (see Figure 8) [93]. The suitability of these immobilized lipase derivatives to carry out the synthesis of biodiesel (methyl oleate) through methanolysis of triolein has been tested in both _tert_-butanol and supercritical carbon dioxide (18.0 MPa, 45 \({}^{\circ}\)C) as reaction media. The use of modified supports with low ionic liquid loads covalently attached to the main polymeric backbone chains provided structured materials that led to high biodiesel yields (up to 95%) and operational stability (85% biodiesel yield after 45 cycles of 8-4 h) in scCO\({}_{2}\) (45 \({}^{\circ}\)C, 18.0 MPa). The presence of _tert_-butanol, as an inert co-solvent, in the scCO\({}_{2}\) phase at the same concentration as triolein was the key to avoid the  
Figure 8: Set-up of a continuous reactor containing immobilized lipase onto Supported Ionic Liquid-Like Phases (SILLPs) for biodiesel synthesis by methanolysis of triolein [93]. T, Temperature indicator; P, pressure indicator; V, valve; BPR, Back-pressure regulator. continuous poisoning of the biocatalyst through the blocking of its active sites by the polar by-product (glycerol) produced in the biodiesel synthesis.