Depending on the kind of catalyst used and the selected operation conditions in the biodiesel production plant, the alcohol to oil molar ratio will present a wide variation. Adding excess alcohol is a common practice, and could serve as reference. However, excess alcohol use implies higher reactant associated costs, especially when the alcohol of choice is ethanol, which is more expensive than methanol. Thus, a more detailed approach to the system optimization in terms of minimal alcohol consumption is needed. Besides, a fine adjustment of the alcohol to oil ratio allows the maximal biodiesel production in the shortest possible time span and with the lowest energy input (Shieh et al., 2003).

The optimization is a relatively simple task when homogeneous catalysts such as sulphuric acid or sodium hydroxide are used to perform the conventional transesterification of vegetable oils with methanol. High yields are achieved with a methanol to oil ratio of 1:1 with an alkaline catalyst (although to improve the yield this proportion rises to 6:1) and a 30:1 ratio when an acid catalyst is used (Zhang et al., 2003).

However, in the case of lipase-catalyzed biodiesel, the situation is more complex and the molar ratio of alcohol to oil varies depending on the type of lipase, the use of an immobilized or free enzyme, and the alcohol used. Similar to the chemical catalysts, an increase of the molar alcohol:oil ratio elevates the efficiency of the reaction, but an excessive alcohol content inhibits and even damages the enzyme, especially when using methanol and free enzymes. Although the lipase-based solvent-free systems are under intensive research, owing to advantages such as the direct saving in solvents and the indirect cost reductions in downstream processes, the utilization of lipases does not necessary mean abandoning the use of a certain amount of solvents. The addition of solvents like f-butanol, diesel oil, hexane or dioxane to the precursors of biodiesel usually allows a better mixing of the reactants. Thus, solvents relieve the problems associated with the different water solubility of lipids and alcohols. In addition, solvents provide a more durable interaction between the enzyme and its substrates, and can favour the circulation of reactants through resins and support pores in immobilized enzyme systems. This improved circulation confers some protection to the lipases against inhibition by substrates and damages by excessive alcohols. However, solvents’ addition has to be carefully studied, since an excess of solvent or an inadequate amount of solvent can affect the enzyme activity and stability. For example, Shieh et al. studied the optimal operation conditions to transesterificate soybean oil with methanol by Rhizomucor miehei lipase immobilized on macroporous weak anionic resin beads. They found that the best transesterification rate was obtained when the methanol:oil molar proportion was 3.4:1 at 36.5°C (Shieh et al., 2003). Raita et al. studied the transesterification of palm oil with ethanol by Thermomyces lanuginosa lipase-coated microcrystals in the presence of f-butanol. In this case, the optimal conditions were ethanol to fatty acids 4:1 molar ratio and f-butanol:tryacylclycerides 1:1 molar ratio, at 45°C (Raita et al., 2010. However, Tongboriboon et al. worked on the solvent-free transesterification of used palm oil with Thermomyces lanuginosa and Candida anfarcfica lipases immobilized in porous polypropylene powder, reporting that the best yield was achieved at an ethanol to oil ratio of 3:1, and the yield decreased when the molar ratio was increased to 4:1 at 45°C (Tongboriboon et al., 2010). These authors pointed to the inhibition of the enzymes by an excessive amount of ethanol, although it is worth emphasizing that they worked on a solvent-free system, so the enzyme was relatively vulnerable to alcohol-driven damage. On the other hand, Shah et al used 4:1 ethanol to oil molar ratio as standard reaction settings in their study about the transesterification of jatropha oil with ethanol at 40°C. The experimental design consisted of a solvent-free system and three different lipases (free and immobilized on Celite), namely Chromobacferium viscosum, Candida rugosa and Porcine pancreas lipases, although they did not try different alcohol to oil molar ratios (Shah et al., 2004).