Methanol is the most commonly used alcohol in biodiesel production, as shown in Table 6.1, mainly because of its high reactivity and relatively low cost. However, sustainable methods of methanol production are currently not economically viable. It is typically produced from syngas that is in turn produced from a non­renewable source, namely natural gas. In addition, methanol is the most toxic and has the most deleterious effect on the biocatalyst activity compared to other alcohols. On the other hand, ethanol can be easily formed from renewable sources by fermentation. Using ethanol that is produced from renewable resources for biodiesel production makes the process entirely ‘green’.

The reason lipase-based biodiesel production has not reached commercial potential at present is the high cost of the enzyme and the loss of its activity. The main reason for the loss of activity is due to the inhibition effect of alcohol. As mentioned in Section 6.3, lipases are inactivated by contact with insoluble methanol that exists as drops in the oil, due to the strong polarity of the methanol that strips the active water from the enzyme’s active site (Lara and Park, 2004). Another potential problem that arises with the use of lipases is the by-product glycerol inhibition of the lipase due to its strong adsorption onto its surface. To overcome the problem of methanol inhibition of lipase, its amount should always be kept below its solubility limits in oil. To achieve this efficiently, a stepwise addition of methanol in a way to keep its amount below its solubility limit has been proposed (Shimada et al., 1999; Shimada et al., 2002). However, this solution does not take into account the problems with glycerol inhibition.

Other ways to overcome the problem include the use of an organic solvent, which are mainly used to dissolve the methanol and eliminate the stripping of the water molecules required for enzyme activation. The use of organic solvents also helps to reduce the effect of the by-product inhibition by dissolving the produced glycerol and to reduce the viscosity of the reaction media. Organic solvents such as и-hexane and ether have been studied (Tweddell et al., 1998; Oliveira and Oliveira, 2001; Al-Zuhair et al., 2008); however the solubility of methanol and glycerol in these solvents is low, and the above problems probably persist. Since the solubility of methanol is higher in 1,4-dioxane, using it as a solvent results in an increased yield of biodiesel production from triolein (Iso et al., 2001). However, large amounts of this solvent that make up 90% of reaction media were required to obtain reasonable conversion. The main disadvantages of using organic solvents are substrate dilution and the requirement of the addition of solvent recovery unit. On the other hand, it was found that when using t-butanol, a long-chain alcohol that does not inhibit the enzyme, as a solvent the enzymatic process is improved (Wang et al., 2006; Royon et al., 2007). t-Butanol dissolves both methanol and glycerol and at the same time it is not a preferred substrate for lipase that does not act as tertiary alcohols. The advantages of using t-butanol with immobilized lipase is further discussed in Section 6.8.

An alternative approach is to replace methanol with a different acyl acceptor such as methyl acetate (Wei et al., 2004). The reaction of triglyceride with methyl acetate is known as interesterification, which is similar to transesterification with the main difference being that the main by-product is triacetyl-glycerol rather than glycerol. Unlike methanol, methyl acetate has no negative effect on enzymatic activity and almost no loss in lipase activity detected even after being continuously used for 100 batches (Du et al., 2004; Wei et al, 2004). However, the main dis­advantage of this approach is that it proceeds in a much slower rate compared to when methanol is used in appropriate concentrations (Wei et al, 2004). In addition, the removal of the by-product tri-acetyl-glycerol is more difficult than glycerol.

Ionic liquids, which are salts that are liquid at room temperature, have also been proposed to replace conventional organic solvents often with improved process performance. Ha et al. (2007) assessed the effectiveness of using several types of ionic liquids in a lipase-catalyzed production of biodiesel from soybean oil and methanol. They reported that higher percent conversions were achieved using hydrophobic ionic liquids as compared to solvent-free systems. The cost of these ionic liquids is expected to hinder their commercial application.