Ecodiesel® is a biofuel incorporating glycerol, produced by enzymatic technology and patented by the University of Cordoba (UCO).37 It is composed of two moles of FAEEs and a mole of MG. Particularly, Ecodiesel® is obtained using pig pancreatic lipase (PPL), in both free and immobilized form, to achieve the 1,3 selective transesterification of TGs to produce the corresponding 2-monoacyl derivatives of glycerol (MG) and two moles of FAEEs. Ethanol is the alcohol employed in the process (Fig. 7.2).

2 RCOOCH2CH3

Fatty acid ethyl ESTERs (FAEEs)

for Ecodiesel®

It is interesting to note that the enzymatic transesterification process can also be carried out with different short-chain alcohol (ethanol, 1- and 2-propanol, 1- and 2-butanol, etc.) and their mixtures, and it is not, in principle, restricted to the use of methanol, as it is normally under conventional chemical reactions (with acidic or basic catalysis).

Many reports on biodiesel preparation using free38 or immobilized lipases can also be found.1117 In particular, PPL has been widely employed in the last decades for the resolution of mixtures of chiral enantiomers, either by enantioselective hydrolysis39,40 or by alcoholysis or transesterification.41

The recent work of Luna et al. and their patents37,42 show the entrapment of the PPL in demineralized sepiolite and its activity in the alcoholysis reaction of TGs contained in sunflower oil. Demineralized sepiolite is a clay mineral (a complex magnesium silicate) with a microporous structure and a channel dimension of

11.5 x 5.3 A. Its structure moves along fibres that confer a high specific surface area to the solid, similar to that of the AlPO-5.43,44 The extraction of the ions (Mg2+, Al+3, etc.) by acid treatment significantly increases the size of the pores, making them comparable to those of amorphous silica45 or even to a mesoporous structure similar to MCM-41.46 These voluminous pores are able to trap some macromolecules including various enzymes.47,48

Results obtained by employing immobilized PPL compared to the free enzyme are reported in Table 7.2. Different temperatures, oil/alcohol ratios and oil/ immobilized PPL ratios have been also investigated and included in Table 7.3.

Note: Data corresponds to the number of reuses (no.) of the biocatalyst, as a continuation of Table 7.2, under different reaction conditions.

The efficiency of the PPL can be obtained by comparing the turn-over frequency (TOF) values of free and immobilized PPL (Table 7.2), both obtained under the same experimental conditions and temperature. The efficiency of PPL was reduced to 42.5% [(24.5/57.7) x 100 = 42.5] after immobilization, due to a potential steric effect of the immobilized enzyme in the reaction and/or to the deactivation of the active sites of the enzyme in the entrapment process.

The TOF values showed that a decrease in the oil/alcohol molar ratio from 1:10 (Table 7.2) to 1:2 (Table 7.3) leads to an increase in the efficiency of the immobilized enzyme, in good agreement with the results obtained for the free enzyme. The results also pointed out that in any case, even with an excess of ethanol, a maximum 66% yield could be obtained, corresponding to a 1,3 selective enzymatic process. Of note was the enzyme stability and recyclability. Although the efficiency was reduced compared to the free form, the immobilization through physical entrapment of the PPL guaranteed the lifespan of the lipases. The free PPL was found to be completely deactivated in 48 hours, whereas the immobilized enzyme was active for several weeks, even after successive reuses preserving over 90% of the initial activity.

Another important advantage of the enzymatic process is the possibility of using various alcohols apart from methanol or ethanol. The effect of different short-chain alcohols on composition, yield, conversion and TOF of Ecodiesel-100, obtained in the alcoholysis of pure and waste frying sunflower oil, is reported in Table 7.4.

The biofuels could smoothly be obtained using the various alcohols employed, obtaining quantitative TGs conversions and selectivity to FAEE higher than 50% in most of the cases. The reaction typically takes 8-12 hours to complete, and the selectivity to FAEE increases with the time of reaction as expected.

A potentially useful biofuel blend of FAEE, MG and traces of DG, in varying proportions (depending on the conversions), can be obtained. The FAEE/MG ratio was around 2:1 molar at quantitative triglyceride conversion.

In conclusion, the alcoholysis of TGs with short-chain alcohols using 1,3-regiospecific lipases can play an advantageous role, compared to the conventional base-catalyzed process, to obtain new biofuels incorporating glycerine and to minimize the waste production by improving the reaction conversion under greener conditions. Milder reaction conditions were employed and a cleaner biofuel (Ecodiesel-100) was obtained. The efficiency of PPL was remarkably increased at a higher pH in contrast with the reported results describing a poor activity of the enzymes at that pH. The immobilized PPL was highly stable, although the efficiency was reduced (42%) compared to the free enzyme. The catalyst can easily be recycled (11 times), almost preserving the initial catalytic activity.