The effect of reaction time on the extent of transesterification reaction was studied for 0-24-h time with immobilized lipase, and the results are shown in Fig. 12.7f. Other reaction parameters remained the same as mentioned in the optimization baseline. A 50% biodiesel yield was obtained at the end of 12 h of reaction time. Complete conversion and 100% yield of ethyl esters were obtained at the end of 24-h reaction time.

In literature, a conversion of 96% of ethyl esters was reported using the same enzyme with a reaction time of 48 h (Hsu et al. 2004). A 71% conversion in 8 h was reported by using ethanol and immobilized Chromobacterium viscosum lipase (Shah et al. 2004a, b). The reaction time mentioned in this study of 24 h can be reduced if we could produce immobilized enzyme with smaller size or increase the mixing intensity.

12.7 Conclusion

Production of biodiesel by chemical catalysts has the disadvantages that the process is energy consuming, requires water treatment, difficulty in recovery of glycerol, and formation of many unwanted products. On the other hand, enzymatic trans­esterification omits these difficulties and can obtain high purity biodiesel at milder working conditions. In order to be economical, the enzyme can be immobilized in some natural matrices, thus cutting down the production cost by reusing the immo­bilized lipase.

Immobilization of lipases is gaining importance due to a broad variety of indus­trial applications they catalyze. In this study, lipase from B. cepacia was first cross — linked with glutaraldehyde followed by entrapment into hybrid matrix of alginate and к-carrageenan polymers. The effect of various parameters like pH, temperature, reusability, enzyme leakage, solvent, and storage stability on immobilized lipase was studied. A higher activity yield of 89.26% was observed after immobilization. The immobilized lipase also retained 84.02% of its initial activity following two weeks of storage in T/Ca buffer at 4°C. Comparative kinetic parameters K and V

m max

values were found to be 0.39 |rM and 10 |rmol/min for free lipase and 0.45 |rM and 9.09 |rmol/min for immobilized lipase, respectively. A significant enzyme leakage reduction of 65.76% was observed as compared to the enzyme immobilized in hybrid matrix without cross-linking. The immobilized lipase also gave better results for hydrolysis of olive oil. Reduced enzyme leakage, higher thermal stability, and better storage stability were the salient features achieved by this method of enzyme immobilization. By this work, an improved entrapment approach of lipase cross­linking followed by entrapment onto a hybrid matrix of alginate and к-carrageenan was studied. This enhanced cross-linked matrix is a step closer in design of a better immobilized lipase for the biofuel industry.

Biodiesel optimization using the immobilized lipase was carried out for J. curcas oil in a stirred tank reactor. The optimal conditions for processing 10 g of Jatropha oil were 35°C, 1:10 molar ratio of oil to ethanol; 1 g water; 5.25 g immobilized lipase; 200 rotations per minute; and 24-h reaction time. At the above-mentioned optimal conditions, 100% yield of biodiesel was obtained. This shows that cross — linked B. cepacia lipase entrapped in a matrix of alginate and k-carrageenan could be a promising biocatalyst in the biofuel industry.

Acknowledgments We would like to acknowledge the Ministry of Science, Technology and Innovation (MOSTI) for the financial support through FRGS Grant (FGRG0244-TK-1/2010).