Applied Biochemistry and Biotechnology

, Volume 186, Issue 3, pp 576–589 | Cite as

Transesterification of Waste Frying Oil and Soybean Oil by Combi-lipases Under Ultrasound-Assisted Reactions

  • Jakeline Kathiele Poppe
  • Carla Roberta Matte
  • Roberto Fernandez-Lafuente
  • Rafael C. Rodrigues
  • Marco Antônio Záchia AyubEmail author


This work describes the use of an ultrasound system for the enzymatic transesterification of oils using combi-lipases as biocatalyst. The reactions were carried out evaluating the individual use of waste oil and fresh soybean oil, and the immobilized lipases CALB, TLL, and RML were used as biocatalysts. It was performed in a mixture design of three factors to obtain the ideal mixture of lipases according to the composition of fatty acids present in each oil, and the main reaction variables were optimized. After 18 h of reaction, ultrasound provided a biodiesel yield of about 90% when using soybean oil and 70% using the waste oil. The results showed that ultrasound technology, in combination with the application of enzyme mixtures, known as combi-lipases, and the use of waste oil, could be a promising route to reduce the overall process costs of enzymatic production of biodiesel.


Transesterification reaction Biodiesel Waste oil Soybean oil Combi-lipase Ultrasound system 



The authors would like to thank Mr. Ramiro Martinez (Novozymes, Spain S.A.) for kindly supplying the enzymes used in this research.

Funding Information

This work was supported by grants from the Brazilian Coordenação de Aperfoiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflicts of interest.

Supplementary material

12010_2018_2763_MOESM1_ESM.docx (31 kb)
ESM 1 (DOCX 31 kb)


  1. 1.
    Kapoor, M., & Gupta, M. N. (2012). Lipase promiscuity and its biochemical applications. Process Biochemistry, 47(4), 555–569.CrossRefGoogle Scholar
  2. 2.
    Zhao, X., Qi, F., Yuan, C., Du, W., & Liu, D. (2015). Lipase-catalyzed process for biodiesel production: enzyme immobilization, process simulation and optimization. Renewable & Sustainable Energy Reviews, 44, 182–197.CrossRefGoogle Scholar
  3. 3.
    Anderson, E. M., Larsson, K. M., & Kirk, O. (1998). One biocatalyst–many applications: the use of Candida antarctica B-lipase in organic synthesis. Biocatalysis and Biotransformation, 16(3), 181–204.CrossRefGoogle Scholar
  4. 4.
    Fernandez-Lafuente, R. (2010). Lipase from Thermomyces lanuginosus: uses and prospects as an industrial biocatalyst. Journal of Molecular Catalysis B: Enzymatic, 62(3-4), 197–212.CrossRefGoogle Scholar
  5. 5.
    Rodrigues, R. C., & Fernandez-Lafuente, R. (2010). Lipase from Rhizomucor miehei as an industrial biocatalyst in chemical process. Journal of Molecular Catalysis B: Enzymatic, 64(1-2), 1–22.CrossRefGoogle Scholar
  6. 6.
    Rodrigues, R. C., & Fernandez-Lafuente, R. (2010). Lipase from Rhizomucor miehei as a biocatalyst in fats and oils modification. Journal of Molecular Catalysis B: Enzymatic, 66(1-2), 15–32.CrossRefGoogle Scholar
  7. 7.
    Tiosso, P. C., Carvalho, A. K. F., de Castro, H. F., de Moraes, F. F., & Zanin, G. M. (2014). Utilization of immobilized lipases as catalysts in the transesterification of non-edible vegetable oils with ethanol. Brazilian Journal of Chemical Engineering, 31(4), 839–847.CrossRefGoogle Scholar
  8. 8.
    Alves, J. S., Vieira, N. S., Cunha, A. S., Silva, A. M., Zachia Ayub, M. A., Fernandez-Lafuente, R., & Rodrigues, R. C. (2014). Combi-lipase for heterogeneous substrates: a new approach for hydrolysis of soybean oil using mixtures of biocatalysts. RSC Advances, 4(14), 6863–6868.CrossRefGoogle Scholar
  9. 9.
    Poppe, J. K., Matte, C. R., do Carmo Ruaro Peralba, M., Fernandez-Lafuente, R., Rodrigues, R. C., & Ayub, M. A. Z. (2015a). Optimization of ethyl ester production from olive and palm oils using mixtures of immobilized lipases. Applied Catalysis A: General, 490, 50–56.CrossRefGoogle Scholar
  10. 10.
    Bergmann, J. C., Tupinambá, D. D., Costa, O. Y. A., Almeida, J. R. M., Barreto, C. C., & Quirino, B. F. (2013). Biodiesel production in Brazil and alternative biomass feedstocks. Renewable & Sustainable Energy Reviews, 21, 411–420.CrossRefGoogle Scholar
  11. 11.
    Geris, R., dos Santos, N. A. C., Amaral, B. A., de S. Maia, I., Castro, V. D., & Carvalho, J. R. M. (2007). Biodiesel de soja: reação de transesterificação para aulas práticas de química orgânica. Quimica Nova, 30(5), 1369–1373.CrossRefGoogle Scholar
  12. 12.
    Poppe, J. K., Fernandez-Lafuente, R., Rodrigues, R. C., & Ayub, M. A. Z. (2015b). Enzymatic reactors for biodiesel synthesis: present status and future prospects. Biotechnology Advances, 33(5), 511–525.CrossRefGoogle Scholar
  13. 13.
    Trentin, C. M., Popiolki, A. S., Batistella, L., Rosa, C. D., Treichel, H., de Oliveira, D., & Oliveira, J. V. (2015). Enzyme-catalyzed production of biodiesel by ultrasound-assisted ethanolysis of soybean oil in solvent-free system. Bioprocess and Biosystems Engineering, 38(3), 437–448.CrossRefGoogle Scholar
  14. 14.
    Yu, D., Tian, L., Wu, H., Wang, S., Wang, Y., Ma, D., & Fang, X. (2010). Ultrasonic irradiation with vibration for biodiesel production from soybean oil by Novozym 435. Process Biochemistry, 45(4), 519–525.CrossRefGoogle Scholar
  15. 15.
    Mostafaei, M., Ghobadian, B., Barzegar, M., & Banakar, A. (2015). Optimization of ultrasonic assisted continuous production of biodiesel using response surface methodology. Ultrasonics Sonochemistry, 27, 54–61.CrossRefGoogle Scholar
  16. 16.
    Ho, W. W. S., Ng, H. K., & Gan, S. (2016). Advances in ultrasound-assisted transesterification for biodiesel production. Applied Thermal Engineering, 100, 553–563.CrossRefGoogle Scholar
  17. 17.
    Lenardão, E. J., Freitag, R. A., Dabdoub, M. J., Batista, A. C. F., & Silveira, C. d. C. (2003). “Green chemistry”: os 12 princípios da química verde e sua inserção nas atividades de ensino e pesquisa. Quimica Nova, 26(1), 123–129.CrossRefGoogle Scholar
  18. 18.
    Alves, J., Garcia-Galan, C., Schein, M., Silva, A., Barbosa, O., Ayub, M., Fernandez-Lafuente, R., & Rodrigues, R. (2014). Combined effects of ultrasound and immobilization protocol on butyl acetate synthesis catalyzed by CALB. Molecules, 19(7), 9562–9576.CrossRefGoogle Scholar
  19. 19.
    Martins, A. B., Schein, M. F., Friedrich, J. L. R., Fernandez-Lafuente, R., Ayub, M. A. Z., & Rodrigues, R. C. (2013). Ultrasound-assisted butyl acetate synthesis catalyzed by Novozym 435: enhanced activity and operational stability. Ultrasonics Sonochemistry, 20(5), 1155–1160.CrossRefGoogle Scholar
  20. 20.
    AOCS (1998). Official methods and recommended practices of the American Oil Chemists Society 1–2, Champaign.Google Scholar
  21. 21.
    EN, 14103 (2001). Fat and oil derivatives - fatty acid methyl esters (FAME) - determination of esters and linolenic acid methyl esters content. European Committee for Standardization.Google Scholar
  22. 22.
    Su, F., Li, G.-L., Fan, Y.-L., & Yan, Y.-J. (2015). Enhancing biodiesel production via a synergic effect between immobilized Rhizopus oryzae lipase and Novozym 435. Fuel Processing Technology, 137, 298–304.CrossRefGoogle Scholar
  23. 23.
    Lee, J. H., Kim, S. B., Kang, S. W., Song, Y. S., Park, C., Han, S. O., & Kim, S. W. (2011). Biodiesel production by a mixture of Candida rugosa and Rhizopus oryzae lipases using a supercritical carbon dioxide process. Bioresource Technology, 102(2), 2105–2108.CrossRefGoogle Scholar
  24. 24.
    Pleiss, J., Fischer, M., & Schmid, R. D. (1998). Anatomy of lipase binding sites: the scissile fatty acid binding site. Chemistry and Physics of Lipids, 93(1-2), 67–80.CrossRefGoogle Scholar
  25. 25.
    Derewenda, Z. S., Derewenda, U., & Dodson, G. G. (1992). The crystal and molecular structure of the Rhizomucor miehei triacylglyceride lipase at 1.9 Å resolution. Journal of Molecular Biology, 227(3), 818–839.CrossRefGoogle Scholar
  26. 26.
    Naik, S., Basu, A., Saikia, R., Madan, B., Paul, P., Chaterjee, R., Brask, J., & Svendsen, A. (2010). Lipases for use in industrial biocatalysis: specificity of selected structural groups of lipases. Journal of Molecular Catalysis B: Enzymatic, 65(1-4), 18–23.CrossRefGoogle Scholar
  27. 27.
    Jachmanián, I., Schulte, E., & Mukherjee, K. D. (1996). Substrate selectivity in esterification of less common fatty acids catalysed by lipases from different sources. Applied Microbiology and Biotechnology, 44(5), 563–567.CrossRefGoogle Scholar
  28. 28.
    Noureddini, H., Gao, X., & Philkana, R. S. (2005). Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil. Bioresource Technology, 96(7), 769–777.CrossRefGoogle Scholar
  29. 29.
    Eguchi, S., Kagawa, S., & Okamoto, S. (2015). Environmental and economic performance of a biodiesel plant using waste cooking oil. Journal of Cleaner Production, 101, 245–250.CrossRefGoogle Scholar
  30. 30.
    Banković-Ilić, I. B., Stojković, I. J., Stamenković, O. S., Veljkovic, V. B., & Hung, Y.-T. (2014). Waste animal fats as feedstocks for biodiesel production. Renewable & Sustainable Energy Reviews, 32, 238–254.CrossRefGoogle Scholar
  31. 31.
    Hama, S., & Kondo, A. (2013). Enzymatic biodiesel production: an overview of potential feedstocks and process development. Bioresource Technology, 135, 386–395.CrossRefGoogle Scholar
  32. 32.
    Deba, A. A., Tijani, H. I., Galadima, A. I., Mienda, B. S., Deba, F., & Zargoun, L. M. (2014). Waste cooking oil: a resourceful waste for lipase catalysed biodiesel production. International Journal of Scientific and Research Publications, 4, 1–12.Google Scholar
  33. 33.
    Yu, C.-Y., Huang, L.-Y., Kuan, I. C., & Lee, S.-L. (2013). Optimized production of biodiesel from waste cooking oil by lipase immobilized on magnetic nanoparticles. International Journal of Molecular Sciences, 14(12), 24074–24086.CrossRefGoogle Scholar
  34. 34.
    Maddikeri, G. L., Pandit, A. B., & Gogate, P. R. (2013). Ultrasound assisted interesterification of waste cooking oil and methyl acetate for biodiesel and triacetin production. Fuel Processing Technology, 116, 241–249.CrossRefGoogle Scholar
  35. 35.
    Subhedar, P. B., & Gogate, P. R. (2016). Ultrasound assisted intensification of biodiesel production using enzymatic interesterification. Ultrasonics Sonochemistry, 29, 67–75.CrossRefGoogle Scholar
  36. 36.
    Liu, Y., Jin, Q., Shan, L., Liu, Y., Shen, W., & Wang, X. (2008). The effect of ultrasound on lipase-catalyzed hydrolysis of soy oil in solvent-free system. Ultrasonics Sonochemistry, 15(4), 402–407.CrossRefGoogle Scholar
  37. 37.
    Kojima, Y., Imazu, H., & Nishida, K. (2014). Physical and chemical characteristics of ultrasonically-prepared water-in-diesel fuel: effects of ultrasonic horn position and water content. Ultrasonics Sonochemistry, 21(2), 722–728.CrossRefGoogle Scholar
  38. 38.
    Batistella, L., Lerin, L. A., Brugnerotto, P., Danielli, A. J., Trentin, C. M., Popiolski, A., Treichel, H., Oliveira, J. V., & de Oliveira, D. (2012). Ultrasound-assisted lipase-catalyzed transesterification of soybean oil in organic solvent system. Ultrasonics Sonochemistry, 19(3), 452–458.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Jakeline Kathiele Poppe
    • 1
  • Carla Roberta Matte
    • 1
  • Roberto Fernandez-Lafuente
    • 2
  • Rafael C. Rodrigues
    • 1
  • Marco Antônio Záchia Ayub
    • 1
    Email author
  1. 1.Biotechnology, Bioprocess, and Biocatalysis Group, Food Science and Technology InstituteFederal University of Rio Grande do SulPorto AlegreBrazil
  2. 2.Department of BiocatalysisICP-CSIC, Campus UAM-CSICMadridSpain

Personalised recommendations