Entre as técnicas estudadas para ruptura celular da levedura Candida sp. LEB-M3, a abrasão por cisalhamento apresentou recuperação de lipídeos de 125%, quando comparada ao método padrão, porém aquelas que empregam HCl e altas temperaturas também apresentaram alta eficiência na ruptura celular, maior facilidade de utilização e menor custo. As diferentes técnicas não influenciaram significativamente no perfil de ácidos graxos, exceto para HPH, que proporcionou maior extração de ácidos graxos saturados, indicando que as condições das técnicas de ruptura, não modificaram o perfil de ácidos
110
graxos. O estudo da extração com CO2 supercrítico, assistido por ultrassom, apesar de apresentar baixos rendimentos, mostrou-se um método promissor para extração de lipídeos microbianos com diversas vantagens operacionais quando comparado aos métodos convencionais utilizados industrialmente.
REFERÊNCIAS
AOAC, Association of Official Analytical Chemists, Official Methods of Analysis, 16th ed., AOAC, Arlington, VA, 1995.
Barba, F.J., Grimi, N., Vorobiev, E., 2014. New Approaches for the Use of Non- conventional Cell Disruption Technologies to Extract Potential Food Additives and Nutraceuticals from Microalgae. Food Engineering Reviews, DOI 10.1007/s12393-014- 9095-6.
Basso, R.C., de Almeida Meirelles, A.J., Batista, E.A.C., 2012. Liquid–liquid equilibrium of pseudoternary systems containing glycerol + ethanol + ethylic biodiesel from crambe oil (Crambe abyssinica) at T/K = (298.2, 318.2, 338.2) and thermodynamic modeling. Fluid Phase Equilibria, 333, 55-62.
Bligh, E.G., Dyer, J.W., 1959. A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911-917.
Callejón, M.J.J., Medina, A.R., Sánchez, M.D.M., Peña, E.H., Cerdán, L.E., Moreno, P.A.G., Grima, E.M., 2014. Extraction of saponifiable lipids from wet microalgal biomass for biodiesel production. Bioresource Technology, 169, 198-205.
111
Cheng, C.H., Du, T.B., Pi, H.C., Jang, S.M., Lin, Y.H., Lee, H.T., 2011. Comparative study of lipid extraction from microalgae by organic solvent and supercritical CO2. Bioresource Technology, 102, 10151-10153.
Chisti, Y., Moo-Young, M., 1986. Disruption of microbial cells for intracellular products. Enzyme Microbiology and Technology, 8, 194-204.
Darani, K.K., Mozafari, M.R., 2009. Supercritical fluids technology in bioprocess industries: A review. Journal Biochemistry Technology, 2, 144-152.
Duarte, S.H., Andrade, C.C.P., Ghiselli, G., Maugeri, F., 2013. Exploration of Brazilian biodiversity and selection of a new oleaginous yeast strain cultivated in raw glycerol. Bioresource Technology, 138, 377-381 (a).
Duarte, S.H., Ghiselli, G., Maugeri, F., 2013. Influence of culture conditions on lipid production by Candida sp. LEB-M3 using glycerol from biodiesel synthesis. Biocatal. Agricult. Biotechnol. 2, 339-343 (b).
Ferraz, T.P.L., Fiúza, M.C., Santos, M.L.A., Carvalho, L.P., Soares, N.M., 2004. Comparison of six methods for the extraction of lipids from serum in terms of effectiveness and protein preservation. Journal of. Biochemical Biophysical Methods, 58, 187-193.
Gao, Y., Nagy, B., Liu, X., Simándi, B., Wang, Q., 2009. Supercritical CO2 extraction of lutein esters from marigold (Tagetes erecta L.) enhanced by ultrasound. The Journal of Supercritical Fluids, 49, 345-350.
112
Goto, M., Kanda, H., Wahyudiono, S.T., 2014. Extraction of carotenoids and lipids from algae by supercritical CO2 and subcritical dimethyl ether. The Journal of Supercritical Fluids, http://dx.doi.org/10.1016/j.supflu.2014.10.003.
Grimi, N., Dubois, A., Marchal, L., Jubeau, S., Lebovka, N.I., Vorobiev, E., 2014. Selective extraction from microalgae Nannochloropsis sp. using diferente methods of cell disruption. Bioresource Technology, 153, 254-259.
Halim, R., Rupasinghe, T.W.T., Tull, D.L., Webley, P.A., 2013. Mechanical cell disruption for lipid extraction from microalgal biomass. Bioresource Technology, 140, 53-63.
Hartman, L., Lago, R.C.A., 1973. Rapid preparation of fatty acids methyl esters. Laboratory Practice, London, 22, 475-476.
Hegel, P.E., Camy, S., Destrac, P., Condoret, J.S., 2011. Influence of pretreatments for extraction of lipids from yeast by using supercritical carbon dioxide and ethanol as cosolvent. The Journal of Supercritical Fluids, 58, 68-78.
Hussain, J., Ruan, Z., Nascimento, I.A., Liu, Y., Liao, W., 2014. Lipid profiling and corresponding biodiesel quality of Mortierella isabellina using different drying and extraction methods. Bioresource Technology, 169, 768-772.
Lim, C.S.Y., Tung, C.H., Rosli, R., Chong, P.P., 2008. An alternative Candida spp. cell wall disruption method using a basic sorbitol lysis buffer and glass beads. Journal of Microbiological Methods, 75, 576-578.
113
Maheshwari, P., Nikolov, Z.L., White, T.M., Hartel, R., 1992. Solubility of Fatty Acids in Supercritical Carbon Dioxide. Journal of the American Oil Chemists Society, 69, 1069- 1076.
Maugeri, F., Hernalsteens, S., 2007. Screening of yeast strains for transfructosylating activity. J. Mol. Catal. B: Enzym. 49, 43-49.
Metcalfe, L.D.; Schmitz, A.A.; Pelka, J.R., 1966. Rapid preparation of fatty acid esters from lipids for gas chromatography. Analytical Chemistry, 38, 514.
Michelon, M., Borba, T.M., Rafael, R.S., Burkert, C., Burkert, J.M., 2012. Extraction of carotenoids from Phaffia rhodozyma: A comparison between different techniques of cell disruption. Food Science and Biotechnology, 21, 1-8.
Milanesio, J., Hegel, P., Medina-González, Y., Camy, S., Condoret, J.S., 2012. Extraction of lipids from Yarrowia lipolytica. Journal of Chemical Technology and Biotechnology, 88, 378-387.
Mouahid, A., Crampon, C., Toudji, S.A.A, Badens, E., 2013. Supercritical CO2 extraction of neutral lipids from microalgae: Experiments and modelling. The Journal of Supercritical Fluids, 77, 7-16.
Santos, P., Aguiar, A.C., Barbero, G.F., Rezende, C.A., Martínez, J., 2015. Supercritical carbon dioxide extraction of capsaicinoids from malagueta pepper (Capsicum frutescens L.) assisted by ultrasound. Ultrasonics Sonochemistry, 22, 78-88.
114
Solana, M., Rizza, C.S., Bertucco, A., 2014. Exploiting microalgae as a source of essential fatty acids by supercritical fluid extraction of lipids: Comparison between Scenedesmus obliquus, Chlorella protothecoides and Nannochloropsis salina. The Journal of Supercritical Fluids, 92, 311-318.
Toma, M., Vinatoru, M., Paniwnyk, L., Mason, T.J., 2001. Investigation of the effects of ultrasound on vegetal tissues during solvent extraction. Ultrasonics Sonochemistry, 8, 137- 142.
Walker, T., Cochran, H., Hulbert, G., 1999. Supercritical carbon dioxide extraction of lipids from Pythium irregulare. Journal of the American Oil Chemists Society, 76, 595-602.
Xiao, A.F., Ni, H., Cai, H.N., Li, L.J., Su, W.J., Yang, Q.M., 2009. An improved process for cell disruption and astaxanthin extraction from Phaffia rhodozyma. World Journal of Microbiology and Biotechnology, 25, 2029-2034.
Zhang, X., Yan, S., Tyagi, R.D., Surampalli, R.Y., Valéro, J.R., 2014. Ultrasonication aided in-situ transesterification of microbial lipids to biodiesel. Bioresource Technology, 169, 175-180.
CAPÍTULO V
ENZYMATIC BIODIESEL SYNTHESIS FROM YEAST OIL USING IMMOBILIZED RECOMBINANT Rhizopus oryzae LIPASE
117
ENZYMATIC BIODIESEL SYNTHESIS FROM YEAST OIL USING
IMMOBILIZED RECOMBINANT Rhizopus oryzae LIPASE
1
Susan Hartwig Duarte, 1Francisco Maugeri and 2Francisco Valero
1
Laboratory of Bioprocess Engineering, Faculty of Food Engineering – UNICAMP, Campinas, Brazil.
2
Deptarment of Chemical Engineering, School of Engineering, Universitat Autònoma de Barcelona – UAB, Barcelona, Espanha.
ABSTRACT
The recombinant Rhizopus oryzae lipase (1-3 positional selective), immobilized on Relizyme OD403, has been applied to the production of biodiesel using single cell oil from
Candida sp. LEB-M3 growing on glycerol from biodiesel process. The composition of
microbial oil is quite similar in terms of saponifiable lipids than olive oil, although with a higher amount of saturated fatty acids. The reaction was carried out in a solvent system, and n-hexane showed the best performance in terms of yield and easy recovery. The strategy selected for acyl acceptor addition was a stepwise methanol addition using crude and neutralized single cell oil, olive oil and oleic acid as substrates. A FAMEs yield of 40.6 % was obtained with microbial oils lower than olive oil 54.3 %. Finally in terms of stability, only a lost about 30% after 6 reutilizations were achieved.
118