Isolation and Screening of Filamentous Fungi Producing Extracellular Lipase with Potential in Biodiesel Production

Sabrina Moro Villela Pacheco¹, Américo Cruz Júnior¹, Ayres Ferreira Morgado¹, Agenor Furigo Júnior¹, Onyetugo Chioma Amadi², José Manuel Guisán³, Benevides Pessela^4,5*

¹Department of Food and Chemistry Engineering, University Campus UFSC, Florianópolis, Brazil.
²Department of Microbiology, Faculty of Biological Science, University of Nigeria, Enugu, Nigeria.
³Departamento de Biocatálises Enzimática, Instituto de Catalisis y Petroleoquimica, ICP (CSIC-UAM), C/Marie Curie 2, Cantoblanco, Campus de la Univerisdad Autónoma de Madrid, Madrid, España.
⁴Instituto de Investigación en Ciencias de la Alimentación CIAL (CSIC-UAM), C/Nicolás Cabrera 9, Cantoblanco, Campus de la Universidad Autónoma de Madrid, Madrid, España.
⁵Departamento de Engenharia e Tecnologías, Instituto Superior Politécnico de Tecnología e de Ciências, ISPTEC, Luanda Sul, Talatona, República de Angola.

DOI: 10.4236/aer.2015.34011                

Abstract

Nineteen fungal strains were isolated from a chicken slaughterhouse effluent and within those, only one showed high values of lipolytic activity in submerged cultures. This fungus was identified as Trametes hirsuta. The crude extract was immobilized in chitosan/clay beads, with an immobilization yield of 80.9%. The analyses of the crude extract and the immobilized derivative at different temperatures, pH (s), solvents, metallic ions and storage showed that the immobilization process increased the enzyme life span. Ethyl esters were obtained in solvent free systems using chicken viscera oil and the enzyme crude extract. For effective comparison, a reaction using viscera oil and commercial lipase Novozym 435 was carried out. The result revealed 35% and 28% esters conversion in the reactions containing chicken viscera oil, using Novozym 435 and the crude extract respectively. The extract was also used in a reaction with soybean oil, traditionally used as starting substrate for biodiesel production.

Keywords

Biodiesel Production, Trametes Hirsuta, Lipase

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Marchetti, J.M., Miguel, V.U. and Errazu, A.F. (2007) Possible Methods for Biodiesel Production. Renewable and Sustainable Energy Reviews, 11, 1300-1311. http://dx.doi.org/10.1016/j.rser.2005.08.006
[2]	Illanes, A. (2008) Enzyme Biocatalysis: Principles and Applications. Springer, Netherlands. http://dx.doi.org/10.1007/978-1-4020-8361-7
[3]	Villeneuve, P., Muderhwa, M., Graille, J. and Haas, M. (2000) Customizing Lipases for Biocatalysis: A Survey of Chemical, Physical and Molecular Biological Approaches. Journal of Molecular Catalysis B: Enzymatic, 9, 113-148. http://dx.doi.org/10.1016/S1381-1177(99)00107-1
[4]	Tan, T., Lu, J., Nie, K., Deng, L. and Wang, F. (2010) Biodiesel Production with Immobilized Lipase: A Review. Biotechnology Advances, 28, 628-634. http://dx.doi.org/10.1016/j.biotechadv.2010.05.012
[5]	Faber, K. (1997) Biotransformations in Organic Chemistry. Springer-Verlag, New York. http://dx.doi.org/10.1007/978-3-662-00431-9
[6]	Kharrat, N., Ali, B.A., Marzouk, S., Gargouri, Y. and Chaabouni, M.K. (2011) Immobilization of Rhizopus oryzae Lipase on Silica Aerogels by Adsorption: Comparison with the Free Enzyme. Process Biochemistry, 46, 1083-1089. http://dx.doi.org/10.1016/j.procbio.2011.01.029
[7]	Minovska, V., Winkelhausen, E. and Kuzmanova, S. (2005) Lipase Immobilized by Different Techniques on Various Support Materials Applied in Oil Hydrolysis. Journal of the Serbian Chemical Society, 70, 609-624. http://dx.doi.org/10.2298/JSC0504609M
[8]	Bayramoglu, G., Kaçar, Y., Denizli, A. and Arica, M.Y. (2002) Covalent Immobilization of Lipase onto Hydrophobic Group Incorporated Poly(2-Hydroxyethyl Methacrylate) Based Hydrophilic Membrane Matrix. Journal of Food Engineering, 52, 367-374. http://dx.doi.org/10.1016/S0260-8774(01)00128-5
[9]	Dave, R. and Madamwar, D. (2006) Esterification in Organic Solvents by Lipase Immobilized in Polymer of PVA-Alginate-Boric Acid. Process Biochemistry, 41, 951-955. http://dx.doi.org/10.1016/j.procbio.2005.10.019
[10]	Yong, Y., Bai, Y., Li, Y., Lin, L., Cui, Y. and Xia, C. (2008) Characterization of Candida rugosa Lipase Immobilized onto Magnetic Microspheres with Hydrophilicity. Process Biochemistry, 43, 1179-1185. http://dx.doi.org/10.1016/j.procbio.2008.05.019
[11]	Cruz, J.C., Pfromm, P.H. and Rezac, M.E. (2009) Immobilization of Candida antarctica Lipase B on Fumed Silica. Process Biochemistry, 44, 62-69. http://dx.doi.org/10.1016/j.procbio.2008.09.011
[12]	Tzialla, A.A., Pavlidis, I.V., Felicissimo, M.P., Rudolf, P., Gournis, D. and Stamatis, H. (2010) Lipase Immobilization on Smectite Nanoclays: Characterization and Application to the Epoxidation of Alfa-Pinene. Bioresource Technology, 101, 1587-1594. http://dx.doi.org/10.1016/j.biortech.2009.10.023
[13]	Ting, W.J., Tung, K.Y., Giridhar, R. and Wu, W.T. (2006) Application of Binary Immobilized Candida rugosa Lipase for Hydrolysis of Soybean Oil. Journal of Molecular Catalysis B: Enzymatic, 42, 32-38. http://dx.doi.org/10.1016/j.molcatb.2006.06.009
[14]	Foresti, M.L. and Ferreira, M.L. (2007) Chitosan-Immobilized Lipases for the Catalysis of Fatty Acid Esterifications. Enzyme and Microbial Technology, 40, 769-777. http://dx.doi.org/10.1016/j.enzmictec.2006.06.009
[15]	Tharanathan, R.N. and Kittur, F.S. (2003) Chitin—The Undisputed Biomolecule of Great Potencial. Critical Reviews in Food Science and Nutrition, 43, 61-87. http://dx.doi.org/10.1080/10408690390826455
[16]	Chang, M. and Juang, R. (2004) Stability and Catalytic Kinetics of Acid Phosphatase Immobilized on Composite Beads of Chitosan and Activated Clay. Process Biochemistry, 39, 1087-1091. http://dx.doi.org/10.1016/S0032-9592(03)00221-8
[17]	Bormans, P. (2004) Ceramics Are More than Clay Alone. Cambridge International Science Publishing, Cambridge.
[18]	Freire, D.M., Teles, E.M., Bon, E.P. and Sant’anna Jr., G.L. (1997) Lipase Production by Penicillium restrictum in a Bench-Scale Fermentor: Effect of Carbon and Nitrogen, Agitation and Aeration. Applied Biochemistry and Biotechnology, 63, 409-421. http://dx.doi.org/10.1007/BF02920442
[19]	Shelley, A.W., Deeth, H.C. and Macrae, I.C. (1987) Review of Methods of Enumeration, Detection and Isolation of Lipolytic Microorganisms with Special Reference to Dairy. Journal of Microbiological Methods, 6, 127-137. http://dx.doi.org/10.1016/0167-7012(87)90008-X
[20]	Raeder, J. and Broda, P. (1985) Rapid Preparation of DNA from Filamentous Fungi. Letters in Applied Microbiology, 1, 17-20. http://dx.doi.org/10.1111/j.1472-765X.1985.tb01479.x
[21]	Thompson, J.D., Higgins, D.G., Gibson, T.J. and Clustal, W. (1994) Improving the Sensitivity of Progressive Multiple Alignment through Sequence Weighting, Positions-Specific Gap Penalties and Weight Matrix Choice. Nucleic Acids Research, 22, 4673-4680. http://dx.doi.org/10.1093/nar/22.22.4673
[22]	Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) Software Version 4.0. Molecular Biology and Evolution, 24, 1549-1599. http://dx.doi.org/10.1093/molbev/msm092
[23]	Kimura, M. (1980) A Simple Model for Estimating Evolutionary Rates of Base Substitutions through Comparative Studies of Nucleotide Sequence. Journal of Molecular Evolution, 16, 111-120. http://dx.doi.org/10.1007/BF01731581
[24]	Saitou, N. and Nei, M. (1987) The Neighbor-Joining Method for Reconstructing Phylogenetic Trees. Molecular Biology and Evolution, 4, 406-425.
[25]	Zeraik, A.E., Souza, F.S., Fatibello-Filho, O. and Leite, O.D. (2008) Desenvolvimento de um Spot Test para Monitora- mento da Atividade da Peroxidase em um Procedimento de Purificação. Química Nova, 31, 731-734. http://dx.doi.org/10.1590/S0100-40422008000400003
[26]	Chiou, S. and Wu, W. (2004) Immobilization of Candida rugosa Lipase on Chitosan with Activation of the Hydroxyl Groups. Biomaterials, 25, 197-204. http://dx.doi.org/10.1016/S0142-9612(03)00482-4
[27]	Bradford, M.M. (1976) A Rapid and Sensitive Method for the Quantification of Microgram Quantities of Protein Dye Binding. Analytical Biochemistry, 72, 248-254. http://dx.doi.org/10.1016/0003-2697(76)90527-3
[28]	Hung, T., Giridhar, R., Chiou, S. and Wu, W. (2003) Binary Immobilization of Candida rugosa Lipase on Chitosan. Journal of Molecular Catalysis B: Enzymatic, 26, 69-78. http://dx.doi.org/10.1016/S1381-1177(03)00167-X
[29]	Abbas, H., Hiol, A., Deyris, V. and Comeau, L.C. (2002) Isolation and Characterization of an Extracellular Lipase from Mucor sp. Strain Isolated from Palm Fruit. Enzyme and Microbial Technology, 31, 968-975. http://dx.doi.org/10.1016/S0141-0229(02)00190-4
[30]	Kamini, N.R., Fujii, T., Kurosu, T. and Iefuji, H. (2000) Production, Purification and Characterization of an Extracellular Lipase from the Yeast, Cryptococcus sp. S-2. Process Biochemistry, 36, 317-324. http://dx.doi.org/10.1016/S0032-9592(00)00228-4
[31]	Pastore, G.M., Costa, V.S.R. and Koblitz, M.G.B. (2003) Production, Partial Purification and Biochemical Characterization of a Novell Rhizopus sp. Strain Lipase. Food Science and Technology, 23, 135-140. http://dx.doi.org/10.1590/S0101-20612003000200006
[32]	Wang, D., Xu, Y. and Shan, T. (2008) Effects of Oils-Related Substrates on the Synthetic Activity of Membrane- Bound Lipase from Rhizopus chinensis and Optimization of the Lipase Fermentation Media. Biochemical Engineering Journal, 41, 30-37. http://dx.doi.org/10.1016/j.bej.2008.03.003
[33]	Lopes, C.R., D’oca, C.D.R.M., Duarte, R.C., Kurz, M.H.S., Primel, E.G., Clementin, R.M., Villarreyes, J.A.M. and D’oca, M.G.M. (2010) Síntese de novas amidas graxas a partir da aminólise de ésteres metílicos. Química Nova, 33, 1335-1341. http://dx.doi.org/10.1590/S0100-40422010000600022
[34]	Tashtoush, G.M., Al-Widyan, M.I. and Al-Jarrah, M.M. (2004) Experimental Study on Evaluation and Optimization of Conversion of Waste Animal Fat into Biodiesel. Energy Conversion and Management, 45, 2697-2711. http://dx.doi.org/10.1016/j.enconman.2003.12.009
[35]	Faccio, C., Rosa, C.D., Amroginski, C., Bender, J.P., Lipke, N., Oliveira, D., Oliveira, J.V., Luccio, M. and Dariva, C. (2005) Optimization of Alkaline Transesterification of Soybean Oil and Castor Oil for Biodiesel Production. Applied Biochemistry and Biotechnology, 122, 553-560. http://dx.doi.org/10.1385/ABAB:122:1-3:0553
[36]	Burkert, J.F.M., Maugeri, F. and Rodrigues, M.I. (2004) Optimization of Extracellular Lipase Production by Geotrichum sp Using Factorial Desing. Bioresource Technology, 91, 77-84. http://dx.doi.org/10.1016/S0960-8524(03)00152-4
[37]	Chaurasia, P.K., Yadav, R.S.S. and Yadava, S. (2014) Purification and Characterization of Yellow Laccase from Trametes hirsuta MTCC-1171 and Its Application in Synthesis of Aromatic Aldehydes. Process Biochemistry, 49, 1647- 1655. http://dx.doi.org/10.1016/j.procbio.2014.06.016
[38]	Couto, S.R. (2007) Decolouration of Industrial Azo Dyes by Crude Laccase from Trametes hirsuta. Journal of Hazardous Materials, 148, 768-770. http://dx.doi.org/10.1016/j.jhazmat.2007.06.123
[39]	Jiang, D., Long, S., Huang, J., Xiao, H. and Zhou, J. (2005) Immobilization of Pycnoporus sanguineus Laccase on Magnetic Chitosan Microspheres. Biochemical Engineering Journal, 25, 15-23. http://dx.doi.org/10.1016/j.bej.2005.03.007
[40]	Morrison, R. and Boyd, R. (1996) Química Organica. Fundação Calouste Gulbenkian, Lisboa.
[41]	Guisan, J.M. (2013) Immobilization of Enzymes and Cells. Third Edition, Methods in Molecular Biology, Vol. 1051, Humana Press, Totowa. http://link.springer.com/book/10.1007%2F978-1-62703-550-7 http://dx.doi.org/10.1007/978-1-62703-550-7
[42]	Desai, P.D., Dave, A.M. and Devi, S. (2006) Alcoholysis of Salicornia Oil Using Free and Covalently Bound Lipase onto Chitosan Beads. Food Chemistry, 95, 193-199. http://dx.doi.org/10.1016/j.foodchem.2004.12.030
[43]	Essamri, M., Deyris, V. and Comeau, L. (1998) Optimization of Lipase Production by Rhizopus oryzae and Study on Stability of Lipase Activity in Organic Solvents. Journal of Biotechnology, 60, 97-103. http://dx.doi.org/10.1016/s0168-1656(97)00193-4
[44]	Klibanov, A.M. (1997) Why Are Enzymes Less Active in Organic Solvents than in Water? Trends in Biotechnology, 15, 97-101. http://dx.doi.org/10.1016/s0167-7799(97)01013-5
[45]	Verger, R. (1997) Interfacial Activation of Lipases: Facts and Artifacts. Trends in Biotechnology, 15, 32-38. http://dx.doi.org/10.1016/S0167-7799(96)10064-0
[46]	Yu, H.W., Chen, H., Yang, Y.Y. and Ching, C.B. (2005) Effect of Salts on Activity, Stability and Enantioselectivity of Candida rugosa Lipase in Isooctane. Journal of Molecular Catalysis B: Enzymatic, 35, 28-32. http://dx.doi.org/10.1016/j.molcatb.2005.05.002
[47]	Demir, B.S. and Tukel, S.S. (2010) Purification and Characterization of Lipase from Spirulina platensis. Journal of Molecular Catalysis B: Enzymatic, 64, 123-128. http://dx.doi.org/10.1016/j.molcatb.2009.09.011
[48]	Dandavate, V., Jinjala, J., Keharia, H. and Madamwar, D. (2009) Production, Partial Purification and Characterization of Organic Solvent Tolerant Lipase from Burkholderia multivorans V2 and Its Application for Ester Synthesis. Bioresource Technology, 100, 3374-3381. http://dx.doi.org/10.1016/j.biortech.2009.02.011
[49]	Mittelbach, M. (1990) Lipase Catalysed Alcoholysis of Sunflower Oil. Journal of the American Chemical Society, 67, 168-169.
[50]	Rodrigues, R.C., Volpato, G., Wada, K. and Ayub, M. (2008) Enzymatic Synthesis of Biodiesel from Transesterification Reactions of Vegetable Oils and Short Chain Alcohols. Journal of the American Oil Chemists’ Society, 85, 925- 930. http://dx.doi.org/10.1007/s11746-008-1284-0
[51]	Maceiras, R., Vega, M., Costa, C., Ramos, P. and Márquez, M.C. (2009) Effect of Methanol Content on Enzymatic Production of Biodiesel from Waste Frying Oil. Fuel, 88, 2130-2134. http://dx.doi.org/10.1016/j.fuel.2009.05.007