Research Article | Volume: 5, Issue: 2, March-April, 2017

Studies on the Optimization of Lipase Production by Rhizopus sp. ZAC3 Isolated from the Contaminated Soil of a Palm Oil Processing Shed

Zainab Adenike Ayinla Adedeji Nelson Ademakinwa Femi Kayode Agboola   

Open Access   

Published:  Mar 20, 2017

DOI: 10.7324/JABB.2017.50205

This study investigated the screening, production and optimization of an extracellular lipase from a fungus isolated from the contaminated soil of a palm oil processing shed. This was with a view to obtaining a strain that can secrete lipase with biochemical properties exploitable for biotechnological applications such as bioremediation of oil contaminated sites. Soil samples were collected from palm oil contaminated sites in Gbogan, Osun State, Nigeria (Latitude N 7°29.1481´ and Longitude E 4°20.7587´). The isolated fungal strains were screened on tributyrin agar for exogenous lipolytic activity. Molecular identification was carried out by amplifications of ITS-1, 5.8S and ITS-2 regions. The effects of incubation time, inducers, pH, temperature, carbon and nitrogen sources were varied for optimal lipase production using one factor at a time approach. Rhizopus oryzae ZAC3 (NCBI accession No: KX035094) was identified as the highest lipase-producing strain. Maximum lipase production was observed on the fourth day, pH 5.0 and a temperature of 45 oC. Olive oil, xylose and yeast extract were the best inducer, carbon and nitrogen sources respectively for lipase production. There was a 2.02 fold increase in lipase production under these optimized conditions. In conclusion, Rhizopus oryzae ZAC3 lipase has properties exploitable for industrial and biotechnological applications.

Keyword:     LipaseRhizopus oryzae ZAC3thermophilic fungiITS regionscreeningoptimization.


Ayinla ZA, Ademakinwa AN, Agboola FK. Studies on the Optimization of Lipase Production by Rhizopus sp. ZAC3 Isolated from the Contaminated Soil of a Palm Oil Processing Shed. J App Biol Biotech. 2017; 5 (02): 030-037. DOI: 10.7324/JABB.2017.50205

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

HTML Full Text

1. Salihu A, Alam MZ. Production and applications of microbial lipases: A review. Scientific Research and Essays. 2012; 7(30):2667-2677.

2. Sharma R, Chisti Y, Banerjee UC. Production, purification, characterization, and applications of lipases. Biotechnology Advances. 2001; 19:627- 662.

3. Saxena RK, Sheoran A, Giri B, Davidson WS. Purification strategies for microbial lipases. Journal of Microbiological Methods. 2003; 52:1-18.

4. Savitha J, Srividya S, Jagat R. Identification of potential fungal strain(s) for the production of inducible, extracellular and alkalophilic lipase. African Journal of Biotechnology. 2007; 6(5):564-568.

5. Laachari F, El Bergadi F, Sayari A, Elabed S, Mohammed I, Harchali EH, Ibnsouda SK Biochemical characterization of a new thermostable lipase from Bacillus pumilus strain. Turkish Journal of Biochemistry. 2015; 40(1):8-14.

6. Jaeger K, Eggert T. Lipases for biotechnology. Current Opinion in Biotechnology. 2002; 13: 390-397.

7. Treichel H, Oliveira DD, Mazuti MA, Luccio MD, Oliveira JV. A review on microbial lipases production. Food Bioprocess Technology. 2010; 3:182-196.

8. Gandhi NN. Applications of Lipases. Journal of the American Oil Chemists’ Society. 1997; 74(6): 621-634.

9. Vakhlu J, Kour A. Yeast lipases: Enzyme purification, biochemical properties and gene cloning. Electronic Journal of Biotechnology. 2006; 9 (1):15.

10. Diaz JCM, Rodriguez JA, Roussos S, Cordova J, Abousalham A, Carriere F. Lipase from the thermotolerant fungus Rhizopus homothallicus is more thermostable when produced using solid state fermentation than liquid fermentation procedures. Enzyme and Microbial Technology. 2006; 39(5):1042-1050.

11. Gupta R, Gupta N, Rathi P. Bacterial lipases: An overview of production, purification and biochemical properties. Applied Microbiology and Biotechnology. 2004; 64:763-781.

12. Souissi N, Bougatef A, Triki-ellouz Y, Nasri M. Production of lipase and biomass by Staphylococcus simulans grown on sardinella (Sardinella aurita) hydrolysates and peptone. African Journal of Biotechnology. 2009; 8(3):451-457.

13. Srivastava ML. Fermentation Technology, Alpha Science, Oxford. United Kingdom, 2008; pp. 404.

14. Rapp P, Backhaus S. Formation of extracellular lipases by filamentous fungi, yeasts and bacteria. Enzyme and Microbial Technology. 1992; 14(11):938-981.

15. Ertugrul S, Donmez G, Takac S. Isolation of lipase producing Bacillus sp. from olive mill wastewater and improving its enzyme activity. Journal of Hazardous Materials. 2007; 149:720-724.

16. Hiol A, Jonzo M, Rugani N, Druet D, Sarda L, Comeau L. Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit. Enzyme and Microbial Technology. 2000; 26(5-6):421-430.

17. Ghorbel S, Souissi N, Triki-Ellouz Y, Dufosse L, Guerard F, Nasri M. Preparation and testing of Sardiella protein hydrolysates as nitrogen source for extracellular lipase production by Rhizopus oryzae. World Journal of Microbiology and Biotechnology. 2005; 21(1):33-38.

18. Nahas E. Control of lipase production by Rhizopus oligosporus under various growth conditions. Journal of General Microbiology. 1988; 134:227-233.

19. Iftikhar T, Niaz M, Afzal M, Haq IU, Rajoka MI. Maximization of intracellular lipase production in a lipase-overproducing mutant derivative of Rhizopus oligosporus DGM 31: a kinetic study. Food Technology and Biotechnology. 2008; 46(4):402-412.

20. Aisaka K, Terada O. Purification and properties of lipase from Rhizopus japonicus. Journal of Biochemistry. 1981; 89(3):817-822.

21. Iwai M, Tsujisaka Y. The purification and the properties of the three kinds of lipase from Rhizopus delemar. Agricultural Biology and Chemistry 1974; 38:1241-1247.

22. Salihu A, Alam MZ, AbdulKarim MI, Salleh HM. Optimization of lipase production by Candida cylindracea in palm oil mill effluent based medium using statistical experimental design. Journal of Molecular Catalysis B: Enzymatic. 2011; 69(1):66-73.

23. Nwuche CO, Aoyagi H, Ogbonna JC. Lipase production from palm oil mill effluent by Aspergillus terreus immobilized on Luffa sponge. Journal of Applied Sciences. 2013; 13:5661-5671.

24. Asih DR, Alam MZ, Salleh MN Salihu A. Pilot-scale production of lipase using palm oil mill effluent as a basal medium and its immobilization by selected materials. Journal of Oleo Science. 2014; 63(8):779-785.

25. Lanka S, Pydipalli M, Latha JNL. Optimization of Process Variables for Extracellular Lipase Production from Emericella nidulans NFCCI 3643 Isolated from Palm Oil Mill Effluent (POME) Dump Sites Using OFAT Method. Research Journal of Microbiology. 2015; 10(2):38-53.

26. Freire DM, Teles EMF, Bom EPS, Sant’Anna JrGL. Lipase production by Penicillium restrictum in a bench-scale fermenter: effect of carbon and nitrogen nutrition agitation and aeration. Applied Biochemistry and Biotechnology. 1997; 63-65:409-421.

27. Colen G, Junqueira RG, Moraes-Santos T. Isolation and screening of alkaline lipase production fungi from Brazil savanna soil. World Journal of Microbial Biotechnology. 2005; 22(8):881-885.

28. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, 1990, pp. 315-322. In MA Innis, DH Gelfand, JJ Sninsky, TJWhite (ed.). PCR protocols: a guide to methods and applications. Academic Press, Inc., New York, N.Y.

29. Ademakinwa AN, Agboola FK. Biochemical characterization and kinetic studies on a purified yellow laccase from newly isolated Aureobasidium pullulans NAC8 obtained from soil containing decayed plant matter. Journal of Genetic Engineering and Biotechnology. 2016;

30. Vorderwulbecke T, Kieslich K, Erdmann H. Comparison of lipases by different assays. Enzyme Miicrobiology Technology. 1992; 14:632-639.

31. Salleh AB, Musani R, Razak CNA. Extra and intracellular lipases from a thermophilic Rhizopus oryzae and factors affecting their production. Canadian Journal of Microbiology. 1993; 39 (10):978-981.

32. Essamri M, Deyris V, Comeau L. Optimization of lipase production by Rhizopus oryzae and study on the stability of lipase activity in organic solvents. Journal of Biotechnology. 1998; 60:97-103.

33. Mukhtar H, Khursheed S, Ul-Haq I, Mumtaz MW, Rashid U, Al-Resayes SI. Optimization of Lipase Biosynthesis from Rhizopus oryzae for Biodiesel Production Using Multiple Oils. Chemical Engineering Technology. 2016; 39:1-10.

34. Musa H, Adebayo-Tayo BC. Screening of microorganisms isolated from different environmental samples for extracellular lipase production. Assumption University. Journal of Technology. 2012;15(3):179-186.

35. Adinarayana K, Raju NB, Zargar MI, Devi RB Lakshmi PJ, Ellaiah P. Optimization of process parameters for production of lipase in solid-state fermentation by newly isolated Aspergillus species. Indian Journal of Biotechnology. 2004; 3:65-69.

36. Cihangir N, Sarikaya E. Investigation of lipase production by a new isolate of Aspergillus sp. World Journal of Microbiology and Biotechnology. 2004; 20(2):193-197.

37. Bigey F, Tuery K, Bougard D, Nicaud J, Moulin G. Identification of triacylglycerol lipase gene family in Candida deformans: Molecular cloning and functional expression. Yeast. 2003; 20(3):233-248.

38. Stern AM, Ordal ZJ, Halvorson HO. Utilization of fatty acids by and lipolytic activities of Mucor mucedo. Journal of Bacteriology. 1953; 68(1):24-27.

39. Abbas H, Hiol A, Deyris V, Comeau L. Isolation and Characterization of an extracellular lipase from Mucor sp strain isolated from palm fruit. Enzyme and Microbial Technology. 2002; 31(7):968-975.

40. Nagy V, Toke ER, Keong LC, Szatzker G, Ibrahim D, Omar IC, Szakacs G, Poppe L. Kinetic resolutions with novel, highly enantioselective fungal lipases produced by solid state fermentation. Journal of Molecular Catalysis B: Enzymatic. 2006; 39:141-148.

41. Leathers TD, Rich JO, Anderson AM, Manitchotpisit P. Lipase production by diverse phylogenetic clades of Aureobasidium pullulans. Biotechnology Letters. 2013; 35(10):1701-1706.

42. Ooijkaas LP, Weber FJ, Buitelaar RM, Tramper J, Rinzema A. Defined media and inert supports: Their potential as solid-state fermentation production systems. Trends in Biotechnology. 2000; 18:356-360.

43. Maia MMD, Heasley A, Morais MMC, Melo EHM, Morais Jr MA, Ledingham WM, Filho JLL. Effect of culture conditions on lipase production by Fusarium solani in batch fermentation. Bioresource Technology. 2001; 76:23-27.

44. Yang X, Wang B, Cui F, Tan T. Production of lipase by repeated batch fermentation with immobilized Rhizopus arrhizus. Process Biochemistry. 2005; 40:2095-2103.

45. Rehman S, Bhatti HN, Bhatti IA, Asgher M. Optimization of process parameters for enhanced production of lipase by Penicillium notatum using agricultural wastes. African Journal of Biotechnology. 2011; 10(84):19580-19589.

46. Amin F, Bhatti HN, Rehman S. Optimization of growth parameters for lipase production by Ganoderma lucidum using response surface methodology. African Journal of Biotechnology. 2011; 10(28):5514-5523.

47. Sun SY, Xu Y, Solid-state fermentation for 'whole-cell synthetic lipase' production from Rhizopus chinensis and identification of the functional enzyme. Process Biochemistry. 2008;4:219-224.

48. Edwinoliver NG, Thirunavukarasu K, Naidu RB, Gowthaman MK, Kambe TN, Kamini NR. Scale up of a novel tri-substrate fermentation for enhanced production of Aspergillus niger lipase for tallow hydrolysis. Bioresource Technology. 2010; 101:6791-6796.

49. Nunes PA, Pires-Cabral P, Guillen M, Valero F, Luna D, Ferreira-Dias S. Production of MLM-Type structured lipids catalyzed by immobilized heterologous Rhizopus oryzae lipase. Journal of the American Oil Chemists’ Society. 2011; 88:473-480.

50. Papanikolaou S, Dimou A, Fakas S, Diamantopoulou P, Philippoussis A, Galiotou-Panayotou M, Aggelis G. Biotechnological conversion of waste cooking olive oil into lipid-rich biomass using Aspergillus and Penicillium strains. Journal of Applied Mirobiology. 2011; 110:1138-1150.

51. Lakshmi BS, Kangueane P, Abraham B, Pennathur G. Effect of vegetable oils in the secretion of lipase from Candida rugosa (DSM 2031). Letters in Applied Microbiology. 1999; 29:66-70.

52. Knothe G, Dunn, RO, Bagby MO. Biodiesel: The use of vegetable oils and their derivatives as alternative diesel fuels. In: Saha B.C., Woodward, J. (Eds.), Fuels and Chemicals from Biomass, ACS symposium series 666, Washington, DC (Chapter 10); 1997.

53. Zouaoui B, Bouziane A, Ghalem BR. Production, Optimization and Purification of Lipase from Pseudomonas aeruginosa. African Journal of Microbiology. 2012; 6(20):4417-4423.

54. Benjamin S, Pandey A. Optimization of Liquid media for lipase production by Candida rugosa. Bioresource Technology. 1996; 55:167-170.

55. Kantak JB, Bagade AV, Mahajan SA, Pawar SP, Shouche YS, Prabhune AA. Isolation, Identification and Optimization of a New Extracellular Lipase producing Strain of Rhizopus sp. Applied Biochemistry and Biotechnology. 2011; 164(7):969-978.

56. Haltrich D, Nidetzky B, Kulbe KD, Steiner W, Zupancic S. Production of fungal xylanases. Bioresource Technology. 1996; 58:137-161.

57. Shulter ML, Kargi F. Bioprocess Engineering Basic Concept, Prentice Hall of India Pvt Ltd, New Delhi, India; 2000.

58. Kader R, Yousuf A, Hoq MM. Optimization of lipase production by a Rhizopus MR12 in shake culture. Journal of Applied Sciences. 2007; 7(6): 855-866.

59. Norouzian D. Effect of different factors on fermentative production of enzymes by fungi. Dynamic Biochemistry, Process Biotechnology and Molecular Biology. 2008; 2(1):14-18.

60. Edwards C. "Thermophiles". In Microbiology of extreme environments. Edited by C. Edwards. McGraw-Hill Publishing Co., New York. 1990; pp. 1-33.

61. Fariha H, Aamer AS, Abdul H. Industrial applications of microbial lipases. Enzyme and Microbial Technology. 2006;39:235-251.

62. Saranya P, Kumari HS, Rao BP, Sekaran G. Lipase production from a novel thermo-tolerant and extreme acidophile Bacillus pumilus using palm oil as the substrate and treatment of palm oil-containing wastewater. Environmental Science and Pollution Research. 2014; 21:3907-3919.

63. Papaparaskevas D, Christakopoulos P, Kekos D, Macris BJ. Optimizing production of extracellular lipase from Rhodotorula glutinis. Biotechnology Letters. 1992; 14:397-402.

64. Bueno PRM, Ferreira de Oliveira T, Caliari M, Castiglioni GL, Soares Junior MS. Selection and Optimization of extracellular lipase production using Agro industrial waste. African Journal of Biotechnology. 2014; 13(4):566-573.

65. Rajendran A, Thangavelu V. Statistical experimental design for evaluation of medium components for lipase production by Rhizopus arrhizus MTCC 2233. LWT-Food Science and Technology. 2009; 42:985-992.

Article Metrics
127 Views 81 Downloads 208 Total



Related Search

By author names

Similar Articles