PRODUCTION OF BIODIESEL BY LIPASE CATALYSED TRANSESTERIFICATION OF Acacia nilotica SEED OIL
PRODUCTION OF BIODIESEL BY LIPASE CATALYSED TRANSESTERIFICATION OF Acacia nilotica SEED OIL
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Date
2016-05
Authors
GARBA, AUWALU
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Abstract
Biodiesel is becoming prominent among the alternatives source of energy due to its economic and environmental values. This work investigated the possibility of using calcium alginate immobilized lipase from Pseudomonas aeruginosa and response surface methodology to optimize the production of biodiesel from Acacia nilotica seed oil. The physicochemical properties of both A. nilotica seed oil and the biodiesel produced were assessed using standard procedure. The purified lipase from P. aeruginosa was immobilized. Both the purified and immobilized lipases were characterized. The oil was yield (13.5%), specific gravity (0.84), refractive index (1.34), viscosity (4.03 Pa. s) calorific value (29.0) MJ/ kg, cetane number (32.9), iodine value (184.0 g I2/ 100 g), acid value (6.25) mg KOH/g, saponification value (189.0 mg KOH/ g) and peroxide value (6.47 meq/kg). A maximum lipase production was achieved after 60 hours of incubation with activity of 8.6 U/ml. The lipase purified by ion exchange chromatography and gel filtration had a specific activity of 147.7 U/mg proteins with 29.8 % recovery and purification fold of 4.63. The molecular weight was found to be 32 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The Km and Vmax were found to be 0.07 mmole-1 and 55.5 µmole/ min respectively. The respective temperature and pH optima of the lipase were in the range of 45°C and 50 °C and pH range of 7.5 and 8.0. The enzyme was activated in the presence of Ca2+, Mg2+, gum arabic, 2-mercaptoethanol, dithiotreitol, n-hexane and methanol with the residual activity of (110.0, 92.3, 104.4 , 90.2, 102.2, 112.3 and 103.4%) respectively, but it was inactivated by Mn2+, Ni2+, CTAB, S.D.S, EDTA, tween-80, triton -X100, ethanol, iso-propanol and butanol (23.8, 26.1, 42.2, 44.6, 34.2, 47.5, 62.9, 26.1, 49.9 and 28.1%) respectively. The immobilized lipase had pH and temperature optima in the range of 7.5 to 8.5 and 45 to 55°C, respectively and was stable at pH (7.5 to 8.5) and the temperature range of 45°C to 60°C. The Km and Vmax were found to be 0.12 mM and 50.0 µmol/ min respectively. The enzyme was activated in the presence of Mg2, Ca2+ and nhexane, and inactivated by Mn2+, Ni2+, ethanol, iso-propanol, butanol and EDTA. From the analysis of the model developed, the quadratic coefficients of lipase had significant (p<0.05) effects on the biodiesel production. All other linear coefficients were not significant (p>0.05) except for incubation time. Verification of experiments confirmed the validity of the predicted model and maximum yield of biodiesel (55.9%) was obtained with oil/methanol ratio (1:10), nhexane (1.20 ml), immobilized lipase (64 U/ ml) in 195 minutes. The methyl esters composition indicated the presence of octadecenoic (69.14%), hexadecanoic (15.47%) and (7.92%) octadecanoic acid as dominant fatty acid methyl esters. Other properties of the biodiesel produced include specific gravity (0.84), refractive index (1.34 0.04), viscosity (3.73 , cetane number (32.81 ), calorific value (29.0), flash point (113oC), cloud point (1.06oC) and pour point (4.0oC) as well as iodine value (187.6±0.60), acid value (5.61±0.01), saponification value (189 ±1.00) and peroxide value (6.60±0.05), which were largely in conformity with the ASTM and EN biodiesel standards. The ability of immobilized lipase to be repeatedly used makes the production of biodiesel from Acacia nilotica seed oil cost effective.
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A THESIS SUBMITTED TO THE SCHOOL OF POST GRADUATE STUDIES, AHMADU BELLO UNIVERSITY, ZARIA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF A DEGREE OF DOCTOR OF PHILOSOPHY IN BIOCHEMISTRY
DEPARTMENT OF BIOCHEMISTRY, FACULTY OF SCIENCE, AHMADU BELLO UNIVERSITY, ZARIA NIGERIA
Keywords
BIODIESEL,, LIPASE,, TRANSESTERIFICATION,, Acacia nilotica,