Synthesis of Biodiesel from Castor Oil and Linseed Oil in Supercritical

Nov 20, 2006 - As these are derived from natural resources, they are biodegradable and nontoxic.1 Natural reserves of petrochemical, coal, and natural...
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Ind. Eng. Chem. Res. 2007, 46, 1-6

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APPLIED CHEMISTRY Synthesis of Biodiesel from Castor Oil and Linseed Oil in Supercritical Fluids Mahesh N. Varma and Giridhar Madras* Department of Chemical Engineering, Indian Institute of Science, Bangalore 560012, India

Biodiesel synthesis (transesterification of triglycerides of higher fatty acid to methyl and ethyl esters) from castor oil and linseed oil using methanol and ethanol was investigated at subcritical and supercritical conditions of methanol and ethanol from 200 to 350 °C at 200 bar. The effect of molar ratio of alcohol to oil, temperature, and time was investigated in supercritical methanol and ethanol. The kinetics of the reaction was first order, and the activation energies were determined from the temperature dependence of the rate coefficients. Biodiesel was also synthesized enzymatically with Novozym 435 in supercritical carbon dioxide (ScCO2). The effect of various parameters such as enzyme loading, alcohol to oil molar ratio, temperature, and time was investigated in these systems. A simplified model, based on the Ping Pong Bi Bi with competitive inhibition mechanism, was proposed to describe the enzymatic transesterification kinetics for castor oil with methanol and ethanol. 1. Introduction Biodiesel is comprised of esters of short chain alcohols made from renewable biological source such as vegetable oil and animal fats and can be used as an alternative diesel fuel. As these are derived from natural resources, they are biodegradable and nontoxic.1 Natural reserves of petrochemical, coal, and natural gas are the current sources of energy and will be exhausted soon at the current usage rate.2 Therefore, the recent focus has been on the renewable energy sources. Oil triglycerides are good alternatives for diesel fuel, but high viscosities, acid composition, and free fatty acid of oil create problems in diesel engines.3 Various methods such as dilution, microemulsions, pyrolysis, catalytic cracking, and transesterification have been considered to overcome the problem associated with direct use of oil. Among all these alternatives, transesterification appears to be the best option.4 In the transesterification of oil, the short chain alcohol is used to replace the glycerol from triglycerides of the oil. The properties of these fatty acid esters are similar to diesel fuels, and methyl and ethyl esters can be used directly in the diesel engine.4 In catalytic transesterification,1-3 different catalysts such as acids (H2SO4, HCl) and alkalis (NaOH, KOH) have been used. Acid and alkali catalyzed processes are energy intensive, the recovery of glycerol is difficult, and the presence of free fatty acid and water interferes with reactions. Therefore, the synthesis of biodiesel by enzyme catalyzed or noncatalyzed methods seems to be a better option. Several researchers have reported that lipases can be effectively used for the enzymatic transesterification of oils.5-13 However, these reactions are mass-transfer limited. Supercritical fluids have densities comparable to that of liquids and diffusivities comparable to that of gases. Among supercritical fluids, carbon dioxidesbeing cheap, nonflammable, and nontoxicsis an obvious choice over organic solvents.12,13 The solubility of higher chain length fatty acid is negligible in carbon dioxide at room temperature and pressure, which makes removal of product * To whom correspondence should be addressed. Fax: 91-080-2360 0683. E-mail: [email protected].

from solvent simple. Thus enzymatic synthesis of biodiesel in supercritical fluids looks promising. The other method is to make the process of synthesis of biodiesel noncatalytic. In this context, the synthesis of biodiesel in supercritical alcohols without catalyst is a promising method to replace the catalytic transesterification process.14,15 While the kinetics of the noncatalytic reaction is slow at low temperatures and pressures,16 rapeseed oil can be converted into its methyl ester15 in less than 5 min at 350 °C and 19 MPa. Thus in this method, free fatty acids can be converted to methyl esters resulting in high yield, and no further purification is required because no separation of catalyst is needed. To the best of our knowledge, this is the first study that investigates the supercritical transesterification of vegetable oils containing primarily ricinoleic acid (castor oil) or linolenic acid (linseed oil). In this study, the synthesis of biodiesel from castor oil and linseed oil in supercritical alcohols without catalyst and in supercritical carbon dioxide (ScCO2) with enzymes was studied. The influence of various parameters such as alcohol to oil molar ratio, temperature, kinetics, and enzyme loading was investigated. For the enzymatic reaction in ScCO2, a Ping Pong Bi Bi mechanism was proposed to model the experimental data. 2. Materials, Methods, and Analysis 2.1. Materials. Castor oil and linseed oil were obtained locally. Specially dried methanol (99.5%) and sulfur free toluene (99%) were purchased from S.D. Fine Chem. Ltd. India. Anhydrous ethanol (99.8%) and glacial acetic acid (99%) were obtained from De Commerce Inc. (Brampton, Ontario) and Merck (India), respectively. Immobilized Novozym 435 was gifted by Novo Nordisk (Denmark). Carbon dioxide from Vinayaka gases (India) was used after dehydrating by passing a gas through a bed of silica gel. All chemicals and distilled deionized water was filtered through microporous membrane before use. Castor oil is a mixture of triglycerides of higher fatty acids of C18 to C20 with 89% of ricinoleic acid (C18) triglycerides. Linseed oil contains C18 triglycerides of oleic, linoleic, and

10.1021/ie0607043 CCC: $37.00 © 2007 American Chemical Society Published on Web 11/20/2006

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primarily linolenic acids. The critical temperature and pressure of methanol4 is 512.2 K and 8.1 MPa, respectively, while that of ethanol4 is 516.2 K and 6.4 MPa. The properties of the methanol and ethanol favor the homogeneous mixing with oil at supercritical conditions because they act as acid catalysts in the supercritical synthesis of biodiesel synthesis.17 2.2. Methods. All the reactions with supercritical ethanol and methanol were performed in a SS 316 reactor of 11 cm3 volume at 200 bar. The required amount of oil and alcohol was calculated using Peng-Robinson equation of state so as to achieve 200 bar pressure at the given temperature. The reactor loaded with reactant was kept in the furnace at the preset temperature maintained within (2 °C. To stop the reaction after the predetermined reaction time, the reactor was quenched by immersing the reactor in a cold water bath. The product was collected, and toluene was used to elute any trace of product left in the reactor. The alcohol and toluene present with product was evaporated at 90 °C, leaving behind the mixture of unreacted oil, ester, and glycerol, and then taken for analysis by gas chromatography. All the enzymatic reactions in ScCO2 were carried out with 7 cm3 tubular batch reactors of SS 316. Each reactor, loaded with reactants and enzyme, was pressurized to an initial pressure of 68 bar at room temperature. The pressurized reactors were then immersed in a water bath maintained at the desired temperature, with fluctuations less than (0.5 °C. All the reactions were conducted at a constant density of CO2 (0.79 g cm-3) for various temperatures. Even though the pressure was higher at every temperature increment, the density of system remained constant. The reactors were equipped with a pressure gauge to ensure that the system operated at the supercritical pressure throughout the reaction. After the desired time, the reactor was depressurized, and the contents were eluted in 3 cm3 of toluene. The enzyme was removed by centrifugation at 4000 rpm for 2 min, and the supernatant reaction mixture was evaporated at 90°C to remove alcohol and toluene and then analyzed by gas chromatography. Many reactions were repeated in triplicate and reproducibility of results was within (2% in conversion to biodiesel. This corresponds to an error of (3 kJ/ mol in the activation energies 2.3. Analysis. The reaction samples were analyzed by gas chromatography (Nucon 5765, India) using a capillary column, BP20 (SGE, Australia; 15 m × 0.53 mm ID × 1 µm film thickness of poly(ethylene glycol)) and a flame ionization detector. Nitrogen was used as the carrier gas at a flow rate of 8 cm3 min-1. The flow rates of oxygen and hydrogen were 8 and 12 cm3 min-1, respectively. The detector and injector temperatures were chosen as 280 °C and 300 °C, respectively. The oven temperature was maintained at a constant temperature of 220 °C. The methyl and ethyl esters for castor oil and linseed oil were synthesized chemically by reacting oils with sodium alkoxide at 60 °C for 20 h with alcohol in excess with 5:1 molar ratio of oil to alcohol. These esters were used for calibration in GC, and a straight line for calibration was obtained. 3. Results and Discussion 3.1. Reaction in Supercritical Methanol and Ethanol. 3.1.1. Effect of Molar Ratio of Alcohol to Oil. The molar ratio of alcohol to oil is an important parameter influencing the conversion of triglycerides to esters in both the catalytic and noncatalytic reactions. In the alkaline catalyzed process, the yield of methyl ester increases with an increase in alcohol-to-oil ratio as the equilibrium shifts toward the product. The optimum ratio of these catalyzed reactions2 is around 6:1, as an excess of

Figure 1. Effect of molar ratio of alcohol to oil for transesterification of (a) castor oil and (b) linseed soil at 200 bar for 40 min. Legends: methanol at 200 °C (9), methanol at 250 °C ([), methanol at 300 °C (2), methanol at 350 °C (b), ethanol at 200 °C (0), ethanol at 250 °C (]), ethanol at 300 °C (4), ethanol at 300 °C (O).

alcohol makes the separation of glycerin from product difficult. A very high molar ratio of 45 is required for complete conversion when oil contains a high amount of free fatty acids.15 For noncatalytic reactions in supercritical alcohol, Saka and Kusdiana15 have suggested that a higher molar ratio of alcohol is required for better transesterification as excess of alcohol increases the contact area between triglycerides and alcohol. In this work, the effect of molar ratio was studied for castor oil and linseed oil with subcritical (200 °C) and supercritical (250-350 °C) methanol and ethanol. The molar ratio of alcohol to oil was varied from 10 to 70 at four different temperatures with reaction time of 40 min. Figure 1a,b shows there is increase in conversion with increase in molar ratio up to 40:1. The results obtained show good agreement with previous work, where maximum conversion was obtained for rapeseed oil15 at a molar ratio of 42:1 and for various vegetable oils18 at molar ratio of 41:1. 3.1.2. Effect of Temperature. The effect of temperature on the synthesis of biodiesel was studied between 200 to 350 °C at fixed molar ratio of 40:1. The results obtained are shown in Figure 2a,b. The conversions obtained at 200 °C after 60 min are 55% and 27%, for transesterification of castor oil with methanol and ethanol, respectively, and 29% and 11% for transesterification of linseed oil with methanol and ethanol, respectively. These conversions are quite low as compared to previous work15 where rapeseed oil was converted 68% and 70% to methyl ester at 200 °C and 230 °C, respectively, in 60 min. A 60% conversion18 was obtained in the case of methyl ester of various vegetable oils at 220 °C in 5 min. The transesterification of soybean oil with methanol16 showed a conversion of only 11% and 20% at 220 and 235 °C. At 350 °C, the reaction is very fast with more than 65% conversion

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Figure 3. Arrhenius plot for transesterification of castor oil with methanol (9), ethanol ([), linseed oil with methanol (2), and ethanol (b)

Figure 2. Kinetics of transesterification reaction for (a) castor oil and (b) linseed oil at 200 bar and 40:1, alcohol to oil molar ratio. See Figure 1 for legends.

achieved within 10 min, and nearly complete conversion is achieved in less than 40 min for both castor oil and linseed oil with both alcohols. At temperatures above 350 °C, decomposition occurs. 3.1.3. Kinetics of Reaction. The conversions obtained for various time intervals at four different temperaturess200, 250, 300, and 350 °Csfor both castor oil and linseed seed oil with methanol and ethanol are shown in Figure 2a,b. The thermal transesterification reaction is divided into three steps. Triglycerides reacts with alcohol to form diglyceride, and then diglyceride reacts to form monoglyceride that further reacts to form alkyl esters and glycerol.16 Each reaction step is assumed to be of first order with respect to each reacting component and irreversible. The complete reaction system can be simplified into a single step,15 and the order of the reaction and the rate coefficient of the reaction can be directly obtained from the intercept and slope of a semi-log plot of the reaction rate with oil concentration.15 The order of the reaction is nearly unity for both castor oil and linseed oil reactions with methanol and ethanol. Figure 3 shows an Arrhenius plot for all four reactions that depicts the variation of the rate coefficients with temperature. The activation energies determined from the slope of the regressed line of Arrhenius plot (Figure 3) are 35, 55, 46.5, and 70 kJ mol-1 for castor oil methyl ester, castor oil ethyl ester, linseed oil methyl ester, and linseed oil ethyl ester, respectively. Activation energies16 of 117, 128, and 29 kJ mol-1 were observed for reaction of triglyceride to diglyceride, diglyceride to monoglycride, and monoglycride to methyl ester, respectively, during the transesterification of soybean oil with methanol. The activation energies obtained in this study are comparable to that of 69 and 39 kJ mol-1 observed for a single step mechanism for the synthesis of rapeseed oil methyl esters15 and various vegetable oil methyl esters,18 respectively.

The rate constants for the transesterification of various vegetable oils13,19-21 and the fatty acid compositions are shown in Table 1. The rate constants are for the transesterification in supercritical methanol at 300 °C at 190-200 bar with a molar ratio of methanol to oil varying between 40 and 43. It is interesting to evaluate how the rate constant is influenced by the composition of the vegetable oil. As the saturated fatty acid triglyceride content decreases in the order of palm oil < palm kernel < coconut, the rate constant also decreases in the same order. Similarly, the monosaturated fatty acid (oleic) triglyceride decreases in the order of sunflower < soybean < groundnut, and the rate constant also decreases in the same order. For the same set, this also can be looked at as an increase in the diunsaturated acid content (linoleic) and a corresponding decrease in the rate constants in the same order. Linseed oil, which contains the highest amount of triunsaturated acid (linolenic), has the slowest rate among all oils investigated. This clearly shows that the transesterification reaction rate is the highest for the triglycerides of saturated fatty acid followed by triglycerides of unsaturated acids. Thus, oils that contain triglycerides of triunsaturated fatty acids will react the slowest compared to triglycerides of diunsaturated or monounsaturated fatty acids, whose reaction rates would be slower than that of triglycerides of saturated fatty acids. Castor oils contain a uniquely high level of hydroxy fatty acid, ricinoleic acid, which is very different from other oil compositions. Thus, the transesterification reaction rates cannot be compared with other vegetable oils. The hydroxyl functionality is rare in plant oils, and the presence of ricinoleic acid, which is a complex fatty acid that contains both a double bond and a hydroxyl group, can impart increased lubricity of the oil and its derivatives as compared to other vegetable oils22,23 and makes it a prime candidate as an additive for diesel fuel. 3.2. Enzymatic Transesterification in Supercritical Carbon Dioxide. All the transesterification reactions of castor oil and linseed oil with methanol and ethanol were also carried out in supercritical carbon dioxide using Novozym 435. The conversion obtained for linseed oil was less than 5-7% and, therefore, not considered for detailed study. 3.2.1. Effect of Enzyme Loading. The effect of enzyme loading was investigated by varying enzyme loading from 5 to 60 mg with 100 µL of oil, 5:1 alcohol molar ratio, at 50 °C for 12 h. Conversion obtained for transesterification of castor oil with methanol and ethanol are plotted in Figure 4a. The results show there is increase in conversion with increasing enzyme loading up to 20 wt % of castor oil, where conversions are 45% and 35%. Further increase in enzyme loading does not show

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Table 1. Fatty Acid Composition (>10%) of Vegetable Oils and the Rate Constant of Transesterification at 300 °C in Supercritical Methanol

oil

yotal saturated acids

monounsaturated acid (oleic)

diunsaturated acid (linoleic)

triunsaturated acid (linolenic)

rate constant k (s-1) × 103

ref

coconut palm kernel palm groundnut soybean sunflower linseed

88.48 85.04 42.6 11.4 14.9 -

8.8 12.56 40.5 48.3 22.1 17.7 18.9

10.1 32.0 55.0 72.9 18.1

55.1

5.9 2.9 1.9 1.85 1.7 0.96 0.69

20 20 21 21 19 13 this study

any further increase in conversion. This is consistent with results reported earlier13 that show maximum conversions for transesterification of sunflower oil for 30 wt % enzymes loading and above. 3.2.2. Effect of Molar Ratio of Alcohol to Oil. Experiments were carried out up to alcohol to oil molar ratio of 10 with 100 µL of castor oil, 10 mg of Novozym 435, 50 °C for 12 h (Figure 4b). The conversion to methyl and ethyl esters of castor oil increases with alcohol to oil molar ratio up to a molar ratio of 5 at which conversions of 45 and 35% are obtained for methyl and ethyl ester, respectively. An initial addition of alcohol increases the conversion as this shifts the reactions toward product side. At higher ratios, the conversion decreases, which can be attributed to excess alcohol distorting the essential water layer and thus destabilizing the enzymes.24 The presence of methanol and ethanol has been shown to have an inhibitory effect on the activity of enzymes.25 The observation of an optimum ratio of 5:1 obtained in this study is consistent to that obtained (6:1) for the alcoholysis of palm kernel oil12 in supercritical carbon dioxide at 40 °C and 73 bar and a ratio of 5:1 for the transesterification of sunflower oil.13

3.2.3. Effect of Temperature. The influence of temperature was investigated by conducting experiments with 100 µL of oil, 10 mg of Novozym 435, 5:1 alcohol molar ratio, for 12 h from 30 to 70 °C. The conversion initially increases with temperature and reaches a maximum. A further increase in temperature leads to a decrease in conversion. The decrease in conversion at high temperatures could be attributed to the deactivation of enzyme. The results (Figure 4c) show an optimum temperature of 50 °C and 45 °C, with a maximum possible conversion of 45% and 38% for transesterification of castor oil with methanol and ethanol, respectively. These results are in good agreement with reported results of optimum temperatures of 40°C for Candida rugosa lipase under highpressure carbon dioxide for hydrolysis of tuna oil26 and of 45°C with Novozym 435 for transesterification of sunflower oil13 with methanol and ethanol. 3.2.4. Kinetics of Reaction. The kinetics of reaction was studied with 100 µL of oil, 10 mg of Novozym 435, 5:1 alcohol to oil molar ratio, at 50 °C for 20 h. Figure 4d shows that 35% and 28% conversions are achieved in the first 4 h for the transesterification in methanol and ethanol, respectively. How-

Figure 4. (a) Effect of enzyme loading during enzymatic transesterification of castor oil in ScCO2 with 100 µL of oil, 5:1 alcohol to oil molar ratio, at 50 °C for 12 h. Legends: methanol (9), ethanol ([). (b) Effect of alcohol to oil molar ratio during enzymatic transesterification of castor oil in ScCO2 with 100 µL of oil, 10 mg enzyme, at 50 °C for 12 h. See part a for legends. (c) Effect of temperature during enzymatic transesterification of castor oil in ScCO2 with 100 µL of oil, 10 mg enzyme, 5:1 alcohol to oil molar ratio for 12 h. See part a for legends. (d) Kinetics of enzymatic transesterification of castor oil in ScCO2 with 100 µL of oil, 10 mg enzyme, 5:1 alcohol to oil molar ratio, at 50 °C. See part a for legends.

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which can be simplified to a single step.15 This has been used for modeling the transesterification of sunflower oil with butanol6 in hexane as a solvent with lipase from Rhizomucor miehei as the catalyst. For this system, the equation of the Ping Pong Bi Bi mechanism is27

Vi )

Vmax CoCa Co Ca CoCa + KaCo 1 + + KoCa 1 + Kio Kia

[ ]

[

]

(1)

where Co and Ca are the initial molar concentration of castor oil and alcohol (methanol or ethanol), Ko and Ka are apparent Michaelis constants, and Kio and Kia are apparent inhibition constants for castor oil and alcohol, respectively, and Vmax is the maximum initial maximum velocity. The kinetic parameters for the equation are determined by fitting the equation to the experimental data for the transesterification of castor oil in methanol and ethanol. In both the cases, the apparent inhibition constant, Kio, is very high, and therefore, the term was neglected and the modified equation is

Vi )

Figure 5. (a) Lineweaver and Burk representation of transesterification of castor oil with methanol, with 10 mg enzyme at 50 °C. Legends represent methanol concentration: 22.28 mmol kg-1 (9), 44.56 mmol kg-1 (2), 111.34 mmol kg-1 ([), 222.78 mmol kg-1 (sideways triangle), 445.6 mmol kg-1 (f), 891.2 mmol kg-1 (b). Solid line represents the corresponding value predicted by the model. (b) Lineweaver and Burk representation of transesterification of castor oil with ethanol, with 10 mg enzyme at 50 °C. Legends represent ethanol concentration. 15.5 mmol kg-1 (9), 31.0 mmol kg-1 (2), 77.5 mmol kg-1 ([), 155.0 mmol kg-1 (sideways triangle), 310.0 mmol kg-1 (f), 620.0 mmol kg-1 (b). Solid line represents the corresponding value predicted by the model. Table 2. Kinetic Parameters for the Ping Pong Bi Bi Mechanism for Transesterification of Castor Oil with Methanol and Ethanol

methanol ethanol

Vmax mmol kg-1 h-1

Ka mmol kg-1

Ko mmol kg-1

Kia mmol kg-1

18.5 13.7

5.9 10.7

32.2 34.5

24.1 24.1

ever, in next 4 h, conversion increased only by 9% and 5%, respectively. Conversion did not increase with further reaction time. The reaction for transesterification of castor oil with methanol and ethanol is slow compared to the ethanolysis of palm kernel oil12 catalyzed by Novozym 435 at 40 °C, and transesterification of sunflower oil with methanol and ethanol.13 The effect of castor oil and alcohol concentration on the initial rate of reaction was investigated. Figure 4d shows the variation of conversion with time is linear for the initial 2-3 h of reaction; hence reaction time of 1 h was selected for calculating initial rates of reactions. The concentration was taken in the range of 0.93-37.33 mmol kg-1 for castor oil, 22.28-891.2 mmol kg-1 for methanol, and 15.5-620 mmol kg-1 for ethanol with an enzyme loading of 10 mg at 50 °C. The presence of alcohol inhibits the activity of enzyme for esterification and transesterification reaction.6,27,28,29 The kinetics of reaction with inhibition can be explained by the Ping Pong Bi Bi reaction mechanism, which has been successfully 6,27,28,29 used for modeling the esterification and transesterification reactions. The transesterification of oil is explained by a three-step mechanism for conversion of triglycerides to corresponding alcohol esters,16

Vmax CoCa

[ ]

Ca CoCa + KaCo + KoCa 1 + Kia

(2)

Figure 5a,b shows the comparison of experimental data and model prediction for the Lineweaver-Burk plot of the reciprocal of the enzymatic reaction velocity (1/Vi) versus the reciprocal of the substrate concentration (1/[s]) for the transesterification of castor oil with methanol and ethanol, respectively. The initial velocities are found to be faster for transesterification with methanol as compared to ethanol, and the parameters of the model are shown in Table 2. 4. Conclusions This study investigated the noncatalytic transesterification of castor oil and linseed oil in supercritical methanol and ethanol. High conversions were obtained at a short time at supercritical conditions. The effect of molar ratio on the conversion was also investigated, and the conversion increases with molar ratio of alcohol to oil up to 40:1. The effect of temperature on the rate coefficients was determined. The activation energies for transesterification of castor oil are 35 and 55 and of linseed oil are 46.5 and 70 kJ mol-1 with methanol and ethanol, respectively. The enzymatic transesterification of castor oil and linseed oil in supercritical carbon dioxide was also investigated. While the conversion of linseed oil was less than 7% in ScCo2, the highest conversion of 50% and 37% was observed for the conversion of castor oil with methanol and ethanol, respectively An optimum alcohol to oil molar ratio of 5:1, enzyme loading of 20 wt % oil, and an optimum temperature of 45-50 °C was observed. A simple model based on the Ping Pong Bi Bi mechanism was able to simulate the experimental data satisfactorily. Acknowledgment The authors thank the ministry of human resource and development, India and department of biotechnology, India for financial support. Literature Cited (1) Ma, F.; Hanna, M. A. Biodiesel Production: A Review. Bioresour. Technol. 1999, 70, 1.

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(2) Meher, L. C.; Vidya Sagar, D.; Naik, S. N. Technical Aspects of Biodiesel Production by Transesterification-A Review. Renew. Sust. Energ. ReV. 2006, 10, 248. (3) Fukuda, H.; Kondo, A.; Noda, H. Biodiesel Fuel Production by Transesterification of Oils. J. Biosci. Bioeng. 2001, 92, 405. (4) Demirbas, A. Biodiesel Production via Non-Catalytic SCF Method and Biodiesel Fuel Characteristics. Energy ConVers. Mgmt. 2006, 47, 2282. (5) Shah, S.; Sharma, S.; Gupta, M. N. Biodiesel Preparation by LipaseCatalyzed Transesterification of Jatropha Oil. Energy Fuel. 2004, 18, 154. (6) Dossat, V.; Combes, D.; Marty, A. Lipase-Catalysed Transesterification of High Oleic Sunflower Oil. Enzyme Micro. Technol. 2002, 30, 90. (7) Lai, C. C.; Zullaikah, S.; Vali, S. R.; Ju, Y. H. Lipase-Catalyzed Production of Biodiesel From Rice Bran Oil. J. Chem. Technol. Biotechnol. 2005, 80, 331. (8) Du, W.; Xu, Y. Y.; Liu, D. H.; Li, Z. B. Study on Acyl Migration in Immobilized Lipozyme TL-Catalyzed Transesterification of Soybean Oil for Biodiesel Production. J. Mol. Catal. B-Enzym. 2005, 37, 68. (9) Athawale, V.; Manjrekar, N.; Athawale, M. Lipase-Catalyzed Synthesis of Geranyl Methacrylate by Transesterification: Study of Reaction Parameters. Tetrahedron Lett. 2002, 43, 4797. (10) Salis, A.; Pinna, M.; Monduzzi, M.; Solinas, V. Biodiesel Production from Triolein and Short Chain Alcohols through Biocatalysis. J. Biotechnol. 2005, 119, 291. (11) Noureddini, H.; Gao, X.; Philkana, R. S. Immobilized Pseudomonas cepacia Lipase for Biodiesel Fuel Production from Soybean Oil. Bioresour. Technol. 2005, 96, 769. (12) Oliveira, J. V.; Oliveira, D. Kinetics of the Enzymatic Alcoholysis of Palm Kernel Oil in Supercritical CO2. Ind. Eng. Chem. Res. 2000, 39, 4450. (13) Madras, G.; Kolluru, C.; Kumar, R. Synthesis of Biodiesel in Supercritical Fluids. Fuel 2004, 83, 2029. (14) Demirbas, A. Biodiesel Fuel from Vegetable Oil via Catalytic and Non-Catalytic Supercritical Alcohol Transesterifications and Other Method: a Survey. Energy ConVers. Mgmt. 2003, 44, 2093. (15) Saka, S.; Kusdiana, D.; Kinetics of Transesterification in Rapseed Oil to Biodiesel Fuel as Treated in Supercritical Methanol. Fuel 2001, 80, 693. (16) Diasakov, M.; Loulodi, A.; Papayannakos N. Kinetics of the NonCatalytic Transesterification of Soybean Oil. Fuel 1998, 77, 1297. (17) Krammer, P.; Vogel, H. Hydrolysis of Esters in Subcritical and Supercritical Water. Supercrit. Fuids. 2000, 16, 189.

(18) Demirbas, A. Biodiesel from Vegetable Oil via Transesterification in Supercritical Methanol. Energy ConVers. Mgmt. 2002, 43, 2349. (19) Han, H.; Cao, W.; Zhang, J. Preparation of Biodiesel from Soybean Oil using Supercritical Methanol and Carbon dioxide as cosolvent. Process Biochem. 2005, 40, 3148. (20) Bunyakiat, K.; Makmee, S.; Sawangkeaw, R.; Ngamprasertsith, S. Continuous Production of Biodiesel via Transesterification from Vegetable Oils in Supercritical Methanol. Energy Fuel. 2006, 20, 812. (21) Rathore, V. Synthesis of Biodiesel in Supercritical Fluids. M. Sc. Thesis, Indian Institute of Science, 2006. (22) Drown, D. C.; Harper, K.; Frame, E. Screening Vegetable Oil Alcohol Esters as Fuel Lubricity Enhancers. JAOCS 2001, 78, 579. (23) Goodrum, J. W.; Geller, D. P. Influence of Fatty Acid Methyl Ester from Hydroxylated Vegetable Oils on Diesel fuel lubricity. Bioresour. Technol. 2005, 96, 851. (24) Selmi, B.; Thomas, D. Immobilized Lipase-Catalyzed Ethanolysis of Sunflower Oil in a Solvent-Free Medium. J. Am. Oil Chem. Soc. 1998, 75, 691. (25) Shimada, Y.; Watanabe, Y.; Sugihara, A.; Tominaga, Y. Enzymatic Alcoholysis for Biodiesel Fuel Production and Application of the Reaction to Oil Processing. J. Mol. Catal. B-Enzym. 2002, 17, 133. (26) Yan, H.; Nagahama, K. Activity of Free Candida rugosa Lipase in Hydrolysis Reaction of Tuna oil under High-Pressure Carbon Dioxide. J. Chem. Eng. Jpn. 2003, 36, 557. (27) Rizzi, M.; Stylos, P.; Riek, A.; Reuss, M. A Kinetic Study of Immobilized Lipase Catalyzing the Synthesis of Isoamyl acetate by Transesterification in n-Hexane. Enzyme Microb. Technol. 1992, 14, 709. (28) Chulalaksananukul, W.; Condoret, J. S.; Combes, D. Geranyl Acetate Synthesis by Lipase Catalyzed Transesterification in Supercritical Carbon Dioxide. Enzyme Microb. Technol. 1993, 15, 181. (29) Marty, A.; Chulalaksananukul, W.; Willemot, R. M.; Condoret, J. S. Kinetics of Lipase Catalyzed Esterification in Supercritical CO2. Biotechnol. Bioeng. 1992, 399, 273.

ReceiVed for reView June 3, 2006 ReVised manuscript receiVed September 18, 2006 Accepted September 23, 2006 IE0607043