Effects of Solvent and Enzyme Source on Transesterification Activity

ARTICLE pubs.acs.org/EF

Effects of Solvent and Enzyme Source on Transesterification Activity Michael D. Gagnon and Palligarnai T. Vasudevan* Department of Chemical Engineering, University of New Hampshire, Durham, New Hampshire 03824, United States ABSTRACT: In this paper, the effect of choice of organic solvent on activity of lipase from three sources, namely Candida antarctica, Pseudomonas cepacia, and Thermomyces lanuginosus is examined. In particular, fifteen hydrophobic and hydrophilic solvents from four organic groups are investigated in the methanolysis of waste soybean oil. The yield of biodiesel (fatty acid methyl ester) is correlated with log P (partition coefficient) of the solvent. The results indicate that the choice of solvent can have a huge effect on both the yield, with values ranging from 2.7 to 87%, with the time to attain equilibrium ranging from 4 h to >24 h. Solvents from the alkane group gave the highest biodiesel yields for all three enzymes, with isooctane and n-hexane being the best performers.

1. INTRODUCTION The majority of biodiesel today is produced by base-catalyzed transesterification of edible oils (triglyceride is the dominant component) with methanol, which results in a relatively short reaction time.1 3 However, the vegetable oil and alcohol must be substantially anhydrous, and the oil must have a low free fatty acid (FFA) content because the presence of water and/or free fatty acid promotes saponification. The soap formed lowers the yield of biodiesel and renders the downstream separation of the products difficult.4,5 Thus, additional steps to remove any water and either the free fatty acids or soap from the reaction mixture are required. Acid-catalyzed processes suffer from a number of drawbacks as well.6 Enzymatic transesterification with lipase as the catalyst eliminates soap formation.7 Unlike alkali-based reactions, the products can easily be collected and separated. Moreover, enzymes require much less alcohol to perform the reaction and can be reused despite some loss in activity at the end of each cycle. Several strains of lipases have been found to have transesterification activity, namely Candida antarctica,8 Pseudomonas cepacia,9 and Thermomyces lanuginosus.9,10 The two primary obstacles in enzyme-catalyzed reactions are (i) the immiscibility of the two substrates, namely, hydrophilic methanol and hydrophobic triglyceride, resulting in the formation of an interface leading to mass transfer resistance; and (ii) the strong polarity of methanol, which tends to strip the active water from the enzyme’s active site leading to enzyme deactivation.11,12 The addition of an organic solvent as the medium to the reaction system simultaneously overcomes the two limitations by enhancing the solubility of oil and methanol in the solvent and by limiting the concentration of methanol surrounding the enzyme. In a previous study, we investigated the effect of organic solvents on the enzymatic transesterification of fresh canola oil.8 Twenty-eight hydrophilic and hydrophobic solvents from seven organic groups were evaluated as possible media in the transesterification of canola oil by Candida antarctica lipase. Iso-octane appeared to be the ideal solvent due to its hydrophobicity and its unique three methyl side-chain molecular structure. Relatively high product yields were also obtained with several hydrophilic solvents possessing high miscibility with methanol, such as dimethoxyethane, methyl iso-propyl ketone (MIPK), and acetone. r 2011 American Chemical Society

In this study, we examined the effect of 15 organic solvents on the enzymatic transesterification of used soybean vegetable oil. Solvents were selected from four distinct functional groups: alkanes, ketones, ethers, and cyclic hydrocarbons. The partition coefficient (log P) was evaluated to correlate hydrophobicity of the solvents with corresponding biodiesel yield. In addition, the effect of solvent on transesterification activity using three different immobilized enzymes (Candida antarctica, Pseudomonas cepacia, and Thermomyces lanuginosus) was also investigated. The use of enzymes to treat oils with a high FFA content is a better process for biodiesel production, and the choice of the right solvent/enzyme to maximize biodiesel yield is critical. This manuscript presents the results of such a study; future studies will involve scale-up and economic analysis.

2. EXPERIMENTAL SECTION Materials. Used soybean vegetable frying oil was obtained from local restaurants and contained 2.3 wt % FFA as determined by titration and NMR analysis.13 Pseudomonas cepacia lipase immobilized on Immobead 150 with an activity of 900 U/g; Thermomyces lanuginosus lipase immobilized on Immobead 150 with an activity of 3000 U/g, and Candida antarctica lipase B immobilized on Immobead 150 with an activity of 5500 U/g, methyl oleate (>98%) (in each case, 1 U corresponds to the amount of enzyme that liberates 1 μmol butyric acid per minute at pH 7.5 and 40 °C with tributyrin, Fluka No. 91010, as substrate).14 ACS grade toluene (99.5+%), 1,2-dimethoxyethane (g99%), 4-heptanol (98%), dodecane (99+%), anhydrous dibutyl ether (99.3%), decane (g99%), HPLC grade 2,2,4-trimethylpentane (isooctane), and 2-butanone were all purchased from Sigma-Aldrich, St. Louis, MO, USA. Reagent grade cyclohexane, ACS grade mixed-xylenes, HPLC grade methanol, and methyl-tert butyl ether (MTBE) were purchased from Fisher Scientific, Pittsburgh, PA, USA. Methyl acetate (98%), extra dry n-hexane (96%), 3-methyl-2-butanone (MIPK), anhydrous diisopropyl ether (99.0%), and 1,4-dioxane (99.0%) were purchased from Acros Organics, Geel, Belgium. Acetone was purchased from EMD Chemicals Inc., Gibbstown, NJ, USA. Synthesis of Biodiesel. The waste oil was heated to 100 °C and passed through a 11.0 cm Whatman 40 filter paper to remove particulates. Received: June 15, 2011 Revised: August 28, 2011 Published: August 30, 2011 4669

dx.doi.org/10.1021/ef2008776 | Energy Fuels 2011, 25, 4669–4674

Energy & Fuels

ARTICLE

Table 1. Log P Values of Solvents alkanes solvent

ketones log P8

solvent

ethers log P8

solvent

cyclic hydrocarbons log P8

solvent

log P8

hexane

3.657

acetone

0.234

dimethoxyethane

0.093

toluene

2.386

iso-octane

3.668

butanone

0.736

MTBE

1.482

xylenes

2.821

decane

5.678

MIPK

0.98

diisopropyl ether

1.775

cyclohexane

3.205

dodecane

6.689

4-heptanone

2.358

dibutyl ether

3.173

Davison molecular sieves with a 4 Å effective pore size (grade 512) were added to the oil to maintain anhydrous conditions. The addition of water can enhance enzyme activity and stability in general. However, in this case, the presence of water can also facilitate the hydrolysis of esters, which will reduce the product yield. The main reason for the use of the molecular sieves was to remove any excess water present in the waste oil. Reactions took place in 40 mL glass vials with PTFE/silicone septa from Kimble, Vineland, NJ, USA. The reaction system contained 500 U of enzyme particles, 4 mL of solvent, 1 mL of used soybean oil, and a molar ratio of methanol to oil of 3.0 (125 μL of methanol). The reaction was maintained at a constant temperature of 40 °C and a stirring speed of 250 rpm. The reaction was stopped after 24 h. Analysis Method. Samples of 30 μL were collected at 4 h, 8 h, and 24 h, respectively, and then diluted with 1 mL of iso-octane for GC analysis.8 The concentration of methyl ester (FAME) was measured by a HP 5890 gas chromatograph with a Restek RTX-1 column (15 m  0.32 mm  3 μm). Helium with a purity of 99.99% was chosen as the carrier gas. The column was initially set at a temperature of 185 °C and ramped up to 200 °C in 1.5 min and then maintained at this value. The temperatures of injector and flame ionization detector were maintained constant at 275 °C. Methyl oleate was employed as the biodiesel standard in the GC analysis. The yield of biodiesel is determined as the mass of FAME produced per mass of oil added (g of biodiesel/g of oil). Selection of Enzymes. The following microbial strains of lipases have been found to have transesterification activity: Candida antarctica15 (CA), Pseudomonas cepacia9 (PC), and Thermomyces lanuginosus10 (TL). The following criteria were used to ensure uniformity in comparison: (i) all three enzymes were purchased from Sigma-Aldrich ensuring similar immobilization procedures and techniques, (ii) immobilization was on the same carrier, Immobead 150, which is a macroporous acrylic polymer bead that is covalently bound to the enzyme and has a particle size of 150 300 μm,16 and (iii) enzymatic activity (500 U) was kept the same in all runs. Selection of Solvents. On the basis of previous work,8 the top performers were chosen for the solvent study. The selection criteria for the solvents were based on the following characteristics: (i) functional group: several solvents were selected from alkanes, ketones, ethers, and cyclic hydrocarbons; (ii) boiling point: typical reaction temperatures for enzymatic processes are 20 60 °C,17 and consequently, the solvents selected had a boiling point above this range, (iii) viscosity: typically, biodiesel has a dynamic viscosity of 4 6.0 cP at 20 to 40 °C,8 and soybean oil has a viscosity of 31.8 cP at 37.8 °C;18 therefore, the desired viscosity for a solvent should be 4 cP or lower, and (iv) partition coefficient (log P) or solvent hydrophobicity. A correlation has been shown to exist between log P value of a solvent and biodiesel yield.8,17,19 Solvents were chosen with log P values between 0 and 7 as shown in Table 1. Solvents with a negative log P value are too hydrophilic and strip away essential water from the enzyme and were eliminated from this study.

3. RESULTS AND DISCUSSION Solvent Effect of Alkanes. These solvents are saturated hydrocarbons that are strongly hydrophobic (high log P), have excellent solubility for organic compounds, and are chemically

stable. We investigated the effect of four alkanes, namely, n-hexane, isooctane, decane, and dodecane, on each of the three lipases. The highest biodiesel yield is obtained with hexane as solvent for all three lipases. However, it is evident from Figures 1a c that the fastest reaction rates are obtained with iso-octane as solvent. For example, in the case of lipase PC (Figure 1b), the reaction levels off at the highest yield in about 4 h or in half the time of 8 h required with other solvents. Of the three enzymes, lipase TL has the best performance with respect to reaction rate and yield for the solvents iso-octane and n-hexane. The lag observed with dodecane may be attributed to its higher viscosity. Lipase CA shows much lower reaction rates with alkane solvents thereby requiring a total reaction time of 24 h to obtain yields equivalent to that of lipase PC and TL. Solvent Effect of Ketones. Solvents with the ketone functional group are widely used in industry as solvents and as biotransformation reaction media.20 The effects of four ketone solvents, namely, acetone, butanone, MIPK, and 4-heptanone on biodiesel yield were investigated. The biodiesel yields with lipase CA for the two solvents MIPK and 4-heptanone are very similar, even though the reaction rate with MIPK is higher (Figure 2a). For lipases PC and TL, the highest biodiesel yields are obtained with 4-heptanone, probably due to its higher log P value (Figures 2b,c). The higher log P value minimizes the solvent’s ability to strip the enzymes of essential water. Solvents like acetone and butanone are hydrophilic (low log P) and have a strong tendency to strip the essential water from the enzyme’s active sites, resulting in a lower yield. This is especially true for lipase TL, which shows very little enzyme activity for transesterification. Solvent Effect of Ethers. Widely used as solvents for a variety of organic compounds and in many reactions, ethers are relatively unreactive.20 The effects of four ether solvents, namely, dimethoxyethane, MTBE, diisopropyl ether, and diisobutyl ether on biodiesel yield were investigated. With lipase CA, the biodiesel yield is in the range of 60 75% indicating that the hydrophobicity of the four solvents does not appear to have much of an effect. The yields for all three lipases using diisopropyl and diisobutyl ethers are fairly similar. However, lipases PC and TL have almost twice the reaction rate of lipase CA (Figures 3a c). For lipases PC and TL, diisopropyl and diisobutyl ethers also give the highest biodiesel yields even though dibutyl ether has a log P value almost double that of diisopropyl ether. Therefore, the structure of the ether molecule may play a more important role than the solvent’s hydrophobicity. It is interesting to note that dimethoxyethane gives a high biodiesel yield with lipase CA (results are similar to what has been reported with fresh canola oil8). However, very low yields are obtained with lipases PC and TL. It indicates that specific biocatalysts have distinct tolerances for the same organic solvent with respect to their particular active sites, but the reason for the difference is still unclear. 4670

dx.doi.org/10.1021/ef2008776 |Energy Fuels 2011, 25, 4669–4674

Energy & Fuels

Figure 1. (a) Effect of alkane solvents on the methanolysis of used soybean oil by Candida Antarctica lipase. Reaction conditions: 1 mL of waste oil, 4 mL of solvent, 500 U of lipase, 40 °C, stir speed of 250 rpm, and 3.0 mol equivalent of methanol (125 μL). (b) Effect of alkane solvents on the methanolysis of used soybean oil by Pseudomonas cepacia lipase. Reaction conditions: 1 mL of waste oil, 4 mL of solvent, 500 U of lipase, 40 °C, stir speed of 250 rpm, and 3.0 mol equiv of methanol. (c) Effect of alkane solvents on the methanolysis of used soybean oil by Thermomyces lanuginosus lipase. Reaction conditions: 1 mL of waste oil, 4 mL of solvent, 500 U of lipase, 40 °C, stir speed of 250 rpm, and 3.0 mol equiv of methanol.

Solvent Effect of Cyclic Hydrocarbons. Industry commonly uses organic solvents that consist of compounds with a six-carbon ring. The effects of three common solvents, two of which are aromatic hydrocarbons (toluene and mixed-xylenes) and the third a cycloalkane (cyclohexane), on biodiesel yield were investigated. For all three lipases, both reaction rates and biodiesel yield are highest with cyclohexane as solvent (Figures 4a c). The performance with cyclohexane is also similar to that of the other alkane solvents (Figure 1a c). With the aromatic solvents, lipase CA shows a small enzyme activity whereas lipase TL shows activity levels similar to the results obtained with other alkanes (Figure 1c). However, this is not true for lipase PC, whose

ARTICLE

Figure 2. (a) Effect of ketone solvents on the methanolysis of used soybean oil by Candida Antarctica lipase. Reaction conditions: 1 mL of waste oil, 4 mL of solvent, 500 U of lipase, 40 °C, stir speed of 250 rpm, and 3.0 mol equiv of methanol. (b) Effect of ketone solvents on the methanolysis of used soybean oil by Pseudomonas cepacia lipase. Reaction conditions: 1 mL of waste oil, 4 mL of solvent, 500 U of lipase, 40 °C, stir speed of 250 rpm, and 3.0 mol equiv of methanol. (c) Effect of ketone solvents on the methanolysis of used soybean oil by Thermomyces lanuginosus lipase. Reaction conditions: 1 mL of waste oil, 4 mL of solvent, 500 U of lipase, 40 °C, stir speed of 250 rpm, and 3.0 mol equiv of methanol.

activity, while similar to lipase TL with alkanes, shows only moderate activity with aromatic solvents. Solvent Effects on Biodiesel Synthesis. Solvents appear to have a significant influence on the yield of biodiesel in the enzymatic transesterification of waste oil with methanol. A wide range of biodiesel yields are observed with all three enzymes used in this study: with lipase CA, the range is 12.5 80.4%; with lipase PC, the range is 18.0 86.9%; and with lipase TL, the range is 2.7 86.6. There appears to be no clear correlation between log 4671

dx.doi.org/10.1021/ef2008776 |Energy Fuels 2011, 25, 4669–4674

Energy & Fuels

ARTICLE

Figure 3. (a) Effect of ether solvents on the methanolysis of used soybean oil by Candida Antarctica lipase. Reaction conditions: 1 mL of waste oil, 4 mL of solvent, 500 U of lipase, 40 °C, stir speed of 250 rpm, and 3.0 mol equiv of methanol. (b) Effect of ether solvents on the methanolysis of used soybean oil by Pseudomonas cepacia lipase. Reaction conditions: 1 mL of waste oil, 4 mL of solvent, 500 U of lipase, 40 °C, stir speed of 250 rpm, and 3.0 mol equiv of methanol. (c) Effect of ether solvents on the methanolysis of used soybean oil by Thermomyces lanuginosus lipase. Reaction conditions: 1 mL of waste oil, 4 mL of solvent, 500 U of lipase, 40 °C, stir speed of 250 rpm, and 3.0 mol equiv of methanol.

Figure 4. (a) Effect of cyclic hydrocarbons solvents on the methanolysis of used soybean oil by Candida Antarctica lipase. Reaction conditions: 1 mL of waste oil, 4 mL of solvent, 500 U of lipase, 40 °C, stir speed of 250 rpm, and 3.0 mol equiv of methanol. (b) Effect of cyclic hydrocarbons solvents on the methanolysis of used soybean oil by Pseudomonas cepacia lipase. Reaction conditions: 1 mL of waste oil, 4 mL of solvent, 500 U of lipase, 40 °C, stir speed of 250 rpm, and 3.0 mol equiv of methanol). (c) Effect of cyclic hydrocarbons solvents on the methanolysis of used soybean oil by Thermomyces lanuginosus lipase. Reaction conditions: 1 mL of waste oil, 4 mL of solvent, 500 U of lipase, 40 °C, stir speed of 250 rpm, and 3.0 mol equiv of methanol.

P and yield with lipase CA (Figure 5a). Lower yields are obtained with hydrophilic solvents (log P < 2), which is probably due to essential water being stripped from the enzyme’s active sites. The highest yields are with hexane and isooctane, which are more hydrophobic (log P > 3). On the contrary, solvents containing benzene rings showed poor yields despite their moderate log P values. A positive correlation between log P of solvent and yield is observed with lipase PC (figure 5b). The more hydrophilic solvents display a lower yield, and the yield increases with log P value and then levels off at a value of 3.2. A positive correlation is also observed with lipase TL (Figure 5c). However, unlike lipase

PC, lipase TL shows significantly adverse effects to solvents that are very hydrophilic (log P < 1). An optimum reaction system should include a solvent that will enhance the rate of transesterification by improving the solubility of methanol, oil, and glycerol byproduct. All three enzymes show an optimum log P between 3.2 and 3.6, which results in the highest biodiesel yield. In general, these hydrophobic solvents protect the enzyme from being denatured by methanol, while still allowing the acyl acceptor to diffuse to the enzyme’s active sites to react. The two primary obstacles in enzyme-catalyzed reactions are (i) the immiscibility of the two substrates, namely, hydrophilic 4672

dx.doi.org/10.1021/ef2008776 |Energy Fuels 2011, 25, 4669–4674

Energy & Fuels

ARTICLE

However, it has been reported that Pseudomonas cepacia is much more 1,3-specific24 than Candida antarctica.25 The theoretical biodiesel yield using a 1,3-specific lipase should only be 66%;26 however, the observed yield of up to 86.6% shows evidence of acyl migration. The mechanisms of the enzymes are very similar; however, there are structural differences in the active sites. It has been reported that Candida antarctica lipase B has a large stiff binding pocket, while Thermomyces lanuginosus lipase has a smaller, more flexible, active site.26 With the exception of aromatic hydrocarbon solvents and to some extent ketone solvents, there are many similarities between the results obtained with lipase PC and lipase TL. The alkane solvents resulted in the highest yields with all three enzymes. The most striking difference is the time that it takes for the reaction to achieve the highest yield for a given solvent. With lipases PC and TL, it is typically around 4 8 h, whereas with lipase CA, it is around 24 h.

Figure 5. (a) Correlation of log Ps of solvents with the corresponding yield of biodiesel produced from the transesterification of triglycerides catalyzed by Candida Antarctica lipase after 24 h. (b) Correlation of log Ps of solvents with the corresponding yield of biodiesel produced from the transesterification of triglycerides catalyzed by Pseudomonas cepacia lipase after 24 h. (c) Correlation of log Ps of solvents with the corresponding yield of biodiesel produced from the transesterification of triglycerides catalyzed by Thermomyces lanuginosus lipase after 24 h.

methanol and hydrophobic triglyceride, resulting in the formation of an interface leading to mass transfer resistance and (ii) the strong polarity of methanol, which tends to strip the active water from the enzyme’s active site leading to enzyme deactivation. The addition of an organic solvent as the medium to the reaction system might simultaneously overcome the two limitations by enhancing the solubility of oil and methanol in the solvent and by limiting the concentration of methanol surrounding the enzyme.8 Enzyme Effects on Biodiesel Synthesis. Lipases are classified by how they catalyze reactions, which refers to their selectivity for the acyl position on the glycerol backbone, also known as regioselectivity. There are two types of regioselectivity commonly found in lipases; 1,3-specific and nonspecific (1,2,3-specific).21 Candida antarctica21 and Pseudomonas cepacia22 are nonspecific lipases and Thermomyces lanuginosus23 is a 1,3-specific lipase.

4. CONCLUSIONS We investigated the effects of 15 hydrophobic and hydrophilic organic solvents with distinct functional groups on the enzymatic transesterification of used soybean oil with three different lipases immobilized on the same support. The results indicate that the choice of solvent can have a huge effect on both the yield, with values ranging from 2.7 to 87%, as well as on the kinetics of the reaction, with the time to attain equilibrium ranging from 4 h to >24 h. Solvents from the alkane group gave the highest biodiesel yields for all three enzymes, with isooctane and n-hexane being the best performers. There appears to be a correlation between the solvent’s hydrophobicity (log P) and biodiesel yield with the enzymes lipase TL and lipase PC. In general, biodiesel yield increases with an increase in log P value, implying that the solvent’s hydrophobicity plays a more important role in enzyme activity than its distinct functional group. However, with certain solvents, the structure of the molecule may play a significant role compared to the hydrophobicity of the solvent alone, and in some instances, a specific biocatalyst may have a distinct tolerance for a certain solvent. The choice of lipase can have a great effect on yield and reaction kinetics. While all three lipases from C. antarctica, P. cepacia, and T. lanuginosus had relatively high biodiesel yields after 24 h with alkane solvents, P. cepacia and T. lanuginosus displayed high reaction rates reaching the final yield in 4 h compared to C. antarctica, which required 24 h. ’ AUTHOR INFORMATION Corresponding Author

*Tel: 603 862 2298. Fax: 603 862 3747. E-mail: [email protected].

’ REFERENCES (1) Alcantara, R.; Amores, J.; Canoira, L.; Fidalgo, E.; Franco, M. J.; Navarro, A. Catalytic production of biodiesel from soy-bean oil, used frying oil and tallow. Biomass Bioenergy 2000, 18 (6), 515–527. (2) Sun, J.; Ju, J. X.; Ji, L.; Zhang, L. X.; Xu, N. P. Synthesis of biodiesel in capillary microreactors. Ind. Eng. Chem. Res. 2008, 47 (5), 1398–1403. (3) Georgogianni, K. G.; Kontominas, A. G.; Pomonis, P. J.; Avlonitis, D.; Gergis, V. Alkaline conventional and in situ transesterification of cottonseed oil for the production of biodiesel. Energy Fuels 2008, 22 (3), 2110–2115. 4673

dx.doi.org/10.1021/ef2008776 |Energy Fuels 2011, 25, 4669–4674

Energy & Fuels

ARTICLE

(4) Vasudevan, P. T.; Briggs, M. Biodiesel production-current state of the art and challenges. J. Ind. Microbiol. Biotechnol. 2008, 35 (5), 421–430. (5) Chen, G.; Ying, M.; Li, W. Enzymatic conversion of waste cooking oils into alternative fuel--biodiesel. Appl. Biochem. Biotechnol. 2006, 129 132, 911–921. (6) Akoh, C. C.; Chang, S. W.; Lee, G. C.; Shaw, J. F. Enzymatic approach to biodiesel production. J. Agric. Food. Chem. 2007, 55 (22), 8995–9005. (7) Vasudevan, P. T.; Gagnon, M. D.; Briggs, M. R. Environmentally Sustainable Biofuels The Case for Biodiesel, Biobutanol and Cellulosic Ethanol. In Sustainable Biotechnology: Sources of Renewable Energy; Harvey, S., Singh, O., Eds.; Springer: New York, 2010; pp 43 62. (8) Fu, B.; Vasudevan, P. T. Effect of Organic Solvents on EnzymeCatalyzed Synthesis of Biodiesel. Energy Fuels 2009, 23 (8), 4105–4111. (9) Deng, L.; Xu, X.; Haraldsson, G.; Tan, T.; Wang, F. Enzymatic production of alkyl esters through alcoholysis: A critical evaluation of lipases and alcohols. J. Am. Oil Chem. Soc. 2005, 82 (5), 341–347. (10) Xu, Y.; Du, W.; Zeng, J.; Liu, D. Conversion of Soybean Oil to Biodiesel Fuel Using Lipozyme TL IM in a Solvent-free Medium. Biocatal. Biotransform. 2004, 22 (1), 45–48. (11) Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Rules for Optimization of Biocatalysis in Organic-Solvents. Biotechnol. Bioeng. 1987, 30 (1), 81–87. (12) Lara, P. V.; Park, E. Y. Potential application of waste activated bleaching earth on the production of fatty acid alkyl esters using Candida cylindracea lipase in organic solvent system. Enzyme Microb. Technol. 2004, 34 (3 4), 270–277. (13) Satyarthi, J. K.; Srinivas, D.; Ratnasamy, P. Estimation of Free Fatty Acid Content in Oils, Fats, and Biodiesel by H NMR Spectroscopy. Energy Fuels 2009, 23, 2273–2277. (14) Sigma-Aldrich Co. http://www.sigmaaldrich.com/united-states. html (accessed May 21, 2011). (15) Chao-Chin, L.; Siti, Z.; Shaik Ramjan, V.; Yi-Hsu, J. Lipasecatalyzed production of biodiesel from rice bran oil. J. Chem. Technol. Biotechnol. 2005, 80, 331–337. (16) ChiralVision BV. http://www.chiralvision.com/immobead.htm (accessed May 21, 2011). (17) Szczesna Antczak, M.; Kubiak, A.; Antczak, T.; Bielecki, S. Enzymatic biodiesel synthesis-Key factors affecting efficiency of the process. Renewable Energy 2009, 34 (5), 1185–1194. (18) Noureddini, H.; Teoh, B.; Davis Clements, L. Viscosities of vegetable oils and fatty acids. J. Am. Oil Chem. Soc. 1992, 69 (12), 1189– 1191. (19) Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Rules for optimization of biocatalysis in organic solvents. Biotechnol. Bioeng. 1987, 30 (1), 81–87. (20) Whittall, J.; Sutton, P. Practical methods for biocatalysis and biotransformations; J. Wiley: Chichester, U.K., 2010. (21) Jegannathan, K. R.; Abang, S.; Poncelet, D.; Chan, E. S.; Ravindra, P. Production of Biodiesel Using Immobilized Lipase—A Critical Review. Crit. Rev. Biotechnol. 2008, 28 (4), 253–264. (22) Chandler, I. Determining the regioselectivity of immobilized lipases in triacylglycerol acidolysis reactions. J. Am. Oil Chem. Soc. 2001, 78 (7), 737–742. (23) Rønne, T.; Pedersen, L.; Xu, X. Triglyceride selectivity of immobilized Thermomyces lanuginosa lipase in interesterification. J. Am. Oil Chem. Soc. 2005, 82 (10), 737–743. (24) Ota, Y.; Itabashi, Y.; Hasuo, M. Measurement of Positional Specificity Index of Microbial Lipases by Chiral Phase High-pressure Liquid Chromatography. Biosci., Biotechnol., Biochem. 1996, 60 (1), 145–146. (25) Chang, Q.-L.; Lee, C.-H.; Parkin, K. L. Comparative selectivities of immobilized lipases from Pseudomonas cepacia and Candida antarctica (fraction B) for esterification reactions with glycerol and glycerol analogues in organic media. Enzyme Microb. Technol. 1999, 25 (3 5), 290–297. (26) 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 (1 6), 68–71. 4674

dx.doi.org/10.1021/ef2008776 |Energy Fuels 2011, 25, 4669–4674