Effect of Organic Solvents on Enzyme-Catalyzed Synthesis of

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Effect of Organic Solvents on Enzyme-Catalyzed Synthesis of Biodiesel Boyi Fu and Palligarnai T. Vasudevan* Department of Chemical Engineering UniVersity of New Hampshire, Durham, New Hampshire 03824 ReceiVed March 7, 2009. ReVised Manuscript ReceiVed May 29, 2009

In this work, the effect of choice of organic solvent on activity of lipase from Candida antarctica is examined. In particular, 28 hydrophobic and hydrophilic solvents from seven organic groups are investigated in the methanolysis of canola oil. The yield of biodiesel (fatty acid methyl ester) is correlated with log P (partition coefficient), dielectric constant (ε), and solubility parameter (δ) of the solvent.

1. Introduction In the United States, oil is the fuel of transportation. Coal, nuclear, hydropower, and natural gas are primarily used for electric power generation. The U.S. with 5% of the world’s population consumes 25% of the world’s petroleum, 43% of the gasoline and 25% of the natural gas. Thus, due to diminishing petroleum reserves and the deleterious environmental consequences of exhaust gases from petroleum diesel, biodiesel has attracted attention during the past few years as a renewable and environmentally friendly fuel. 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 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 A relatively new and promising development in the production of biodiesel is via enzymatic transesterification with lipase as the catalyst. 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. 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 * To whom correspondence should be addressed. Tel: 603 862 2298. Fax: 603 862 3747. E-mail: [email protected]. (1) Alcantara, R.; Amores, J.; Canoira, L.; Fidalgo, E.; Franco, M. J.; Navarro, A. Biomass Bioenergy 2000, 18, 515–527. (2) Sun, J.; Ju, J. X.; Ji, L.; Zhang, L. X.; Xu, N. P. Ind. Eng. Chem. Res. 2008, 47, 1398–1403. (3) Georgogianni, K. G.; Kontominas, M. G.; Tegou, E.; Avlonitis, D.; Gergis, V. Energy Fuels 2008, 22, 1353–1357. (4) Vasudevan, P. T.; Briggs, M. J. Ind. Microbiol. Biotechnol. 2008, 35, 421–430. (5) Chen, G. Y.; Ying, M.; Li, W. Z. Appl. Biochem. Biotechnol. 2006, 132, 911–921. (6) Akoh, C. C.; Chang, S. W.; Lee, G. C.; Shaw, J. F. J. Agric. Food. Chem. 2007, 55, 8995–9005.

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.7,8 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 (Figure 1). Several organic solvents have been employed in the synthesis of biodiesel catalyzed by lipase from different sources. Results suggest that enzymes typically show high activities in the presence of hydrophobic solvents9-12 and a few hydrophilic solvents.13-15 This work reports the results of a systematic study of solvent effects based on distinct functional groups on the enzymatic transesterification reaction. In this study, 28 hydrophilic and hydrophobic solvents from seven organic groups (alkane and cycloalkane, ketone, ether, ester, alcohol, nitrile, and derivatives) were evaluated as possible media in the methanolysis of canola oil by immobilized Candida antarctica lipase, to gain a more comprehensive understanding of solvent effects on the transesterification reaction. In addition, solvent parameters such as hydrophobicity (log P), polarity (dielectric constant), and solubility (Hildebrand Solubility Parameter), were correlated with the corresponding biodiesel yields, with the aim of investigating their potential relationship to enzyme activity. 2. Experimental Section Materials. Fresh canola oil was purchased from ConAgra Foods, Inc., Omaha, NE, USA. Candida antarctica lipase B immobilized (7) Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Biotechnol. Bioeng. 1987, 30, 81–87. (8) Lara, P. V.; Park, E. Y. Enzyme Microb. Technol. 2004, 34, 270– 277. (9) Soumanou, M. M.; Bornscheuer, U. T. Enzyme Microb. Technol. 2003, 33, 97–103. (10) Zhao, X. Y.; El-Zahab, B.; Brosnahan, R.; Perry, J.; Wang, P. Appl. Biochem. Biotechnol. 2007, 143, 236–243. (11) Nie, K. L.; Xie, F.; Wang, F.; Tan, T. W. J. Mol. Catal. B: Enzym. 2006, 43, 142–147. (12) Lu, J.; Nie, K. L.; Wang, F.; Tan, T. W. Bioresour. Technol. 2008, 99, 6070–6074. (13) Du, W.; Liu, D. H.; Li, L. L.; Dai, L. M. Biotechnol. Prog. 2007, 23, 1087–1090. (14) Royon, D.; Daz, M.; Ellenrieder, G.; Locatelli, S. Bioresour. Technol. 2007, 98, 648–653. (15) Su, E.; Wei, D. Z. J. Mol. Catal. B: Enzym. 2008, 55, 118–125.

10.1021/ef900187v CCC: $40.75  2009 American Chemical Society Published on Web 07/06/2009

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Figure 1. The micro reaction environment with and without solvent, showing the effect of methanol on active water in the presence/absence of a solvent.

on macroporous resin (Novozyme 435) with an approximate activity of 10 000 units/g, methyl oleate (>98 wt. %), ethyl oleate (>98 wt. %) and iso-octane (HPLC grade) were all purchased from SigmaAldrich, St. Louis, MO, USA. HPLC grade methanol, hexane, octane, acetone, methyl tert-butyl ether (MTBE), acetonitrile, and iso-propanol were all purchased from Fisher Scientific, Pittsburgh, PA, USA. Pure tert-butanol was purchased from J. T. Baker Chemical. Co., Phillipsburg, NJ, USA. 99% pure decane, dodecane, methyl acetate, and diisopropyl ether were bought from Acros Organics, Geel, Belgium. Analytical grade butanone, methyl isopropyl ketone (MIPK), 3-pentanone, methyl iso-butyl ketone (MIBK), dimethoxyethane, 1,2-dichloroethane, tetrachloromethane, chloroform, ethyl acetate, 1-propanol, 1-butanol, and iso-butanol were obtained from excess inventory at the University of New Hampshire (UNH). 4-Heptanone and tetrahydrofuran (THF) with purities of 99% and iso-amyl alcohol (ACS certified grade) were also obtained from UNH. Synthesis of Biodiesel. Several 40-mL glass vials with PTFE/ silicone septa from Kimble, Vineland, NJ, USA were used as reactors to produce biodiesel. The reaction system contained 0.075 g (750 U) enzyme particles, 4 mL solvent, and 1 mL canola oil. To minimize the denaturation of lipase caused by methanol, exactly 62 µL methanol (molar ratio of methanol to oil ) 1.5) was added stepwise to the reactor at 0 and 10 h, respectively. Hence, the total molar ratio of methanol to oil was kept at 3:1 (144 µL methanol). The vial was sealed by Parafilm after the addition of reactants to prevent vaporization of methanol and organic solvent. The reaction was carried out in a constant temperature water bath at 40 °C and a stirring speed of 250 rpm. The reaction was stopped after 21 h. Analysis Method. Samples of 30 µL were collected at 0, 3, 5, 10, 12, and 21 h, respectively, and then diluted with 1 mL isooctane for GC analysis. The concentration of methyl ester, ethyl ester, and other mono fatty acid esters (MFAE) were 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 and ethyl oleate were employed as biodiesel standards in the GC analysis. Biodiesel in this study was defined as methyl ester (or ethyl ester from ethyl acetate), and excluded MFAEs produced by other alcohols (MFAEs are defined as mono fatty acid esters from the transesterifications of triglyceride with higher alcohols. Higher alcohols in this study indicate propanol, iso-propanol, butanol, isobutanol, tert-butanol, and iso-amyl alcohol; MFAEs refer to 1-propyl ester, iso-propyl ester, 1-butyl ester, iso-butyl ester, tert-butyl ester, and iso-amyl ester). The yield of biodiesel was determined as the mass of methyl ester (or ethyl ester) produced per initial mass of oil (g biodiesel/g oil).

Selection of Solvents. The preliminary screening of solvents was based on solvent characteristics and reaction conditions (i) log PsSolvents with low log P values exhibit strong hydrophilicity, triggering enzyme deactivation.7 Thus, solvents with log P less than zero, such as dimethyl sulfoxide (DMSO), diglyme, and 1,4-dioxane were not chosen in this study; (ii) Boiling pointsThe typical reaction temperature of enzymatic biodiesel synthesis is 35-45 °C.10,11,16 Hence, the rational boiling points of solvents should be higher than this range, which resulted in the elimination of solvents like dimethyl ether, diethyl ether, and pentane from further consideration; (iii) ViscositysOne essential aim of adding solvent is to reduce mass transfer resistance by decreasing viscosity of the reaction mixture. The dynamic viscosity of biodiesel is in the range of 4 to 6 cP at 20-40 °C;17-19 thus, the desirable value for solvent viscosity should be less than 4 cP, based on which, solvents in the alcohol group with a carbon number greater than five were not considered; (iv) Toxicology qualitys In keeping with the trend of green synthesis, toxic solvents like benzene, toluene, and chlorobenzene, were not evaluated in this study. Several physical properties of substrates and solvents used in this study together with the corresponding final yields of biodiesel at 21 h are listed in Table 1. One of the parameters, the Hildebrand Solubility Parameter, δ, was calculated using the following equation:20

δ)




∆Hvap - RT Vm

(1)

where ∆Hvap is the enthalpy of vaporization of liquid;21 R equals 8.314 J/mol.K, and Vm is the molar volume of liquid. A derived parameter, σ, was introduced to demonstrate the similarity of δ values between solvent and triglyceride:

σ ) |δsolvent - δtriglyceride |

(2)

(16) Sanchez, F.; Vasudevan, P. T. Appl. Biochem. Biotechnol. 2006, 135, 1–14. (17) Tate, R. E.; Watts, K. C.; Allen, C. A. W.; Wilkie, K. L. Fuel 2006, 85, 1010–1015. (18) Knothe, G.; Steidley, K. R. Fuel 2007, 86, 2560–2567. (19) Tyson, K. S.; McCormick, R. L. Biodiesel Handling and Use Guidelines, 3rd ed.; National Renewable Energy Laboratory: Golden, CO, 2006. (20) Burke, J. Solubility Parameters: Theory and Application. In The Book and Paper Group Annual (Online); The American Institute for Conservation: Washington, D.C., 1984; Vol. 3. (21) Lide, D. R. CRC Handbook of Chemistry and Physics, 87th ed.; CRC Press/Taylor and Francis: Boca Raton, FL, 2006. (22) Chefson, A.; Auclair, K. ChembioChem 2007, 8, 1189–1197.

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Table 1. Physical Properties of Substrates and Solvents, As Well As the Final Yields of Biodiesel Based on Specific Solvents at 21 h name

ηa

b.p.b

log Pc

εd

δe

σ

yieldf

triolein methyl oleate cyclohexane (CY) hexane (HE) iso-octane (isoOC) octane (OC) decane (DE) dodecane (DO) acetone (AC) butanone (BU) MIPK 3-pentanone (PE) MIBK 4-heptanone (HP) dimethoxyethane (DME) THF MTBE diisopropyl ether (DIPE) methyl acetate ethyl acetate acetonitrile (ACN) 1,2-dichloroethane (DI) chloroform (CH) tetrachloromethane (TE) methanol 1-propanol iso-propanol 1-butanol iso-butanol tert-butanol iso-amyl alcohol

64.860 5.661 0.894 0.300 0.473 0.508 0.838 1.383 0.306 0.405 0.346 0.444 0.545 0.730 2.471 0.480 0.360 0.379 0.364 0.423 0.345 0.840 0.562 0.966 0.544 1.945 2.038 2.544 3.950 4.312 3.692

237.0 218.0 80.7 68.7 98.0 125.7 174.2 216.3 56.5 80.0 94.0 101.0 117.5 145.0 85.0 66.0 55.2 69.0 56.9 77.1 81.6 83.5 61.2 76.7 64.7 97.1 82.3 117.7 108.0 82.4 132.0

10.775 8.334 3.205 3.657 3.668 4.668 5.678 6.689 0.234 0.736 0.980 1.239 1.511 2.358 0.093 0.700 1.482 1.775 0.384 0.760 0.474 1.524 2.091 2.754 –0.320 0.559 0.420 1.118 0.803 0.866 1.333

3.200 3.211 2.017 1.885 1.937 1.942 1.990 2.006 20.514 18.303 10.171 16.816 12.852 12.356 7.227 7.528 2.600 3.880 6.879 5.927 35.970 10.119 4.705 2.231 32.289 20.340 19.094 17.295 16.775 12.392 15.441

7.433 8.598 8.219 7.246 6.792 7.575 7.747 7.882 9.628 9.285 8.754 9.028 8.530 8.824 8.837 9.326 7.409 7.090 9.468 8.978 11.807 9.945 9.238 8.204 14.368 11.987 11.582 11.412 11.094 10.551 10.794

0.786 0.187 0.640 0.142 0.314 0.450 2.195 1.852 1.321 1.596 1.097 1.391 1.404 1.893 0.024 0.343 2.035 1.545 4.374 2.512 1.805 0.771 6.935 4.555 4.149 3.979 3.662 3.118 3.362

0.382 0.407 0.831 0.478 0.501 0.493 0.416 0.342 0.513 0.072 0.301 0.090 0.667 0.019 0.384 0.354 0.254 0.242 0.354 0.022 0.011 0.010 0.104 0.117 0.047 0.071 0.442 0.072

a Dynamic viscosity with unit of cP at 25 °C from ref 21 (iso-octane from ref 32 and DME from ref 33). b Boiling point with unit of °C at 760 mmHg from ref 21 (triolein at 18 mmHg and methyl oleate at 20 mmHg). c Partition coefficient (from ref 34). d Dielectric constant at 25 °C from ref 21 (methyl oleate from ref 35 diisopropyl ether from ref 36 and acetonitrile from ref 37). e Hildebrand solubility parameter with unit of cal0.5 · cm-1.5 at 25 °C. f Final biodiesel yield at 21 h (g/g oil).

A low value of σ is desirable since it indicates better miscibility between the solvent and triglyceride.

3. Results and Discussion Solvent Effect of Alkanes and Cycloalkanes. Alkanes and cycloalkanes have strong hydrophobicity (high log P), excellent stability, and solubility for organic compounds.22 Six solvents in this group (cyclohexane, hexane, iso-octane, octane, decane, and dodecane) were employed in this study. As shown in Figure 2, with the sole exception of iso-octane, biodiesel yield increased with increase in log P from cyclohexane (38.2%) to decane (50.1%). The reason that dodecane (49.3%) had a biodiesel yield similar to decane is probably due to its relatively high viscosity (Table 1). The highest yield (83.1%) was obtained with isooctane as solvent, and may be due to its unique side-chain molecular structure containing three methyl groups. This could have resulted in better mixing with triglyceride and methanol compared to linear and cyclic alkanes. It is important to note that methanol was added stepwise initially and at 10 h in all experiments in this study. The plot for iso-octane in Figure 2 indicates that after 5 h, almost all of the methanol added at 0 h was converted to methyl ester; therefore, the yield showed a lower rate of increase in the subsequent period. At 10 h, an additional 1.5 mol equivalent of methanol (62 µL) was added to the reactor, which resulted in a larger rate of increase in the yield. However, for other solvents, the low biodiesel yields at 10 h demonstrate that not all the methanol was consumed. Thus, stepwise addition of methanol at 10 h resulted in the inhibition of the enzyme followed by a concomitant reduction in the rate of increase in yield for about 2 h following the addition, as is evident in Figure 2.

Figure 2. Effect of alkane and cyclohexane solvents on methanolysis of canola catalyzed by Candida antarctica lipase. Reaction conditions: 1 mL canola oil, 4 mL solvent, 0.075 g (750 U) lipase, 40 °C temperature, 250 rpm. 1.5 mol equivalent methanol (62 µL) was added into the reaction system at 0 h, 10 h, respectively. “2nd MeOH” refers to the second addition of 62 µL methanol.

Figure 3 shows the volume effect of solvent on biodiesel production, which suggests that the optimal volume ratio of solvent to oil is 4:1. It is also interesting to note that stepwise addition of methanol resulted in considerably higher yields than a single addition of methanol at the start of the experiment. Hence, stepwise addition of methanol is necessary even in the presence of solvent media. Solvent Effect of Ketone. Solvents in the ketone group are typical reaction media for biotransformation.23,24 But none has been utilized in the biocatalytic synthesis of biodiesel except

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Figure 3. Volume effect of solvent on biodiesel yield. Three ml, 4 mL, and 5 mL iso-octane indicate that 3, 4, and 5 mL isooctane were added to the reactor, respectively. “MeOH stepwise” denotes that 1.5 mol equivalent methanol (62 µL) was added to the reactor at 0 h and 10 h. “MeOH single addition” refers to 3 mol equiv. methanol addition (144 µL) at 0 h. Other reaction conditions: 1 mL canola oil, 0.0750 g (750 U) lipase, 40 °C temperature, 250 rpm stirring.

Figure 4. Effect of ketone solvents on methanolysis catalyzed by Candida antarctica lipase. Reaction conditions: 1 mL canola oil, 4 mL solvent, 0.075 g (750 U) lipase, 40 °C temperature, 250 rpm stirring. 1.5 mol equiv. methanol (62 µL) was added to the reaction system at 0 h, 10 h, respectively. The data for decane were employed in this graph for comparison.

acetone. Hence six solvents, namely acetone, butanone, MIPK, 3-pentanone, MIBK, and 4-heptanone, were evaluated in this study. The results shown in Figure 4 reveal that the biodiesel yield of 51.3% at the end of the run with MIPK, a hydrophilic and polar aprotic solvent that does not have a strong tendency to strip the water from the enzyme’s active site, is comparable to the highest yield (50.1%) obtained with decane in the alkane group of solvents barring iso-octane. Similarly, acetone (41.6%) and hexane (40.7%) show similar results. Furthermore, the plots in Figure 4 indicate that biodiesel yields during the initial 10 h period with MIPK and acetone are higher than that of decane. It is conceivable that methanol is better dispersed in the aprotic ketone solvent thereby minimizing the concentration of methanol near the enzyme’s active site,15 or that the carbonyl functional (23) Graber, M.; Irague, R.; Rosenfeld, E.; Lamare, S.; Franson, L.; Hult, K. Biochim. Biophys. Acta, Proteins Proteomics 2007, 1774, 1052–1057. (24) Vega, M.; Karboune, S.; Kermasha, S. Appl. Biochem. Biotechnol. 2005, 127, 29–42.

Fu and VasudeVan

Figure 5. Effect of ether solvents on methanolysis catalyzed by Candida antarctica lipase. Reaction conditions: 1 mL canola oil, 4 mL solvent, 0.075 g (750 U) lipase, 40 °C temperature, 250 rpm stirring. 1.5 mol equiv. methanol (62 µL) was added into the reaction system at 0 and 10 h, respectively.

group in ketone may partially modify the configuration of lipase and improve its flexibility, similar to DMSO with protease, lipase25 and carboxylesterase.26 Solvent Effect of Ether. Figure 5 shows the results of biodiesel synthesis based on four solvents in the ether group, namely dimethoxyethane, THF, MTBE, and diisopropyl ether. The reaction with dimethoxyethane displayed the highest yield of biodiesel (66.7%) in this group though dimethoxyethane has the strongest polarity (log P ) 0.093) among the four solvents. This yield was also the second highest among all 28 solvents used in this study, only slightly lower to iso-octane. The probable reason may again be attributed to the improved miscibility of the solvent with the acyl acceptor, methanol, and/ or the effect of the ether functional group of dimethoxyethane on enzyme configuration. Even though MTBE possesses three methyl side-chain molecules similar to iso-octane, its corresponding biodiesel yield was much lower than the latter. Almost no product was detected when THF was used as the solvent suggesting that the enzyme was completely inhibited in the presence of THF. This result is in agreement with previous studies on lipases from Pseudomonas fluorescens10 and Candida antarctica,15 but is in contradistinction with that of Candida sp.99-125.12 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. The unique heterocyclic structure of THF may trigger the deactivation of Candida antarctica lipase. Solvent Effect of Alcohols. Higher alcohols (C2-C5) are normally considered as solvents or cosolvents in enzymatic synthesis.27,28 Six higher alcohols namely, 1-propanol, isopropanol, 1-butanol, iso-butanol, tert-butanol, and iso-amyl alcohol, were utilized in this study. As shown in Figures 6, these six alcohols served not only as solvents, but also as acyl acceptors in the transesterification reaction with triglyceride. Moreover, the yields of MFAEs such as iso-propyl ester and iso-butyl ester, were higher than that of methyl ester, resulting (25) Almarsson, O.; Klibanov, A. M. Biotechnol. Bioeng. 1996, 49, 87– 92. (26) Sehgal, A. C.; Tompson, R.; Cavanagh, J.; Kelly, R. M. Biotechnol. Bioeng. 2002, 80, 784–793. (27) Chen, Y.; Xu, J. H.; Pan, J.; Xu, Y.; Shi, J. B. J. Mol. Catal. B: Enzym. 2004, 30, 203–208. (28) Hansen, T. V.; Waagen, V.; Partaliv, V.; Anthonsen, H. W.; Anthonsen, T. Tetrahedron: Asymmetry 1995, 6, 499–504.

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Figure 7. Alcohols and esters as alternative acyl-acceptors in the transesterification of triglyceride catalyzed by Candida antarctica lipase. Reaction condition: 1 mL canola oil, 4 mL solvent, 0.075 g (750 U) lipase, 40 °C temperature, 250 rpm stirring. No methanol was added.

Figure 6. Effect of alcohols on methanolysis catalyzed by Candida antarctica lipase. Parts a, b, c, d, e, and f refer to the solvent effect of 1-propanol, iso-propanol, 1-butanol, iso-butanol, tert-butanol, and isoamyl alcohol, respectively. Reaction condition: 1 mL canola oil, 4 mL solvent, 0.075 g (750 U) lipase, 40 °C temperature, 250 rpm stirring. 1.5 mol equiv. methanol (62 µL) was added into the reaction system at 0 h, 10 h, respectively. Table 2. Selectivities toward Methyl Ester Based on Various Alcohol Solvents at 10 h and 21 h alcohol solvents

selectivity in 10 ha

selectivity in 21 hb

1-propanol iso-propanol 1-butanol iso-butanol tert-butanol iso-amyl alcohol

0.12 0.13 0.05 0.06 1.15 0.06

0.16 0.15 0.08 0.08 2.19 0.09

a

Selectivity of methyl ester to MFAE at 10 h:

Selectivity ) b

Yield of Methyl Ester Yield of MFAE

(3)

Selectivity of methyl ester to MFAE at 21 h.

in selectivities toward methyl ester of less than 1 as shown in Table 2 (selectivity is defined as mass of methyl ester to mass of other MFAEs). The σ values of these alcohols are lower than that of methanol as shown in Table 1. The only alcohol that had a selectivity greater than 1 was tert-butanol (Table 2). Thus, when both methanol and tert-butanol are present, the methanolysis reaction is favored. This may be ascribed to the steric hindrance due to the presence of the three methyl side groups in tert-butanol (as an acyl-acceptor) compared to only one methyl group in methanol. Other studies13,14 indicate that tert-butanol is an excellent solvent in enzymatic methanolysis. Nevertheless, our study does not support this conclusion as the yield of methyl ester based on tert-butanol (44.2%) was much lower than with iso-octane (83.1%). The experiment was repeated twice and the yields of methyl ester were 43.5% and 44.3%. One explanation is that

about 20% of the oil was converted to tert-butyl ester as shown in Figure 6e. An interesting phenomenon was observed when methanol was added again at 10 h. The yield of tert-butyl ester initially decreased, which suggests some decomposition of the tert-butyl ester. The observation was substantiated in two trials. If the desired objective in solvent choice is to advance methanolysis without participation in the reaction, then these higher alcohols are not appropriate candidates. Considering their high reaction activities with triglyceride as shown in Figure 6, lipasecatalyzed transesterification with alcohols (1-propanol, iso-propanol, 1-butanol, iso-butanol, tert-butanol, and iso-amyl alcohol) were conducted in the absence of methanol (Figure 7). It can be seen that alcohols with side-chain molecular structures have higher yields of MFAEs (g90%) compared to the linear ones with the exception of tert-butanol. This may be attributed to the differences in miscibility with triglyceride. Table 1 shows that for alcohol molecules with the same carbon numbers, the branched ones have better miscibility with triglyceride than the linear isomers as indicated by the lower σ values. Two traditional substituted acylacceptors to methanol, methyl acetate,29 and ethyl acetate, were also investigated in this study for comparison. The results in Figure 7 indicate that for the same reaction conditions, the corresponding MFAE yields (methyl oleate and ethyl oleate) are lower. Since methyl- and ethyl acetate are derived from fossil fuels, 1-propanol and 1-butanol are better reagents for synthesis of biodiesel in solvent-free systems due to the fact that they can be obtained from biomass through fermentation, and also give higher MFAE yields. Effects of Other Common Solvents. Tests were conducted with acetonitrile as solvent. An obvious phase interface between the oil and acetonitrile was observed during the experiment suggesting a high mass transfer resistance, which might be the reason for the low yield of biodiesel (35.4%, Table 1). Three derivatives, namely tetrachloromethane, 1,2-dichloroethane, and chloroform, were also utilized in this study. Unfortunately, very little product was detected ( 3) contains hydrophobic solvents, while the while region (0 < log P < 3) contains hydrophilic solvents. The nomenclature of solvents follows Table 1.

Figure 9. Correlation of Dielectric Constant (ε) of solvents with the corresponding yields of biodiesel produced from the transesterification of triglyceride catalyzed by Candida antarctica lipase.

surprising. The yields of biodiesel varied greatly from 1.0% to 83.1%, and the study indicates that iso-octane appears to be the best choice based solely on biodiesel yield. Relationship of Solvent Parameters with Enzyme Activity. As stated earlier, the primary effect of alcohols and esters (methyl acetate and ethyl acetate) is to serve as acyl-acceptors rather than as solvents. Thus, these are excluded in the following discussion. Figure 8 exhibits the relationship between log Ps of solvents and the corresponding biodiesel yields. Solvents can be classified into two categories (i) hydrophobic solvents (alkane and cycloalkane) with log P > 3, where the results clearly indicate a positive correlation between log P and yield (with the exception of iso-octane). The rational explanation is that solvents with higher log P have better miscibility with triglyceride and stronger hydrophobicity to protect the enzyme from deactivation;7 and (ii) hydrophilic solvents having specific functional groups (>CdO, -O-, -CN, and -Cl) with log P ε(0,3), where a negative correlation is observed between log P and biodiesel yield in general except for certain solvents. This conclusion is in disagreement with previous reports that suggest that enzymatic reactions in hydrophilic media result in lower yields.7,22

Fu and VasudeVan

Figure 10. Correlation of Hildebrand Solubility Parameter (δ) of solvents with the corresponding yields of biodiesel produced from the transesterification of triglyceride catalyzed by Candida antarctica lipase.

Figure 11. Correlation of σ of solvents with the corresponding yields of biodiesel produced from the transesterification of triglyceride catalyzed by Candida antarctica lipase.

The reason for the higher yields in our study may be ascribed to the enhanced miscibility that some of these solvents have with methanol,15 which reduces the concentration of methanol surrounding the enzyme resulting in lower deactivation of the enzyme. These hydrophilic solvents are polar aprotic solvents that do not tend to have a strong tendency to strip water from the enzyme.30 In addition, some solvents like MTBE, MIBK, and 1,2-dichloroethane with very similar log P values but different functional groups have obviously distinct biodiesel yields as shown in Figure 8. The ε, δ, and σ of every solvent were correlated with the corresponding biodiesel yields in Figures 9, 10, and 11, respectively. However, similar to the results reported in previous studies,7,11,31 no clear conclusion emerges from these illustrations. Particularly in Figure 9, some solvents with very similar (30) Wang, D.; Xu, Y.; Teng, Y. Bioprocess. Biosyst. Eng. 2007, 30, 147–155. (31) Yang, F. X.; Weber, T. W.; Gainer, J. L.; Carta, G. Biotechnol. Bioeng. 1997, 56, 671–680. (32) Pa´dua, A. A. H.; Fareleira, J. M. N. A.; Calado, J. C. G.; Wakeham, W. A. J. Chem. Eng. Data 1996, 41, 1488–1494. (33) Muhuri, P. K.; Hazra, D. K. J. Chem. Eng. Data 1994, 39, 375– 377. (34) Molinspiration Home Page. http://www.molinspiration.com/cgi-bin/ properties/ (accessed February 28, 2009).

Effect of SolVents on Synthesis of Biodiesel

values of ε have quite different yields, e.g., iso-octane and octane. Therefore, ε, δ, and σ do not appear to be reasonable parameters to assess the solvent effect on enzyme activity. 4. Conclusions This study reports the effects of 28 solvents with distinct functional groups on the enzyme-catalyzed synthesis of biodiesel. The results indicate that the yields of biodiesel based on specific solvents vary dramatically from 1.0% to 83.1%, with iso-octane appearing to be the best solvent based solely on biodiesel yield. (35) Gouw, T. H.; Vlugter, J. C. J. Am. Oil Chem. Soc. 1964, 41, 675– 678. (36) WohlfarthC. Dielectric Constant of Diisopropyl Ether. In LandoltBo¨rnstein IV/17: Static Dielectric Constants of Pure Liquids and Binary Liquid Mixtures (Supplement to IV/6); Landolt-Bo¨rnsteinsGroup IV Physical Chemistry; Springer: Berlin, Heidelberg, 2008, p 363. (37) Wohlfarth, C. Dielectric constant of Acetonitrile. In LandoltBo¨rnstein IV/17: Static Dielectric Constants of Pure Liquids and Binary Liquid Mixtures (Supplement to IV/6); Landolt-Bo¨rnsteinsGroup IV Physical Chemistry; Springer: Berlin, Heidelberg, 2008; pp 117-121.

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In the case of hydrophobic solvents such as alkanes and cycloalkanes, there appears to be a positive correlation between biodiesel yield and increasing log P values (iso-octane being an exception); while for hydrophilic solvents, a negative correlation between log P and biodiesel yields was observed in general, with the exception of certain solvents. Solvents with similar log P but distinct functional groups display different yields of biodiesel. This implies that specific functional groups also have an effect on enzyme activity. There appears to be no clear-cut correlation between polarity (ε), solubility (δ, σ), and biodiesel yield. Higher alcohols appear to inhibit the formation of methyl ester. These alcohols have the potential to serve both as acyl acceptors and as solvents, similar to methyl and ethyl acetate. Interestingly, higher yields of MFAE were obtained with these alcohols compared to methyl and ethyl acetate, even when no methanol was present. EF900187V