Energy Fuels 2010, 24, 1269–1273 Published on Web 11/06/2009
: DOI:10.1021/ef9009313
Improvement of Immobilized Lipase-Catalyzed Methanolysis of Tributyrin Using Methyl Acetate Burak V. Kabasakal*,† and Arif Caglar Department of Chemical Engineering, Hacettepe University, Beytepe, Ankara, 06532, Turkey. †Present address: Department of Chemistry, University of California, Davis, CA 95616. Received August 27, 2009. Revised Manuscript Received October 24, 2009
Transesterification by methanol and interesterification by methyl acetate were performed simultaneously, and the advantages of using methyl acetate with methanol were investigated in the enzymatic production of methyl ester from tributyrin. It was observed that 100% excess amount (1:6 mol ratio) of methanol/methyl acetate makes no negative effect on the enzyme activity. The initial reaction rate decreases as the tributyrin concentration increases. Consequently, tributyrin causes an inhibition at high concentrations. When the tributyrin concentration was used above the stoichiometric ratio (1:3 mol ratio), it decreased the activity of the enzyme. Michaelis-Menten parameters (Km) for tributyrin and methanol/methyl acetate were calculated as 0.1 and 50 M, respectively. An uncompetitive substrate inhibition constant for tributyrin was determined as 22.42 M. Experimental results were found to correlate well with the results of the kinetic model according to the ping-pong bi-bi mechanism. High conversions up to 90% were observed in a semicontinuous fluidized bed with low enzyme levels (1%). Conversions decreased with increasing flow rates.
and the used organic solvent.3 Hydrophobic organic solvents, such as heptane, hexane, toluene, benzene, 1,4-dioxane, isooctane, and cyclohexane, are generally used as reaction media. The highest transesterification yield was achieved by hexane,4-7 heptane,8,9 and toluene10 in the previous studies. Enzyme-catalyzed methanolysis reactions face some problems, such as methanol inhibition. To increase the methyl ester yield, an excess amount of methanol needs to be used; hence, the conversion of all of the oil in the reaction medium to methyl ester can be obtained. However, methanol inhibition is observed when it is used in stoichiometric amounts with oil and even below these amounts. Enzyme activity decreases when the methanol/oil molar ratio is above (1.5:1.0)5 or (1.0:1.0).7,11 The excess amount of methanol remains within the oil without dissolving, and this might cause the deactivation of the enzyme because of changes in its tertiary structure. Some methods have been developed to avoid enzyme deactivation resulting from methanol inhibition. For instance, Fukuda et al.,5 Nie et al.,7 and Shimada et al.12 used the stepwise addition of methanol to the reaction medium and achieved yields up to 99% with a long reaction time (48 h). In
Introduction Methyl esters, which are the source of biodiesel, are produced by transesterification catalyzed with either a chemical catalyst or an enzyme. Lipases are hydrolyase enzymes affecting the carboxylic ester bonds. The physiological role of lipases is the hydrolysis of triglycerides for the production of diglyceride, monoglyceride, fatty acid, and glycerol. Lipases not only hydrolyze the carboxylic ester bonds but also catalyze esterification, interesterification, and transesterification reactions in non-aqueous media.1 The transesterification reaction is defined by three consecutive reversible equations triglyceride þ ROHTdiglyceride þ R1 COOR diglyceride þ ROHTmonoglyceride þ R2 COOR monoglyceride þ ROHTglycerol þ R3 COOR
ð1Þ
The first step is the conversion of triglycerides to diglycerides, and the following is the conversion of diglycerides to monoglycerides and then the conversion of monoglycerides to glycerols. A total of 1 mol of methyl ester is generated for each mole of glyceride at each step.2 This reaction is called interesterification when an ester is used instead of alcohol as an acyl (COOR) acceptor. Theoretically, the transesterification reaction is an equilibrium reaction, and it shifts to the right when an excess amount of methanol is used, which then allows for the desired product, methyl ester, to be generated more. The most important parameters affecting the reaction rate and conversion are the reaction temperature, alcohol/oil molar ratio, catalyst type and concentration, agitation rate, purity of reactants,
(3) Srivastava, A.; Prasad, R. Renewable Sustainable Energy Rev. 2000, 4, 111–133. (4) Dossat, V.; Combes, D.; Marty, A. Enzyme Microb. Technol. 1999, 25, 194–200. (5) Fukuda, H.; Kondo, A.; Noda, H. J. Biosci. Eng. 2001, 92, 405– 416. (6) Soumanou, M. M.; Bornscheuer, U. T. Enzyme Microb. Technol. 2003, 33, 97–103. (7) Nie, K.; Xie, F.; Wang, F.; Tan, T. J. Mol. Catal. B: Enzym. 2006, 43, 142–147. (8) Yadav, G. D.; Trivedi, A. H. Enzyme Microb. Technol. 2003, 32, 783–789. (9) Yadav, G. D.; Devi, K. M. Chem. Eng. Sci. 2004, 59, 373–383. (10) Yadav, G. D.; Lathi, P. S. J. Mol. Catal. B: Enzym. 2005, 32, 107– 113. (11) Kaieda, M.; Samukawa, T.; Kondo, A.; Fukuda, H. J. Biosci. Bioeng. 2001, 91, 12–15. (12) Shimada, Y.; Watanabe, Y.; Samukawa, T.; Sugihara, A.; Noda, H.; Fukuda, H.; Tominaga, Y. J. Am. Oil Chem. Soc. 1999, 76, 789–793.
*To whom correspondence should be addressed. E-mail: bvkabasakal@ ucdavis.edu. (1) Houde, A.; Kademi, A.; Leblanc, D. Appl. Biochem. Biotechnol. 2004, 118 (1-3), 155–170. (2) Barnwal, B. K.; Sharma, M. P. Renewable Sustainable Energy Rev. 2005, 9, 363–378. r 2009 American Chemical Society
1269
pubs.acs.org/EF
Energy Fuels 2010, 24, 1269–1273
: DOI:10.1021/ef9009313
Kabasakal and Caglar
another study, methanol was used in stoichiometric amounts and nearly 90% conversion was obtained after 7 h. However, a large amount of enzyme was required (30 g of enzyme for 100 g of oil).13 Another method is adding water to the reaction media at specific proportions. In the study by Kaieda et al.,11 lipase deactivation by methanol was prevented using water, whereas the hydrolysis rate was obtained as the highest one among the methanolysis, hydrolysis, and esterification rates. The reaction favors hydrolysis at higher water contents, and the transesterification efficiency decreases. For the constant methanol amount, as the water content decreased, the methanolysis rate increased. The methanol/oil ratio was increased up to 2 and 3, and high methyl ester yields were obtained. The byproduct of methanolysis, glycerol, which is generated during the reaction, inhibits the reaction by accumulating on the enzyme and restricting the substrate and product diffusion. Glycerol, which is insoluble in oil and organic solvents, is removed using silica gel or other adsorbents.6,14 In another study, transesterification of sunflower oil with butanol in a continuous fixed-bed reactor filled with immobilized lipase was investigated. The initial conversion was decreased because of the accumulation of glycerol. To obtain a high conversion, silica gel was used in a packed-bed reactor and some of the glycerol was adsorbed on the silica gel; also, a polar solvent, such as acetone, was used with hexane to increase the polarity of the reaction medium and the solubility of glycerol. However, the reaction rate decreased by one-third from the reaction rate that was obtained with only hexane. Additionally, the enzyme support was washed and fed with fresh substrate solution during the process. This prolonged the process time.4 Methanolysis reactions are usually carried out in organic solvent media, such as heptane, 1,4-dioxane, isooctane, cyclohexane, toluene, hexane, and petroleum ether.4,6-8 Hydrophilic solvents are known as unsuitable for biocatalysis because of their damage to the enzyme active site by lacking water around the enzyme.7 Even though hydrophobic solvents, such as hexane, are preferred by virtue of high enzyme activity and stability, insoluble hydrophilic substrates and products cause low reaction rates by hanging polar molecules on hydrophilic enzyme supports.15,16 In transesterification reactions, not only are organic solvents used but also solvent-free media have been studied to reduce the reaction time and volume and also to eliminate the cost of organic solvents. Despite that, the same problems take place in solvent-free systems.6,13 Methyl esters from oils can also be obtained by interesterification, in which the acyl acceptor is an ester, such as methyl acetate.17 In this study, an excess amount of methyl acetate (12:1 methyl acetate/oil mole ratio) made no negative effect on the enzyme activity and high conversions were obtained. High concentrations of methyl acetate make the reaction medium dilute and avoid the oil to inhibit enzyme activity. The byproduct, triacetin, does not block the enzyme active site and renders the enzyme active and stable. Because the
interesterification rate with methyl acetate is lower than that of transesterification with methanol, high amounts of enzyme need to be used (30% of the oil amount). We have offered a novel reaction system in which methanol inhibition and the lack of activity because of glycerol are not observed. Transesterification of tributyrin with methanol and interesterification with methyl acetate were carried out simultaneously in a solvent-free medium with a low enzyme concentration. Experimental Section Novozym 435, immobilized in polyacrylate resin, Candida antarctica lipase from Novozymes (Denmark), was used as an enzyme. Reactants were tributyrin (BDH, U.K.), methanol (Merck, Germany), and methyl acetate (BDH, U.K.). Transesterification and interesterification reactions are given by eqs 2 and 3, respectively. Batch system experiments were carried out in 10 mL tubes on an orbital shaker. Reaction volumes change between 2 and 7 mL. A semi-continuous system was obtained using a glass cylinder, which is 15 cm in length and 1 cm in diameter. The reactor was filled up with immobilized lipase (1% of total reactant mass) providing fluidized-bed conditions, and the substrate solution was recycled continuously. A semi-continuous fluidized-bed system is given in Figure 1. For batch experiments, immobilized lipase was added as particular percentages of the total reactant mass to the homogeneous reaction medium, in which any additional organic solvent was not used. Methyl acetate plays both reactant and solvent roles. Samples were analyzed using high-performance liquid chromatography (HPLC) (Agilent Technologies model 1100) at 210 nm in approximately 10 min with a 0.8 mL/min flow rate. Hichrom C18 (10 cm 4.6 mm inner diameter) was used as a reversed phase column, and the mobile phase was methanol/water (90:10) (v/v). Methyl acetate, tributyrin, and methyl butyrate left the column, consecutively. Methyl butyrate, which is the main product of tributyrin transesterification/interesterification, was confirmed by comparing mass spectra in the library of gas chromatograph/mass spectrometer (GC/MS) (Agilent Technologies model 6890 GC system 5973 MSD).
Results and Discussion Determination of the Methanol/Methyl Acetate Mole Ratio. To determine the proportions of methanol and methyl acetate, which is the additional acyl acceptor, the transesterification rate of tributyrin with methanol and the interesterification rate of tributyrin with methyl acetate were compared. Tributyrin was used in an excess amount. According to Figure 2, the transesterification rate with methanol is higher than the interesterification rate of methyl acetate. The ratio of initial reaction rates was calculated as 5. The consumed amount of methanol within the tributyrin reaction is 5 times that carried out with methyl acetate in unit time. Therefore, the methanol/methyl acetate mixture was used as a 5 mol of methanol/1 mol of methyl acetate ratio for the rest of the transesterification/ interesterification reactions.
€ T€ (13) K€ ose, O.; uter, M.; Aksoy, H. A. Bioresour. Technol. 2002, 83, 125–129. (14) Stevenson, D. E.; Stanley, R. A.; Furneaux, R. H. Enzyme Microb. Technol. 1994, 16, 478–484. (15) Balcao, V. M.; Paiva, A. L.; Malcata, F. X. Enzyme Microb. Technol. 1996, 18, 392–416. (16) Castillo, E.; Dossat, V.; Marty, A.; Condoret, J. S.; Combes, D. J. Am. Oil Chem. Soc. 1997, 74, 77–85. (17) Du, W.; Xu, Y.; Dehua, L.; Zeng, J. J. Mol. Catal. B: Enzym. 2004, 30, 125–129.
1270
Energy Fuels 2010, 24, 1269–1273
: DOI:10.1021/ef9009313
Kabasakal and Caglar
Figure 1. Semi-continuous fluidized-bed system.
Figure 2. Determination of the methanol/methyl acetate mole ratio. Reaction conditions: 6 mmol of tributyrin, 6 mmol of methanol or methyl acetate, 5% immobilized enzyme, 25 °C, and a 150 rpm mixing rate. (2) Transesterification of tributyrin with methanol. (9) Interesterification of tributyrin with methyl acetate.
Figure 3. Effect of the mixing rate on tributyrin conversion. Reaction conditions: 5 mmol of tributyrin, 15 mmol of methanol/methyl acetate, 5% immobilized enzyme, and 25 °C.
Effect of the Mixing Rate. The optimum mixing rate for the transesterification/interesterification of tributyrin was determined by observing conversion at various mixing rates changing between 100 and 250 rpm (Figure 3). At high mixing rates, the enzyme activity was decreased because of shear forces or foam formation. A 200 rpm was obtained as the mixing rate at which the maximum conversion was observed. Effect of the Methanol/Methyl Acetate Concentration. Reactions were carried out at different tributyrin/methanol/methyl acetate mole ratios (1:3, 1:4, 1:5, and 1:6), while the tributyrin mole number was kept as 5 mmol. In Figure 4, tributyrin conversions are shown for different methanol/methyl acetate concentrations. As the concentration increases, the initial reaction rate increases without inhibition. A 100% excess amount of methanol/methyl acetate did not make a negative effect on the enzyme activity. Effect of the Tributyrin Concentration. Reactions were carried out at different tributyrin/methanol/methyl acetate mole ratios (1:3, 2:3, 3:3, and 4:3), while the methanol/methyl
Figure 4. Effect of methanol/methyl acetate concentration on tributyrin conversion. Reaction conditions: 5 mmol of tributyrin, 15, 20, 25, and 30 mmol of methanol/methyl acetate, 5% immobilized enzyme, 25 °C, and a 200 rpm mixing rate.
1271
Energy Fuels 2010, 24, 1269–1273
: DOI:10.1021/ef9009313
Kabasakal and Caglar
Figure 5. Effect of the tributyrin concentration on methanol/methyl acetate conversion. Reaction conditions: 15 mmol of methanol/ methyl acetate, 5, 10, 15, and 20 mmol of tributyrin, 5% immobilized enzyme, 25 °C, and a 200 rpm mixing rate.
Figure 7. Tributyrin inhibition. Reaction conditions: (2) 5 mmol of tributyrin and 15, 20, and 25 mmol of methanol/methyl acetate, (9) 15 mmol of tributyrin and 15, 20, and 30 mmol of methanol/methyl acetate, 5% enzyme, 25 °C, and a 200 rpm mixing rate (A = methanol/methyl acetate).
Determination of the Tributyrin Inhibition Constant. Michaelis-Menten kinetics is defined in the following equation for uncompetitive substrate inhibition:18 rm ½S r ¼ ð4Þ 2 0 Km þ ½S þ ½S KSI where rm is the maximum reaction rate, Km0 is the Michaelis-Menten constant for tributyrin, and KSI is the tributyrin inhibition constant. After the initial reaction rates are obtained at different tributyrin concentrations, the tributyrin inhibition constant, KSI, was determined as 22.42 M using Systat 12 software by fitting experimental data with the above equation (Figure 7). Kinetic Model. The mechanism of transesterification reactions is described by a ping-pong bi-bi mechanism. According to this model, reactions take place in series by which a product is generated and then another substrate attaches to the enzyme.3 The reaction rate with the presence of inhibition of A is r0 ½A0 ½B0 ð5Þ ¼ ½A rmax KmB ½A0 1 þ 0 þ KmA ½B0 þ ½A0 ½B0
Figure 6. Lineweaver-Burk plot for methanol/methyl acetate. Reaction conditions: 5 mmol of tributyrin, 15, 20, and 25 mmol of methanol/methyl acetate, 5% enzyme, 25 °C, and a 200 rpm mixing rate (A = methanol/methyl acetate). The rate is defined in terms of the tributyrin concentration.
acetate mole number was kept as 15 mmol (Figure 5). Initial reaction rates decreased with increasing tributyrin concentrations; therefore, tributyrin inhibits the enzyme activity at high concentrations. Tributyrin concentrations that are above the stoichiometric value (1:3 tributyrin/methanol/methyl acetate) decrease the enzyme activity. In transesterification reactions, oil is known to be the limiting reactant and not used in an excess amount. Thus, this inhibition is not a problem practically. Determination of Michaelis-Menten Parameters. The Michaelis-Menten constant (Km) for tributyrin was determined using a constant methanol/methyl acetate concentration and various tributyrin concentrations at non-inhibitory conditions (data not shown) and vice versa for methanol/methyl acetate. Km values for tributyrin and methanol/methyl acetate are 0.1 and 50 M, respectively. The lipase enzyme has a high affinity toward tributyrin rather than methanol/methyl acetate. Investigation of Tributyrin Inhibition. The presence and type of tributyrin inhibition was investigated by obtaining the reaction rates at various methanol/methyl acetate concentrations for two constant tributyrin concentrations. In Figure 6, the reaction rate decreased with an increasing tributyrin concentration. This shows the presence of an inhibition. According to Lineweaver-Burk plots, the inhibition is uncompetitive.
KIA
where r0 is the initial reaction rate, rmax is the maximum reaction rate, [A0] is the initial oil concentration, [B0] is the initial alcohol concentration, KmA is the Michaelis-Menten constant for oil, KmB is the Michaelis-Menten constant for alcohol, and KIA is the inhibition constant for oil.19 Experimental results obtained from different tributyrin and methanol/methyl acetate concentrations were compared to rate values resulting from the kinetic model, and they were found to be consistent with the ping-pong bi-bi mechanism (Figure 8). The slope of the graph that is plotted between experimental and theoretical rate values is nearly 1, and this confirms that the kinetics can be described with this mechanism. Fluidized-Bed Experiments. Substrate conversion was observed at constant substrate and enzyme concentations and different flow rates in a semi-continuous fluidized bed, regardless of whether the transesterification/interesterification of
(18) Shuler, M. L.; Kargi, F. Bioprocess Engineering: Basic Concepts, 2nd ed.; Prentice-Hall International Editions: New York, 2002; pp 57-67.
(19) Rizzi, M.; Stylos, P.; Riek, A. Enzyme Microb. Technol. 1992, 15, 367–382.
1272
Energy Fuels 2010, 24, 1269–1273
: DOI:10.1021/ef9009313
Kabasakal and Caglar
Figure 8. Comparison of the experimental data to data obtained from the ping-pong bi-bi kinetic model.
Figure 9. Fluidized-bed experiments. Reaction conditions: 0.6 mol of tributyrin, 0.36 mol of methanol/methyl acetate (1:6 tributyrin/ methanol/methyl acetate), 35 mL of reaction volume, 320 mg of enzyme (1%), and 25 °C.
tributyrin is applicable to continuous systems. Flow rates that are 5, 10, 15, and 20 mL/min were adjusted with a peristaltic pump (Figure 9). Fixed- and packed-bed reactors were previously used for transesterification reactions.4 In such systems, activity loss resulting from accumulation of glycerol on the immobilized enzyme is usually observed. The proposed fluidized-bed system provides the enzyme to move continuously in the reactor, and this prevents the accumulation of glycerol on the enzyme support because of shear forces. Glycerol is dissolved by excess methanol. The highest conversion (91%) was obtained at 5 mL/min in 4 h. As the flow rate increases, conversion decreases because of low retentions, which allow for the interaction between the enzyme and substrate. Low retentions cause a high local concentration of substrate around the enzyme, because there is an exchange with a relatively fresh substrate solution continuously. High local concentrations of oil affect the reaction negatively for a particular amount of enzyme by
the fact of oil inhibition. This results rather inhibited the reaction and deactivated the enzyme. Conclusion Transesterification of tributyrin with methanol and interesterification with methyl acetate were carried out simultaneously in a solvent-free reaction medium. Methyl acetate avoids the methanol inhibition and provides the usage of methanol in high amounts. It is also a secondary reactant, which enhances the conversion of tributyrin. It increased the solubility of methanol in oil by means of its solubility in oil and miscibility with methanol. A 100% excess amount of methanol/methyl acetate mixture not only causes no inhibition but also prevents the activity loss coming from glycerol. Experimental data are consistent with the kinetic model based on the ping-pong bi-bi mechanism with substrate inhibition.
1273
