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Improved Method for Efficient Production of Biodiesel from Palm Oil† M. M. Rahman Talukder,* K. L. Min Beatrice, O. Puay Song, S. Puah, J. Chuan Wu, C. Jae Won, and Y. Chow Institute of Chemical and Engineering Sciences, 1 Pesek Road, Jurong Island, Singapore 627833, Singapore ReceiVed May 27, 2007. ReVised Manuscript ReceiVed August 27, 2007
Biodiesel (methyl esters of long-chain fatty acids) can be produced by methanolysis of vegetable oils using lipase as a biocatalyst. However, the lipase, such as immobilized Candida antarctica lipase B (Novozym 435), is poisoned as a result of the contact with insoluble methanol, reducing the lipase activity. To minimize the problem, a salt-solution-based controlled release system for methanol is developed. The developed method can easily minimize the problem by dissolving methanol in the salt solution and keeping an acceptable methanol concentration in vegetable oil, thereby increasing the lipase activity. The results of methanolysis of palm oil in the salt-solution-based methanol release system are compared to those in a salt-solution-free system, where methanol is added by the traditional method of three successive additions of methanol. The maximum biodiesel yields in both systems are the same (97%), but methanolysis in the developed method progresses 4-fold faster than that in the traditional method. It is found that a LiCl-saturated solution stabilizes Novozym 435 against heat-induced inactivation. The results suggest that the salt-solution-based methanol release system can be an acceptable substitution for the traditional method and provides an efficient method for biodiesel production.
Introduction It is known that there is a finite amount of fossil fuels, which will last for only a few more decades. Developing renewable resources of energy is therefore necessary. Among the renewable sources for the production of alternative fuels, fats and oils have attracted much attention. Among vegetable oils, palm oil ranks among the best in terms of availability and cost. Methyl ester of long-chain fatty acids, known as biodiesel (BD), is an environmentally friendly alternative to fossil fuels. BD can be produced by methanolysis of vegetable oils. BD is industrially produced via chemical catalysis using strong bases as a catalyst. A strong base process suffers from several drawbacks, such as the need for the removal of a base catalyst from products and the treatment of alkaline wastewater. Enzymatic production of BD has been reported as an effective means of circumventing the aforementioned problems.1,2 Lipase, Novozym 435 (Candida antarctica lipase B immobilized on acrylic resin), has been shown to be the most effective for methanolysis of vegetable oils in producing BD.3–5 Although at least a molar equivalent of methanol is required for the complete conversion of vegetable oils to BD, this lipase is poisoned by adding more than a one-third molar equivalent of † Presented at the International Conference on Bioenergy Outlook 2007, Singapore, April 26–27, 2007. * To whom correspondence should be addressed. Fax: +65-63166182. E-mail:
[email protected]. (1) Fukuda, H.; Kondo, A.; Noda, H. J. Biosci. Bioeng. 2001, 92, 405– 416. (2) Nelson, L. A.; Foglia, T. A.; Marmer, W. N. J. Am. Oil. Chem. Soc. 1996, 73, 1191–1195. (3) Du, W.; Xu, Y.; Liu, D.; Zeng, J. J. Mol. Catal. B: Enzym. 2004, 30, 125–129. (4) Shimada, Y.; Watanabe, Y.; Samukawa, T.; Sugihara, A.; Noda, H.; Fukuda, H.; Tominaga, Y. J. Am. Oil. Chem. Soc. 1999, 76, 789–793. (5) Xu, Y.; Du, W.; Liu, D.; Zeng, J. Biotechnol. Lett. 2003, 25, 1239– 1241.
methanol because of its poor solubility in vegetable oils.5–7 The insoluble methanol droplets attach to the surface of Novozym 435, thereby blocking the access of the substrate to the active site of the enzyme.8 The low solubility of glycerol in BD is also a problem; a deposit of glycerol coating the immobilized lipase is formed during the process, reducing the activity.9 To overcome the problem, the stepwise addition of methanol has been employed to maintain a low concentration of methanol,4,7,10 but the method is not suitable for large-scale productions.8 Esters have been used as an alcohol substitute in the BD production;5 however, the process generates a significant amount of byproducts and is costly. Organic solvents have been used for methanolysis of soybean oil,2 but the reaction rate is relatively low when compared to the stepwise methanol addition. For the forgoing reasons, there is a need to develop an efficient enzymatic method for the production of BD. The method should provide a system for having a high enzymatic activity with a minimized methanol poison. In this study, a salt-solution-based controlled release system for methanol is developed to meet the aforementioned needs. Because Novozym 435 is poisoned because of contact with insoluble methanol, the method could easily minimize the problem by keeping the acceptable concentration of methanol in the oil phase while methanol is released according to its partitioning coefficient between the oil and salt-solution phases. Furthermore, once glycerol is produced during the process, it (6) Shimada, Y.; Watanabe, Y.; Sugihara, A.; Tominaga, Y. J. Mol. Catal. B: Enzym. 2002, 76, 133–142. (7) Talukder, M. M. R.; Puah, S. M.; Wu, J. C.; Choi, W. J.; Chow, Y. Biocatal. Biotransform. 2006, 24, 257–262. (8) Chen, J. W.; Wu, W. T. J. Biosci. Bioeng. 2003, 95, 466–469. (9) Dossat, V.; Combes, D.; Marty, A. Enzyme Microb. Technol. 1999, 25, 194–200. (10) Samukawa, T.; Kaieda, M.; Matsumoto, T.; Ban, K.; Kondo, A.; Shimada, Y.; Noda, H.; Fukuda, H. J. Biosci. Bioeng. 2000, 90, 180–183.
10.1021/ef700277r CCC: $40.75 2008 American Chemical Society Published on Web 09/26/2007
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is dissolved in the salt-solution phase, eliminating the glycerol deposition on the immobilized lipase. The results in the developed method are compared to those in the traditional method of three successive additions of methanol. It is found that the developed method is more promising in terms of efficiency and enzyme thermostability. Experimental Section Materials. Novozym 435 (C. antarctica lipase B immobilized on acrylic resin), palm oil, MgCl2, LiCl, and standard methyl esters were purchased from Sigma, St. Louis, MO. The saponification value of palm oil was 200. Methanol and hexane were bought from J.T. Baker, Phillipsburg, NJ. All chemicals were analytical-grade, except high-performance liquid chromatography (HPLC) solvents: hexane, isopropanol, and methanol, and were used as received. Methanolysis. Methanol was dissolved in a salt (1.1 g/mL MgCl2 and 1.08 g/mL LiCl) saturated solution at 25 °C, and the appropriate amount of mixture based on a specified methanol/palm oil molar ratio was then added to 10 g of palm oil. Methanol is miscible with a salt solution at any ratio. The reaction was initiated by adding 0.4 g of Novozym 435. The mixer was mixed by a water bath shaker at 180 rpm and a specified reaction temperature. Upon mixing, methanol was diffused from the salt-solution/methanol phase to the palm oil phase and the biphasic system is formed. Novozym 435 was mostly located at the interface. In the salt-solution-free system, methanol was directly added. The specific activity of lipase was expressed as grams of BD produced min–1 (g of Novozym 435)–1. Recycling of Novozym 435. The immobilized lipase (Novozym 435) was filtered after the reaction and directly used for the next cycle of methanolysis without any treatment. The reaction time for each cycle was kept constant at 8 and 32 h for the salt-solutionbased methanol release system and the traditional method of the three-step addition of methanol, respectively. Analysis. The sample after a specified reaction time was mixed with hexane (80 mL) and filtered to separate Novozym 435. The filtrate was then centrifuged at 5000 rpm to obtain an upper layer. The methyl esters (BD) content in the upper layer was analyzed by HPLC with a UV detector at 210 nm.8,11 A prevail-C18 5u column (4.6 × 250 mm, Altech, Inc., Birmingham, AL) was employed. The mobile phase consisted of three different components: hexane, isopropanol, and methanol. Reservoir A contained methanol, and reservoir B contained a mixture of isopropanol and hexane (5:4, v/v). The gradient went from 100% A to 50% A plus 50% B linearly over 30 min. The flow rate of the mobile phase was 1 mL/min, and the sample injection volume was 10 µL. The water activity (aw) of the salt solution was measured at 25 °C by an AquaLab water activity meter (Decagon Devices, Inc., Pullman, WA).
Figure 1. BD yield at different methanol/palm oil molar ratios. Reaction conditions: 10 g of palm oil, 0.4 g of lipase (Novozym 435), reaction time of 30 h, temperature at 40 °C, MgCl2-saturated solution/methanol ratio of 0.33:1 (v/v), agitation speed at 190 rpm, and premixing for 1 h. The methanol concentration is varied by increasing the volume of MgCl2-saturated solution/methanol mixture.
Figure 2. Effect of added water on Novozym 435 activity in a saltsolution-free system. Reaction conditions: 10 g of palm oil, 0.4 g of lipase (Novozym 435), reaction time of 2 h, temperature of 60 °C, methanol/palm oil ratio of 1:1, agitation speed at 190 rpm, and premixing for 1 h.
Effect of the Methanol/Palm Oil Molar Ratio. At least a stoichiometric amount of methanol (methanol/palm oil ratio of 3:1) is required for the complete conversion of palm oil to BD. However, the BD yield in a salt-solution-free system decreases by adding more than 1/2 of the stoichiometric amount, equivalent to a ratio of 1.5:1, and the reaction ceased at a ratio g3:1 (Figure 1). At a higher ratio, Novozym 435 is easily poisoned as a result of the contact with insoluble methanol.5–7 The insoluble methanol droplets attach to the solid support (acrylic resin) used for lipase immobilization, and the access of the substrate to the lipase active site is blocked, causing the reaction to stop.8 Shimada et al.6 have reported that the maximum solubility of methanol in a mixture of soybean and rapeseed oils is 1/2 of the stoichiometric amount.
It is evident from Figure 1 that methanol poisoning to Novozym 435 can be minimized by the salt-solution-based methanol release system, giving a 97% yield at a ratio g3:1. Because the release of methanol from the salt solution to oil phase depends upon the solubility of methanol in palm oil, the possible methanol concentration in the palm oil phase is within the soluble limit. Therefore, the salt-solution-based controlled release system for methanol can easily minimize the problem by keeping an acceptable concentration of methanol in palm oil. The motivation of using a salt solution instead of pure water is to reduce the water activity (aw) because the presence of water significantly decreases the enzyme activity (Figure 2). Water is a competitive inhibitor for lipase-catalyzed transesterification.12 Effect of the Water Activity (aw) of the Salt Solution. In addition to the water content, it is often desirable to reduce water activity (aw) for reducing unfavorable lipase-catalyzed side reactions. To understand the effect of the water activity (aw) on Novozym-435-catalyzed methanolysis of palm oil, a LiCl salt solution was selected because it provides a broad range of
(11) Holcapek, M.; Jandera, P.; Fischer, J.; Prokes, B. J. Chromatogr. 1999, 858, 13–31.
(12) Valivety, R. H.; Johnston, G. A.; Suckling, C. J.; Halling, P. J. Biotechnol. Bioeng. 1991, 38, 1137–1143.
Results and Discussion
BD from Lipase-Catalyzed Methanolysis
Figure 3. Effect of the water activity (aw) of the LiCl solution on Novozym 435 activity. Reaction conditions: 10 g of palm oil, 0.4 g of lipase (Novozym 435), reaction time of 2 h, methanol/palm oil ratio of 3.2:1, temperature at 60 °C, LiCl-saturated solution/methanol ratio of 0.43:1 (v/v), agitation speed at 190 rpm, and premixing for 1 h. The mixing ratio between the salt-saturated solution/methanol mixture and palm oil is 0.233:1 (v/v).
aw (0.113–1.0). The aw of 1.0 and 0.113 represents the pure water and LiCl-saturated solution, respectively. The solution of different aw is prepared by diluting the LiCl-saturated solution. Figure 3 shows that the activity of Novozym 435 increases with the decrease in aw and reaches a maximum at aw ) 0.113. Since water inhibits the lipase-catalyzed transesterification, the maximum activity is observed at a minimum aw of 0.113. The result indicates that the activity might be further improved by using a salt-saturated solution having an aw of below 0.113. Because the aw of the LiCl-saturated solution is relatively lower (0.113) and, except for MgCl2 and LiCl, others salts (such as NaCl, KCl, NaBr, K–acetate, and KCO3) are precipitated when methanol was added to their saturated solutions, the LiClsaturated solution was used in subsequent experiments. Effect of the Reaction Temperature. The melting point of palm oil is about 37 °C. It is thus necessary to keep the reaction temperature higher than the melting point of palm oil. The results reported in Figure 4 describe the effect of the temperature on the enzyme activity. The maximum activity in the salt-solutionfree system is observed at about 50–60 °C, after which the enzyme undergoes inactivation, while the activity in the saltsolution-containing system increases up to 80 °C. The results indicate that the presence of the salt solution stabilizes immobilized C. antarctica lipase against heat-induced inactivation. The stabilization by the salt solution could be attributed to the decrease in enzyme dissociation. It has been reported that the high salt concentration (5.3 M LiCl) stabilizes the enzyme covalently immobilized in porous glass beads.13 The C. antarctica lipase is physically adsorbed onto macroporous acrylic resin, thereby dissociable at higher temperatures. The increase in the temperature may help to release methanol from the salt solution to oil phase and dissolve glycerol in the salt solution. Hence, the rate of activity increase with the temperature in the salt-solution-based methanol release system is higher than that in the salt-free system. Effect of Premixing. Because of the poor solubility of methanol in palm oil, it is important to premix the methanol and palm oil before initiating the reaction by adding Novozym 435. Figure 5 shows that the premixing time influences the enzyme activity in both systems, but the effect in the salt(13) Klibanov, A. M. Anal. Biochem. 1979, 93, 1–25.
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Figure 4. Effect of the reaction temperature on Novozym 435 activity. Reaction conditions: 10 g of palm oil, 0.4 g of lipase (Novozym 435), reaction time of 2 h, methanol/palm oil ratio of 3.2:1 (solution containing LiCl) and 1:1 (LiCl-free solution), LiCl-saturated solution/ methanol ratio of 0.43:1 (v/v), agitation speed at 190 rpm, and premixing for 1 h. The mixing ratio between the salt-saturated solution/ methanol mixture and palm oil is 0.233:1 (v/v).
Figure 5. Effect of the premixing time on Novozym 435 activity. Reaction conditions: temperature at 60 °C and other conditions are the same as those mentioned in Figure 4.
solution-based methanol release system is more significant than that in the salt-solution-free system. The activity in the saltsolution-containing system reaches a plateau after premixing for 50 min, lower than that in the salt-solution-free system (30 min). This is not surprising because methanol first defuses from the salt solution to palm oil phase and then takes part in the reaction while in salt-solution-free system; methanol gets direct contact with palm oil. Time-Course Production of BD. Figure 6 shows that the maximum BD yield (97%) in the LiCl-saturated-solution-based methanol release system is same as that in the salt-solutionfree system, in which methanol is added by three successive additions.4,7,10 However, the reaction in the case of the single addition of methanol ceases after a short period (
