Energy & Fuels 2009, 23, 1347–1353
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Immobilized Lipase on Fe3O4 Nanoparticles as Biocatalyst for Biodiesel Production Wenlei Xie* and Ning Ma School of Chemistry and Chemical Engineering, Henan UniVersity of Technology, Zhengzhou 450052, P. R. China
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ReceiVed August 7, 2008. ReVised Manuscript ReceiVed December 3, 2008
In this work, magnetic Fe3O4 nanoparticles treated with (3-aminopropyl)triethoxysilane were used as immobilization material. Lipase was covalently bound to the amino-functionalized magnetic nanoparticles by using glutaraldehyde as a coupling reagent with the activity recovery up to 70% and the enzyme binding efficiency of 84%. The binding of lipase to the magnetic particles was confirmed by enzyme assays, transmission electron microscopy, X-ray powder diffraction, and Fourier transform infrared spectra. Moreover, the immobilized lipase was found to be able to catalyze the transesterification of soybean oil with methanol to produce fatty acid methyl esters (better known as biodiesel). Besides, it was determined that the conversion of soybean oil to biodiesel fuels reached over 90% by the three-step addition of methanol when 60% immobilized lipase was employed. Further study showed that the immobilized lipase could be used four times without significant decrease of activity.
Introduction
and the purification of fatty acid methyl esters is simple to accomplish.7-9
The limited reserves of fossil fuels, the increasing prices of crude oils, and environmental concerns have spurred the development of alternative renewable energy sources. Biodiesel, which is a mixture of monoalkyl ester produced by transesterification of vegetable oils, has attracted considerable attention in the recent past as a renewable, biodegradable, and nonpolluting fuel.1,2 For industrial biodiesel production, homogeneous basic catalysts, including potassium hydroxide, sodium hydroxide, as well as potassium and sodium alkoxides, are commonly used for the transesterification of vegetable oils with methanol to produce fatty acid methyl esters. However, the base-catalyzed process suffers from several drawbacks, such as difficulty in recycling catalyst and environmental pollution.3 To circumvent homogeneous process problems, attempts to use heterogeneous catalysts in the transesterification of vegetable oils have been made. These catalysts can be easily separated at the end of reaction and may also be reused. Several heterogeneous catalysts for the production of biodiesel have been reported in the literature, mainly based on basic alkaline and alkaline-earth compounds, such as calcined hydrotalcites, Li/CaO, and MgO · MgAl2O4.4-6 Recently, the enzymatic transesterification of vegetable oils has become more attractive for biodiesel production, since the byproduct glycerol can be recovered easily
However, the utilization of an enzyme for biodiesel production is often hampered by its reusability, because it lacks longterm stability under processing conditions, and the difficulty in its recovery and recycling from the reaction mixture. Thus, immobilized-lipase-mediated transesterifications for biodiesel production have advantages over free lipase in terms of choice of batch or continuous processes, ease of lipase removal from the reaction mixture, and adaptability to various engineering designs.10-14 Currently, nanosized magnetic particles used widely in the immobilization of protein, peptide, and enzyme have received considerable attention.15-18 The use of magnetic nanoparticles as supports for enzyme immobilization is endowed with the following advantages: (1) higher specific surface favors the binding efficiency, (2) lower mass transfer resistance and less fouling, (3) the selective separation of immobilized enzymes
* To whom correspondence should be addressed. Telephone: +86-37167756302. Fax: +86-371-67756718. E-mail:
[email protected]. (1) Sharma, Y. C.; Singh, B.; Upadhyay, S. N. Fuel 2008, 87, 2355– 2373. (2) Sharma, Y. C.; Singh, B. Fuel 2008, 87, 1740–1742. (3) Ma, F.; Hanna, M. A. Bioresour. Technol. 1999, 70, 1–15. (4) Xie, W.; Peng, H.; Cheng, L. J. Mol. Catal. A: Chem. 2006, 246, 24–32. (5) Watkins, R. S.; Lee, A. F.; Wilson, K. Green Chem. 2004, 6, 335– 340. (6) Wang, Y.; Zhang, F.; Xu, S.; Yang, L.; Li, D.; Evans, D. G.; Duan, X. Chem. Eng. Sci. 2008, 63, 4306–4312.
(7) Vembanur, R. S.; Lakshmi, N. S.; Karuppan, M. Bioresour. Technol. 2008, 99, 3975–3881. (8) Nelson, L. A.; Foglia, T. A.; Marmer, W. N. J. Am. Oil Chem. Soc. 1996, 73, 1191–1195. (9) Shimada, Y.; Watanabe, Y.; Samukawa, T.; Sugihara, A.; Noda, H.; Fukuda, H.; Tominaga, Y. J. Am. Oil Chem. Soc. 1999, 76, 789–793. (10) Shah, S.; Sharma, S.; Gupta, M. N. Energy Fuels 2004, 18, 154– 159. (11) Tan, T.; Nie, K.; Wang, F. Appl. Biochem. Biotechnol. 2006, 128, 109–116. (12) Xu, Y.; Du, W.; Liu, D.; Zeng, J. Biotechnol. Lett. 2003, 25, 1239– 1241. (13) Du, W.; Xu, Y.; Liu, D.; Li, Z. J. Mol. Catal. B: Enzym. 2005, 37, 68–71. (14) Kumari, V.; Shah, S.; Gupta, M. N. Energy Fuels 2007, 21, 368– 372. (15) Huang, S. H.; Liao, M. H.; Chen, D. H. Biotechnol. Prog. 2003, 19, 1095–1100. (16) Akgo¨l, S.; Kacar, Y.; Denizli, A.; Arica, M. Y. Food Chem. 2001, 74, 281–288. (17) Tong, X. D.; Xue, B.; Sun, Y. Biotechnol. Prog. 2001, 17, 134– 139. (18) Guo, Z.; Sun, Y. Biotechnol. Prog. 2004, 20, 500–506.
10.1021/ef800648y CCC: $40.75 2009 American Chemical Society Published on Web 01/21/2009
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under a magnetic field and hence lower operation cost, and (4) the application of a continuous biocatalysis system.17-19 Covalent coupling methods have been adopted to immobilize enzyme onto different supports. In this way, an enzyme with an amino acid residue can be site-directly immobilized by the formation of a covalent bond between the amino acid residue and an active group on the support.20-22 Comparatively, the covalent immobilization can eliminate or significantly reduce leakage of enzyme through increased bond strength. In the present work, magnetic Fe3O4 nanoparticles treated with (3aminopropyl)triethoxysilane (APES) were activated via the glutaraldehyde method. The activated magnetic carries could effectively couple with lipase by covalent bond with higher activity recovery. The immobilization was confirmed by Fourier transform infrared (FT-IR) spectrum measurements, and the size and structure of the particles before and after lipase binding were characterized by transmission electron microscopy (TEM) and X-ray powder diffraction (XRD) techniques. Besides, the factors affecting the activity recovery and immobilization efficiency were investigated. Moreover, the magnetically immobilized lipase was used to produce biodiesel fuels from soybean oil. The effects of various transesterification reaction parameters on the enzymatic conversion of soybean oil were also evaluated. Experimental Section Materials. Lipase (Lipozyme-TL) from Thermomyces lanuginosa was a generous gift from Novozymes. APES was obtained from Jingzhou Chemical Reagent Co. Soybean oil was purchased from a local firm having the following composition in fatty acids (wt %): 12.3% palmitic acid, 5.8% stearic acid, 26.5% oleic acid, 49.4% linoleic acid, and 5.9% linolenic acid, with 874 g/mol average molecular weight. All other materials were of analytical grade and supplied from Shanghai Chemical Reagent Co. Preparation of Magnetic Nanoparticles. Magnetic Fe3O4 nanoparticles were prepared by coprecipitating Fe2+ (FeSO4 · 7H2O) and Fe3+ (FeCl3 · 6H2O) ions in ammonia solution and treating under hydrothermal conditions.15,22 A 2.78 g portion of FeSO4 · 7H2O and 5.4 g of FeCl3 · 6H2O (molar ratio 1:2) were dissolved in 100 mL of distilled water at a final concentration of 0.3 M iron ions. By adding 75 mL of NH4OH solution into the above mixed solution, solid precipitates were formed at 25 °C under vigorous stirring. The particles obtained here were black in color and exhibited a strong magnetic response. Subsequently, the solution was heated at a constant temperature of 80 °C for 30 min and then filtered and washed with distilled water and ethanol. Finally, the resultant precipitates were dried in a vacuum oven at 60 °C and stored for future use. Lipase Immobilization. The procedure used to prepare immobilized lipase was based on the methods developed before, where glutaraldehyde was used as a spacer to covalently immobilize Lipozyme-TL onto Fe3O4.22 To prepare reactive magnetic supports having aldehyde groups on their surfaces, 0.5 g of Fe3O4 nanoparticles containing hydroxyl groups was dispersed in 9.7 mL of ethanol by sonication and then 0.3 mL of APES was added to this solution. Afterward, the reaction mixture was sonicated sufficiently followed by shaking overnight at room temperature. The APES-bound magnetic nanoparticles were thus obtained. The supernatant was removed by magnetic separation and the precipitates were washed several times with distilled water (19) Deng, J.; Peng, Y.; He, C.; Long, X.; Li, P.; Chan, A. S. C. Polym. Int. 2003, 52, 1182–1187. (20) Liu, X.; Ma, Z.; Xing, J.; Liu, H. J. Magn. Magn. Mater. 2004, 270, 1–6. (21) Liu, X.; Xing, J.; Guan, Y.; Shan, G.; Liu, H. Colloid Surf. A 2004, 238, 127–131. (22) Shaw, S. Y.; Chen, Y. J; Ou, J. J.; Ho, L. Enzyme Microb. Technol. 2006, 39, 1089–1095.
Xie and Ma and ethanol. Thereafter, 20 mL of glutaraldehyde, as a coupling reagent, was added to the precipitates of APES-coated magnetic nanoparticles, and the reaction lasted at room temperature for 2 h. After 2 h, the glutaraldehyde-activated particles were separated by magnetic decantation and subsequently washed with distilled water. The activated magnetic carriers thus produced had reactive aldehyde groups that can react with the amino groups of lipase to give covalent bonds. Lipase immobilization was performed as follows. A 10 mL aliquot of lipase solution (0.1 M phosphate buffer, pH 7.0) was mixed with the above activated carriers. The mixture was then shaken at room temperature for the required time. After completion of the reaction, the unbounded enzyme was removed under a magnetic field, and the precipitates were recovered and washed carefully with phosphate buffer (0.1 M phosphate buffer, pH 7.0) for several times until no lipase was detected in the supernatant and then directly used for the activity measurements. The amount of lipase protein immobilized on magnetic nanoparticles was determined by measuring the initial and final concentration of lipase in the immobilization medium using the Bradford method and standardized with bovine serum albumin (BSA).23 A calibration curve constructed with BSA was used in the concentration determination of lipase. The immobilization efficiency of lipase onto the magnetic supports was determined from the following equation
q ) (Ci - Cf)V1 ⁄ CiV2 (%)
(1)
where q is the immobilization efficiency (%), Ci and Cf are the concentrations of the initial soluble lipase, and the final lipase concentrations in the supernatant after immobilization, respectively (mg mL-1), and V1 and V2 are the solution volume (mL). All data in this formula are averages of duplicate experiments. It is worth noting that, in the immobilization processes, there were probably nonspecific sorption (such as ionic and hydrophchic bond) besides mainly covalent binding. However, nonspecific sorption lipase could be removed by buffer washing. Thus, the main interactions between carrier and lipase were the covalent binding. Enzyme Assays. The enzymatic activity of immobilized lipase and free lipase was assayed by the method described previously.24,25 The hydrolytic activities of both forms of the lipase were tested with olive oil emulsion containing 3% (w/v) PVA. To 4 mL of the emulsion and 5 mL of the phosphate buffer (0.025 M, pH 7.5) was added a certain amount of the free or immobilized lipase, and hydrolysis reactions were carried out at 40 °C for 15 min. The quantity of fatty acid liberated was measured by titration with 0.05 M NaOH solution. One unit of enzyme activity was defined as the amount of lipase that liberates 1 µmol of fatty acids per minute under the assay conditions.26 The activity recovery (%) remaining after immobilization was the ratio between the activity of immobilized lipase and the total activity of lipase added in the initial immobilization solution. Characterizations. The size and morphology of magnetic nanoparticles were observed by transmission electron microscopy (TEM) using a JEOL model JEM-1200EX at 80 kV. X-ray powder diffraction measurements were recorded on a Rigaku D/max-3B X-ray diffractometer (Tokyo, Japan) employing Cu KR radiation (γ ) 0.1542 nm). The KBr pellet technique was used for determining the FT-IR spectra of magnetic nanoparticles, free lipase, and lipase-bound nanoparticles. Enzymatic Transesterification Reaction. Transesterification reactions were carried out at 50 °C in a 50 mL shaking flask on a reciprocal shaker. A typical reaction mixture consisted of 9.65 g of soybean oil, a weighed amount of the supported lipase, and a three-step addition of methanol with 0.35 g of methanol in each step. Once the transesterification reaction had finished, the reaction mixture was recovered by magnetic separation and the residual (23) Bradford, M. M. Anal. Biochem. 1976, 72, 248–254. (24) Cho, S. W.; Rhee, J. S. Biotechnol. Bioeng. 1993, 41, 204–210. (25) Kwon, D. Y.; Rhee, J. S. Korean J. Chem. Eng. 1984, 1, 153–158. (26) Abramic, M.; Lescic, I.; Korica, T.; Vitale, L.; Saenger, W.; Pigac, J. Enzyme Microb. Technol. 1999, 25, 522–529.
Immobilized Lipase for Biodiesel Production
Figure 1. Effect of lipase amounts on the immobilization efficiency and activity recovery. Immobilization conditions: coupling reaction time, 6 h; reaction temperature, 25 °C; glutaraldehyde concentration, 10%.
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Figure 2. Effect of coupling time on the immobilization efficiency and activity recovery. Immobilization conditions: lipase amount, 25 mg; reaction temperature, 25 °C; glutaraldehyde concentration, 10%.
methanol was distilled off completely with the help of a rotary evaporator at 60 °C under vacuum. The conversion of soybean oil to methyl esters was determined by measuring hydroxyl content on the transesterified soybean oil as previously described by us in the literature.27 Reusability Assay. To test the stability of immobilized lipase in repeated use, batch transesterification of soybean oil and methanol was conducted by the addition of immobilized lipase. The reaction conditions were the same as described above. The immobilized lipase was reused with fresh substrates for each cycle.
Results and Discussion Factors Affecting the Activity Recovery and Immobilization Efficiency for Lipase Immobilization. Lipase can be covalently immobilized on the magnetic nanoparticles Fe3O4 by forming a Schiff base linkage between the aldehyde group of glutaraldehyde and the terminal amino group of lipase. Various factors can affect the interactions, including lipase concentration, coupling time, coupling temperature, and glutaraldehyde concentration. In this study, five different lipase weights between 12.5 and 62.5 mg were used for immobilization on 500 mg of Fe3O4 nanoparticles at pH 7 to determine the proper lipase weight in immobilization solution. The relationship of lipase weights with the immobilization efficiency and the activity recovery is shown in Figure 1. From this figure, it can be seen that the immobilization efficiency was decreased when the lipase weight rose from 12.5 to 62.5 mg. However, an optimum lipase weight of 25 mg for the activity recovery was observed in Figure 1, and thus, the low lipase weight could improve the activity recovery, but lipase weights of >25 mg caused the activity recovery to decrease. By drawing on the results, the proper lipase weight of 25 mg could be used for the lipase immobilization under the assay conditions. The immobilization efficiency and activity recovery of immobilized lipase as a function of immobilizing time are indicated in Figure 2. It was found that, with increasing the coupling time from 0 to 2 h, the percentage of immobilized lipase increased; the immobilization efficiency on the magnetic nanoparticles remained almost constant as the immobilization time was increased beyond 2 h. It can be explained that at the initial stage, the coupling reaction is rapid, and with further increase in immobilization time, the residual active aldehyde (27) Xie, W.; Li, H. J. Am. Oil Chem. Soc. 2006, 83, 869–872.
Figure 3. Effect of glutaraldehyde concentration (vol %) on the immobilization efficiency and activity recovery. Immobilization conditions: coupling reaction time, 6 h; reaction temperature, 25 °C; lipase amount, 25 mg.
group on the surface of magnetic particles for enzyme immobilization was decreased. This result may suggest that the immobilization is chemical binding, rather than physical adsorption, due to formation of the covalent linkages between the aldehyde group of nanoparticles and the amino group of enzymes. Herein, the activity recovery of the immobilized lipase increased with an increase in coupling time, and the highest activity recovery was obtained if the immobilization was allowed to proceed for 6 h. However, the activity recovery decreased when the coupling time was longer. Long coupling time increases the number of covalent bounds between lipase and carrier. Most probably, the stronger binding seems to decrease the lipase activity, and mostly due to this, the activity recovery decreases, depending on the coupling time. Given these results, it can be inferred that the optimum coupling time for the immobilization process was 6 h. In order to extend the spacer and facilitate the covalent attachment of lipase, the amino functional group on the surface of the magnetic particles was transferred to aldehyde groups by the glutaraldehyde method and was subsequently coupled with the amino group of lipase. Figure 3 shows the effect of glutaraldehyde concentration on the immobilization efficiency and activity recovery. As can be seen, the highest activity recovery and binding efficiency were obtained by using 10% glutaraldehyde as a cross-linking reagent. Any glutaraldehyde concentration less than 10% resulted in insufficient activation of the surface of magnetic carrier, while higher concentrations caused the decline of activity recovery and immobilization efficiency, probably owing to the excessive self-cross-linking
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Figure 4. Effect of coupling temperature on the immobilization efficiency and activity recovery. Immobilization conditions: lipase amount, 25 mg; reaction time, 6 h; glutaraldehyde concentration, 10%.
Figure 5. FT-IR spectra of the magnetic nanoparticles with (a) and without (b) bound lipase and the pure lipase (c).
of glutaraldehyde, which might have a steric effect for lipase immobilization. Thus, the optimal glutaraldehyde concentration was considered to be 10% for the surface activation of magnetic supports. The immobilization efficiency and activity recovery for different immobilization temperatures between 25 and 45 °C were determined, and the results are given in Figure 4. It was found that the immobilization temperature did not affect the immobilization efficiency obviously if the immobilization time is long enough. However, as illustrated in Figure 4, the higher the immobilization temperature was, the lower the activity recovery was. It is a known phenomenon that the thermal deactivation of enzyme could be realized during the immobilization, and consequently, the enzymatic activity is decreased. The highest activity recovery was observed when the immobilization was carried out at room temperature. Characteristics of Magnetic Nanoparticles. Figure 5 shows the FT-IR spectra of the lipase-bound nanoparticles, naked Fe3O4, and pure lipase. The sample of bound lipase for FT-IR spectrum measurement was washed with phosphate buffer (0.1 M phosphate buffer, pH 7.0) until protein in the washing could not be detected before being dried. This procedure guaranteed that no free enzyme was adsorbed on the support. As shown in Figure 5, for the pure lipase, the IR absorption peaks of 1659 and 1540 cm-1 were characteristic peaks of lipase (spectrum c). The strong absorption band around 590 cm-1, for the naked Fe3O4, was ascribed to the Fe-O bond of Fe3O4 (spectrum b), while the absorption peak of hydroxyl groups was observed at 3445 cm-1. After immobilization of lipase on the magnetic
Xie and Ma
particles, the sample showed the characteristic bands of both lipase and Fe3O4 (spectrum a). The weak characteristic peaks of lipase for the enzyme-bound nanoparticles should be owing to the low lipase loading. No additional band in the spectrum for the immobilized lipase was observed, since the covalent bond between the lipase and carrier is of the same nature as typical protein bonds. The glutaraldehyde, used as activation reagent in the immobilization procedure, is a bifunctional reactive compound capable of reacting with the amine groups of enzyme and the support, resulting in amide bonds similar to those present in free lipases. Accordingly, the lipase was indeed bound to the surface of magnetic nanoparticles successfully. The high-resolution transmission electron micrographs for the magnetic nanoparticles without and with bound lipase are shown in Figure 6. According to the TEM pattern, the naked Fe3O4 particles were essentially fine and almost spherical, with the average diameter of 11.2 nm. They are polydisperse and some of them are aggregated due to the magneto-dipole interactions between particles. Furthermore, the particles with bound lipase remained polydisperse discrete and had a mean diameter of 12.9 nm, which was similar to that of unbound ones. This showed that the immobilization process did not significantly result in agglomeration and change in the size of particles, suggesting that the reaction occurred only on the particle surface. The XRD patterns of magnetic nanoparticles without and with bound lipase indicated that, when the lipase was covalently bound to the amino-functionalized magnetic nanoparticles, the sample had XRD patterns identical to that of Fe3O4 carrier (figure not shown). For the Fe3O4 carrier, six characteristic peaks of Fe3O4 were registered at 2θ of 30.1°, 35.5°, 43.1°, 53.4°, 57.0°, and 62.6°, respectively, implying that the particles were pure Fe3O4 with a spinel structure (JCPDS database file, No.851436). For magnetic particles with bound lipase, the same six characteristic peaks were observed. This suggested that the binding process did not result in the phase change of Fe3O4. As a result, the magnetic particles could preserve their magnetic properties during the separation process, which is suitable for application in bioseparations. Properties of the Bound Lipase. Immobilized and free lipases were incubated in the phosphate buffer (pH 7.5) with olive oil emulsion as substrate for 15 min at different temperatures, ranging from 35 to 65 °C. The data in Figure 7 showed that the optimum temperature for the native lipase was 45 °C and the immobilized lipase played the highest hydrolytic activity at 50 °C. Moreover, the immobilized lipase was less sensitive to the change of temperature than the native lipase when the temperature ranged from 45 to 65 °C. Most probably, the increase in optimum temperature was caused by the changing physical and chemical properties of enzyme by immobilization. As a matter of fact, the covalent bond formation might also have reduced the conformational flexibility, as immobilized enzymes need a higher temperature to form the proper conformation to recognize and bind substrate molecules, thus leading to a higher thermoresistance compared to free enzyme.15 To find the pH value of the reaction medium at which maximum hydrolytic activity of lipase is obtained, the influence of different pH values between 1 and 10 on the enzyme activity was investigated. From the data reported in Figure 8, it was observed that the curve of the relationship of immobilized lipase activity versus pH was shifted to the right when compared with that of native lipase, and the optimum pH of immobilized lipase was 7.5, higher than that of free lipase (pH 7.0). Besides, the immobilized lipase retained higher hydrolytic activities when the pH was far from the optimal pH in comparison to its soluble
Immobilized Lipase for Biodiesel Production
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Figure 6. TEM images of magnetic nanoparticles with (a) and without (b) bound lipase.
Figure 7. Effect of temperature on hydrolytic activities of the free and immobilized lipases. The maximum was defined as 100% activity.
Figure 8. Effect of medium pH on hydrolytic activities of the free and immobilized lipases. The maximum was defined as 100% activity.
counterpart. It is likely that the lipase was fixed on the surface of carriers, thus resulting in an increase of the lipase tolerability to the pH variance in its surroundings. Therefore, the resulting immobilized lipase holds excellent adaptability in a wider pH region comparable to native lipase. Immobilized Lipase-Catalyzed Transesterification for Biodiesel Production. The catalytic activity of nanoparticles Fe3O4 and the lipase-bound Fe3O4 in the transesterification reaction of soybean oil was examined. As expected, the Fe3O4 as prepared did not present particular catalytic activities in the reaction at 1:3 oil/menthol molar ratios at 50 °C for 12 h. But after immobilization of lipase on Fe3O4 nanoparticles, these catalysts were active and exhibited a high catalytic activity. As
Figure 9. Effect of oil/methanol molar ratio on the enzymatic transesterification of soybean oil. Reaction conditions: immobilized lipase, 40% (w/w oil); reaction temperature, 50 °C.
a consequence, the catalyst activity is generated by loading the Fe3O4 with the lipase. Since the transesterification of vegetable oil is reversible, an increase in the amount of one of the reactants will lead to higher conversions of oils. In the work, we investigated the role of substrate molar ratio in the transesterification reaction in solventfree medium, conducting the reactions at 1:0.5, 1:1, 1:1.5, 1:2, and 1:3 oil/methanol molar ratios at 50 °C for 12 h with the immobilized lipase. As indicated in Figure 9, the highest soybean oil conversion could be achieved at the oil/methanol molar ratio of 1:1.5. Both higher (1:2 and 1:3) and lower (1:1 and 1:0.5) methanol concentrations would result in the decreased conversion to some degree. Similar results were also obtained by Shimada, who found that immobilized Candida antarctica lipase was inactivated in mixtures containing greater than 1.5 molar equiv of methanol in oil.28 It is well-known that excessive shortchain alcohols such as methanol might have negative effects on lipase activity.29 However, at least 3 molar equiv of methanol is required for the complete conversion of the oil to its corresponding methyl esters. For this adverse effect of methanol, the result aforementioned above was thus obtained. The deactivation of the lipase at higher methanol concentrations (1:2 and 1:3) was due to the low solubility of methanol in the oil phase, and any methanol to oil ratio above 1.5:1 caused a serious loss of lipase activity.28 Moreover, Watanabe and Shimada reported the stepwise addition of methanol through which over 90% conversions were attained in the transesterification of
(28) Shimada, Y.; Watanabe, Y.; Sugihara, A.; Tominaga, Y. J. Mol. Catal. B: Enzym. 2002, 17, 133–142. (29) Watanabe, Y.; Shimada, Y.; Sugihara, A.; Tominaga, Y. J. Am. Oil Chem. Soc. 2001, 78, 703–707.
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Figure 10. Effect of various amounts of lipase on the enzymatic transesterification of soybean oil. Reaction conditions: three-step addition of methanol; methanol/oil 1:1 in each step; reaction temperature, 50 °C.
Figure 11. Effect of water content on the enzymatic transesterification of soybean oil. Reaction conditions: immobilized lipase, 40% (w/w oil); three-step addition of methanol; methanol/oil 1:1 in each step; reaction temperature, 50 °C.
vegetable oil.28,30 With three-step addition of methanol, by which methanol concentration in medium is kept low, hence eliminating enzyme deactivation, a high conversion could be attained. Therefore, to reduce inactivation of immobilized lipases, a threestep addition of methanol, with an oil/methanol molar ratio of 1:1 at each step, was adopted for the enzymatic transesterification in biodiesel production, though the highest soybean oil conversion was achieved at an oil/methanol molar ratio of 1:1.5 with one-step addition of methanol. The effect of immobilized lipase dosages on the enzymatic transesterification in a solvent-free system was also investigated. The variation of conversion when different amounts of this immobilized lipase were used is presented in Figure 10. It was shown that the more immobilized lipase that was used for the transesterification, the higher the conversion to methyl esters could be. During the three-step transesterification for immobilized lipase-catalyzed biodiesel production, the highest conversion was 94% when using 60% immobilized lipase (based on oil weight). Amount of water present in the reactants is an important parameter that is known to influence the lipase activity in nonaqueous media.31 Therefore, the effect of water content on the activity of bound lipase was examined. The results displayed in Figure 11 revealed that addition of water to the reactants afforded a reverse effect on the bound lipase activity. With an increasing amount of water from 0.1% to 0.5%, the soybean (30) Watanabe, Y.; Shimada, Y.; Sugihara, A.; Tominaga, Y. J. Mol. Catal. B: Enzym. 2002, 17, 151–155. (31) Triantafyllou, A. O.; Wehtje, E.; Adlercreutz, P.; Mattiasson, B. Biotechnol. Bioeng. 1997, 54, 67–76.
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Figure 12. Cyclic use of the immobilized lipase. Reaction conditions: three-step addition of methanol; methanol/oil 1:1 in each step; reaction temperature, 50 °C; immobilized lipase, 40% (w/w oil).
oil conversion decreased almost linearly from 87% to 75%. Lipases possess the unique feature of acting at the interface between an aqueous and an organic phase. Activation of the enzyme involves unmasking and restructuring of active sites through conformational changes of the lipase molecule, which requires the presence of an oil-water interface.32 Since there is always some water in the immobilized lipase, water contained in the immobilized lipase is enough to accelerate the transesterification reaction. However, the higher water content might make the lipase aggregate, which could result in a loss of lipase activity. An alternative reason for the decline of bound lipase activity may be due to the hydrolysis side reaction occurring when too much water was contained in the feedstock. The stability of the immobilized lipase was also assessed by reusing it five times in the transesterification reaction. The spent immobilized lipase was recovered by magnetic separation, washed three times with phosphate buffer (pH 7.5), and then used again for a fresh transesterification reaction. The assay conditions remained the same as described above. Figure 12 shows the residual activity as a function of operational time from which about 89% residual activity could be observed when the immobilized lipase was recycled four times. However, the activity of the recovered catalyst to the transesterification reaction was significantly decreased if it was reused more than four times, as illustrated in Figure 12. This indicated that the as-immobilized lipase had a better reusability and can be used over four times. Immobilized lipase can be separated by the process of decantation, after the transesterification reaction, and does not require a troublesome method of separation. Obviously, the immobilized lipase was identified as an effective biocatalyst for the transesterification reaction of soybean oil. The conversion to methyl esters in excess of 90% was obtained using the biocatalyst. Several other immobilized lipases are also reported in the literature. For example, Lipozyme TL IM immobilized on hydrotalcite gave a yield of 92.8% in the transesterification reaction for biodiesel production.33 Watanabe et al. reported that three-step transesterification successfully converted 93.8% of degummed soybean oil to methyl esters by using immobilized lipase, and the lipase could be reused for 25 cycles.30 Using immobilized C. antarctica lipase, a 98% conversion of vegetable oil to its corresponding methyl esters could be achieved.34 Although the immobilized lipase prepared (32) Iso, M.; Chen, B. X.; Eguchi, M.; Kudo, T.; Shreestha, S. J. Mol. Catal. B: Enzym. 2001, 16, 53–58. (33) Yagiz, F.; Kazan, D.; Akin, A. N. Chem. Eng. J. 2007, 134, 262– 267. (34) Shimada, Y.; Watanabe, Y.; Samukawa, T.; Sugihara, A.; Noda, H.; Fukuda, H.; Tominaga, Y. J. Am. Oil Chem. Soc. 1999, 76, 789–793.
Immobilized Lipase for Biodiesel Production
in our study showed a high catalytic activity under the employed conditions, the biocatalyst loading seems to be so high for a good conversion of soybean oil. However, the approach is meaningful because of the magnetic nanoparticles used as a carrier for immobilized lipase. Besides, some other researchers also reported the transesterification of vegetable oil, in which >40% catalyst loading of immobilized lipase was employed for the adequate yield of biodiesel.35-37 Conclusions Enzymatic transesterification of vegetable oils offers an environmentally more attractive option to biodiesel production. The lipase from T. lanuginosa was immobilized onto APES(35) 286. (36) Enzym. (37) 1241.
Herna´ndez-Martı´n, E.; Otero, C. Bioresour. Technol. 2008, 99, 277– Salis, A.; Pinna, M.; Monduzzi, M.; Solinas, V. J. Mol. Catal. B: 2008, 54, 19–26. Xu, Y.; Du, W.; Liu, D.; Zeng, J. Biotechnol. Lett. 2003, 25, 1239–
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coated magnetic nanoparticles via glutaraldehyde coupling reaction. The analyses of TEM and XRD patterns showed that the size and structure of magnetic nanoparticles had no significant change after lipase binding. The optimal conditions for lipase immobilization were dependent on the immobilization time, temperature, the concentration of glutarahyde, and the ratio of lipase to magnetic carrier. After being immobilized on the support, the lipase exhibited good pH tolerance and thermostability. It was determined that the lipase immobilized on magnetic nanoparticles could catalyze the transesterification of soybean oil with methanol to produce biodiesel fuels, with the oil conversion being over 90%. The present study also demonstrated that, in the enzymatic process, the immobilized lipase allowed for its recovery and reuse. Acknowledgment. This work was sponsored by Program for Science & Technology Innovation Talents in Universities of Henan Province in China (HASTIT). EF800648Y
