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Enzymatic transesterification of soybean oil by using immobilized lipase on magnetic nano-particles
biomass and bioenergy 34 (2010) 890–896
Available at www.sciencedirect.com
http://www.elsevier.com/locate/biombioe
Enzymatic transesterification of soybean oil by using immobilized lipase on magnetic nano-particles Wenlei Xie*, Ning Ma School of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou 450001, PR China
article info
abstract
Article history:
Lipase was covalently immobilized onto magnetic Fe3O4 nano-particles by using 1-ethyl-3-
Received 21 December 2009
(3-dimethylaminopropyl) carbodiimide (EDAC) as an activating agent, and the bound lipase
Received in revised form
was used to catalyze the transesterification of vegetable oils with methanol to produce
18 January 2010
fatty acid methyl esters. The binding of lipase to magnetic particles was confirmed by
Accepted 19 January 2010
enzyme assays, transmission electron microscopy (TEM) and Fourier transform infrared
Available online 6 February 2010
(FT-IR) spectra. It was determined that the immobilized lipase exhibited better resistance to temperature and pH inactivation in comparison to free lipase. Using the immobilized
Keywords:
lipase, the major parameters affecting the transesterification reaction, such as the alcohol/
Transesterification
oil molar ratio, enzyme loading and free fatty acid present in reactants were investigated to
Biodiesel
obtain the optimum reaction condition. The conversion of soybean oil to methyl esters
Immobilization
reached over 90% in the three-step transesterification when 40% immobilized lipase was
Magnetic nano-particles
used. Moreover, the lipase catalyst could be used for 3 times without significant decrease of
Thermomyces lanuginosa
the activity. ª 2010 Elsevier Ltd. All rights reserved.
Vegetable oil Glycine max
1.
Introduction
The search for alternative fuels has gained much attention in the recent past because of the potential exhausting and increasing price of petroleum together with environment issues caused by the combustion of fossil fuels. Biodiesel fuel, consisting methyl esters of long chain fatty acids produced by transesterification of vegetable oils or animal fats with methanol, is a promising alternative fuel to mineral-based fossil 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 transesterification of vegetable oils with methanol to produce fatty acid methyl esters. However, the homogeneous processes have major drawbacks due to the difficulties in
catalyst recovery and wastewater treatment, thereby increasing biodiesel production cost [3,4]. To overcome these challenges, heterogeneous transesterification techniques using solid catalysts have been developed and provide clean technology with a simplified downstream process [5–7]. During recent years, the immobilized lipase-mediated transesterification reaction for biodiesel production has been extensively investigated because of its easy recovery from the reaction mixture facilitating its repeated use [8–11]. The supports used for the lipase immobilization are quite important since their interaction with enzyme molecules may influence the activity and stability of immobilized lipase [12]. Considering the facile and fast separation of immobilized enzymes from the reaction mixture, magnetic nano-particles are employed as carriers for enzyme immobilization [13–15]. Moreover, the
* Corresponding author. Tel.: þ86 371 67756302; fax: þ86 371 67756718. E-mail address: [email protected] (W. Xie). 0961-9534/$ – see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biombioe.2010.01.034
biomass and bioenergy 34 (2010) 890–896
covalent binding methods have been recently adopted to immobilize enzyme onto different supports. In this way, an enzyme molecule having an amino acid residue can be sitedirectly immobilized by forming a covalent bond between the amino acid residue and an active group on the support [16–18]. In the present work, the covalent binding of lipase onto the magnetic nano-particles Fe3O4 via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) activation was investigated. The immobilization was confirmed by Fourier transform infrared (FT-IR) spectrum measurement. The size and structure of the resultant particles before and after binding to lipase were characterized by transmission electron microscopy (TEM) and X-ray diffractometer (XRD). The factor affecting the activity recovery and immobilization efficiency was investigated to obtain the optimum condition for lipase immobilization. Dependence of pH and temperature on the hydrolytic activity of the bound lipase was studied. Moreover, the immobilized lipase was tested as a catalyst for the transesterification reaction to produce biodiesel fuels from soybean oil. The effects of various reaction parameters on the transesterification of soybean oil were estimated regarding the conversion of soybean oil to methyl esters.
2.
Experimental
2.1.
Materials
Lipase (Lipozyme-TL) from Thermomyces lanuginosa was a generous gift from Novozymes. Soybean oil was purchased from a local firm having the following composition in fatty acids (wt.%): 12.3% palmitic, 5.8% stearic, 26.5% oleic, 49.4% linoleic and 5.9% linolenic, with 874 g mol1 average molecular weight. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) was purchased from Sigma and used as received. All other chemicals were of analytical grade and used without further purification.
2.2.
Preparation of magnetic nano-particles
Magnetic Fe3O4 nano-particles used for immobilization of lipase were prepared by co-precipitation of Fe2þ (FeSO4$7H2O) and Fe3þ (FeCl3$6H2O) ions in an ammonia solution and treating under hydrothermal conditions [13,18]. Briefly, a 2.78 g portion of FeSO4$7H2O and 5.4 g of FeCl3$6H2O (molar ratio 1:2) were dissolved in 100 cm3 distilled water at a final concentration of 0.3 mol dm3 iron ions. Chemical precipitation was achieved at 25 C by adding NH4OH solution under vigorous stirring. During the reaction process, the pH was maintained at about 10. After incubation for 30 min at 80 C, the mixture was cooled to room temperature with stirring, and the resulting magnetic Fe3O4 nano-particles were separated magnetically and washed three times with distilled water. The product thus obtained was dried in a vacuum oven at 60 C and stored for future use.
2.3.
Lipase immobilization
The use of carbodiimide activation has been an effective method for enzyme immobilization [13]. For preparation of
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lipase-bound magnetic nano-particles, 200 mg of Fe3O4 nano-particles was first dispersed in 2 cm3 of phosphate buffer (0.003 mol dm3 phosphate, pH 6, 0.1 mol dm3 NaCl), and then a solution of carbodiimide (0.5 cm3, 2.5 mg cm3) was added. Afterward, the suspension was treated by sonication at a frequency of 40 kHz for 10 min. Next, 2.5 cm3 of lipase solution (2 mg cm3) was added, and the reaction mixture was sonicated sufficiently for 30 min. The immobilization process was carried out at a constant temperature of 4 C [12,13]. The resulting lipase-bound nano-particles were subsequently recovered from the mixture by magnetic decantation, washed with phosphate buffer (0.003 mol dm3 phosphate, pH 6, 0.1 mol dm3 NaCl) for several times until no free lipase was detected in the supernatant. Finally, the bound lipase was freeze–dried and stored at 4 C for future use. The supernatant was used for enzyme determination according to the method of Bradford by using bovine serum albumin (BSA) as a standard [19]. The immobilized amount of enzyme was calculated by subtracting the amount of unimmobilized enzyme from the total amount of the lipase used for immobilization. The immobilization efficiency of lipase onto the magnetic supports was determined from the following equation. q ¼ Ci Cf V1 =Ci V2 ð%Þ
(1)
where q represents the immobilization efficiency (%), Ci and Cf are the amount-of-substance concentration of the initial soluble enzyme and the final amount-of-substance lipase concentration in the supernatant after immobilization, respectively (mg cm3), and V1 and V2 are the solution volume (cm3). All data in this formula are averages of at least duplicated experiments.
2.4.
Enzyme activity assays
The hydrolytic activity of free and immobilized lipase was tested by olive oil hydrolysis [20]. To 4 cm3 of olive oil emulsion and 5 cm3 of the phosphate buffer (0.025 mol dm3, pH 7.5), a predetermined amount of the free or immobilized lipase was added and the hydrolysis reaction was carried out at 40 C for 15 min. The resultant fatty acids were measured by titration with 0.05 mol dm3 NaOH solution using a phenolphthalein indicator. One unit of lipase activity was defined as the amount of lipase which liberates 1 mmol fatty acid per minute under the assay conditions [21]. The activity recovery (%) remaining after immobilization was the ratio between the activity of bound lipase and the total activity of lipase added in the initial immobilization solution.
2.5.
Characterizations
The size and morphology of magnetic nano-particles were observed by transmission electron microscopy (TEM) using a JEOL model JEM-1200EX at 80 kV. The sample for TEM analysis was obtained by placing a drop of well-dispersed nanoparticles solution onto a copper grid coated with a Formval film, followed by drying the sample at ambient temperature. Once evaporated, the sample was loaded into the microscope for imaging. The accelerating voltage used was 100 kV.
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biomass and bioenergy 34 (2010) 890–896
Enzymatic transesterification reaction
Transesterification reactions were carried out at 45 C in a 50 cm3 capped flask on a shaking incubator. A typical reaction mixture consisted of 9.65 g soybean oil, a weighed amount of the immobilized lipase, and a three-step addition of methanol with 0.35 g methanol in each step. Once the transesterification reaction had completed, the residual methanol was separated with the help of a rotary evaporator under reduced pressure. 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 [22].
2.7.
Reusability assay
To test the stability of immobilized lipase, the immobilized lipase was separated magnetically, washed with phosphate buffer (0.003 mol dm3 phosphate, pH 6, 0.1 mol dm3 NaCl) and tert-butanol, and finally freeze–dried. The recovered immobilized lipase was used in the next batch transesterification with fresh substrates. The assay condition was same as described above.
3.
Results and discussion
3.1. Variables affecting immobilization efficiency and activity recovery for lipase immobilization
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Immobilization efficiency(%)
2.6.
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Activity recovery
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Amounts of lipase(mg) Fig. 1 – Effect of lipase amounts on the immobilization efficiency and activity recovery. Immobilization conditions: immobilization time, 45 min; immobilization temperature, 4 8C.
a constant lipase amount of 5 mg, the immobilization efficiency increased from 48.2% to 90.4%. However, the immobilization efficiency on the magnetic nano-particles almost kept constant when the immobilization time prolonged beyond 45 min. It is explained that at the initial stage, immobilization is rapid; and with further increase in immobilization time, the surface of magnetic particles is saturated with lipase. Besides, as shown in Fig. 2, the activity recovery of the immobilized lipase increased with increasing immobilization time and the highest activity recovery was obtained at immobilization time of 45 min. However, the activity recovery started to decrease as the immobilization time was longer than 45 min. Therefore, the optimal immobilization time was considered to be 45 min for the immobilization of lipase on the magnetic Fe3O4 particles.
3.2.
Characteristics of magnetic particles
Immobilization efficiency(%)
Immobilization is normally considered to be an important method to improve the stability of enzyme. In this work, the lipase from T. lanuginosa was covalently bound to the Fe3O4 100
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Immobilization efficiency Activity recovery
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20 10
Activity recovery (%)
The lipase was immobilized onto the magnetic Fe3O4 nanoparticles using EDAC as an activating agent. This method is notable owing to its simplicity and high efficiency. In this work, the immobilization was carried out under different variables to determine the optimum condition for the lipase immobilization. In order to determine the proper lipase amount for the immobilization, different lipase weights ranging from 1 to 9 mg were used for immobilization on 200 mg Fe3O4 nanoparticles. The experimental results are given in Fig. 1. It was shown that the immobilization efficiency decreased slightly from 96.2% to 90.3% with the increase of lipase amount from 1 to 5 mg, and then decreased sharply with the further increase of lipase amount from 5 to 9 mg. Although a higher amount of lipase binding occurred as the low lipase amount was used for immobilization, there was substantial loss of enzyme activity. For the activity recovery, it reached its maximal value of 70.8% at a lipase amount of 5 mg. After that, it remained nearly constant with further increasing the lipase amount to 9 mg. By drawing on the results, the proper lipase amount for the immobilization was 5 mg. The immobilization efficiency and activity recovery as a function of time are indicated in Fig. 2. It was found that, with increasing the immobilization time from 10 to 45 min at
40
Immobilization efficiency
30
Activity recovery(%)
XRD measurement was performed on a Rigaku D/max-3B X-ray diffractometer using Cu Ka radiation (l ¼ 0.1542 nm). The KBr pellet technique was used for determining the FT-IR spectra of magnetic nano-particles, free lipase, and lipasebound nano-particles. Spectra were recorded on a Shimadzu IR-Prestige-21 spectrometer with 4 cm1 resolution.
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Time(min) Fig. 2 – Effect of immobilization time on the immobilization efficiency and activity recovery. Immobilization conditions: lipase amount, 5 mg; immobilization temperature, 4 8C.
biomass and bioenergy 34 (2010) 890–896
nano-particles by use of EDAC activation. The binding proof of lipase on the Fe3O4 nano-particles was confirmed by FT-IR spectrum measurement and TEM observation. Besides, the immobilized lipase exhibited excellent susceptibility to applied magnetic field, and thus could be readily separated by magnetic decantation. Fig. 3 shows the FT-IR spectra of the pure lipase (a), the naked Fe3O4 (b), and lipase-bound magnetic particles (c). For the naked Fe3O4, the characteristic absorption peak at 578 cm1 was ascribed to the Fe–O stretching vibrations of Fe3O4, while the hydroxyl group absorption peak was observed at 3441 cm1. For the pure lipase, the IR absorption peaks of 1657 cm1 and 1542 cm1 were characteristic peaks of lipase (spectrum a in Fig. 3) [14]. In the FT-IR spectrum of lipase-bound magnetic particles (spectrum c in Fig. 3), the sample showed the characteristic peaks of both lipase and Fe3O4. As a result, the lipase was successfully bound to the magnetic Fe3O4 nano-particles. Moreover, it is worth noting that the weak characteristic bands of lipase for the enzymebound particles should be owing to the low lipase loading. The TEM micrographs for the magnetic nano-particles without and with bound lipase are illustrated in Fig. 4. According to the TEM pattern, the magnetic Fe3O4 nanoparticles were almost spherical or ellipsoidal with a mean particle size of 11.2 nm, and some aggregation occurred due to interactions between magnetic particles. The particles with bound lipase remained discrete and had the average diameter of 12.7 nm which was similar to that of unbound ones. Therefore, the immobilization process did not significantly result in agglomeration and change in the size of the particles after lipase binding, suggesting that the reaction occurred only on the particle surface. The XRD patterns for naked Fe3O4 nano-particles and lipase-bound Fe3O4 nano-particles are shown in Fig. 5. It can be observed from this figure that the lipase-bound magnetic particles exhibited the same XRD patterns as the magnetic Fe3O4. For both samples, six characteristic peaks for Fe3O4 occurred at 2q of 30.1 , 35.5 , 43.1 , 53.4 , 57.0 , and 62.6 , which are marked by their corresponding indices (220), (311), (400), (422), (511) and (440), respectively, implying that both the magnetic particles before and after binding lipase were pure Fe3O4 [13,14]. Given the results, it can be inferred that the immobilization of lipase did not cause the phase change of Fe3O4 particles.
Fig. 3 – FT-IR spectrum of the magnetic nano-particles with (c) and without (b) bound lipase and the pure lipase (a).
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Fig. 4 – TEM images of magnetic nano-particles without (a) and with (b) bound lipase.
3.3.
Properties of immobilized lipase
The hydrolytic activities of free and immobilized lipase were measured at various temperatures, ranging from 35 C to 65 C, and the results are shown in Fig. 6. As indicated in this
Fig. 5 – XRD patterns for magnetic nano-particles with (a) and without (b) bound lipase.
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biomass and bioenergy 34 (2010) 890–896
immobilized lipase retained higher activity when the pH was far from the optimal pH in comparison to its soluble counterpart, suggesting that the immobilization improved the pH stability of the enzyme, in accordance with the results reported [12].
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Relative activity (%)
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3.4. Enzymatic transesterification of soybean oil for biodiesel production 60
Free lipase Immobilized lipase 45
30 35
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Temperature (ºC) Fig. 6 – Effect of temperature on hydrolytic activities of the free and immobilized lipases. The maximum was defined as 100% activity.
figure, the activity was shown to be strongly dependent on the temperature for both free and bound lipase. Meanwhile, the free and bound lipase displayed the highest activity at approximately 45 C. Besides, the immobilized lipase was less sensitive to the change of temperature than the free enzyme as the temperature ranged from 35 C to 65 C. For example, at a temperature of 55 C the immobilized enzyme exhibited 82% of relative activity, while the free enzyme just showed 51%. Thus, the immobilized lipase exhibited a better thermoresistance compared to its free one. The pH dependence of hydrolytic activity for free and immobilized lipase at pH range between 6 and 9 was also investigated. As illustrated in Fig. 7, it can be seen that the maximum hydrolytic activity was observed at pH 7.0 for both free and immobilized lipase. This indicated that the optimal pH value for enzyme activity had no evident changes after the lipase was immobilized to the support. Furthermore, the
One of the important parameters affecting the enzymatic transesterification reaction is the molar ratio of methanol to oil. Fig. 8 graphically illustrates the change of the conversion to methyl esters in solvent-free medium under the assay conditions as a function of the molar ratio of methanol/oil. The enzymatic transesterification reaction was carried out at 45 C for 12 h, with the methanol/oil molar ratio of 1:0.5, 1:1, 1.5:1, 2:1 and 3:1. As indicated in Fig. 7, the conversion to methyl esters increased by an increase in the molar ratio of methanol/oil from 1:0.5 and reached the maximum value at the molar ratio of 1.5:1. However, the further increase in the molar ratio from 1.5:1 to 3:1 resulted in the decreased conversion to some degree. A similar trend, that an increase in the molar ratio of methanol/oil beyond 1.5:1 decreased the oil conversion to methyl esters catalyzed by lipases, was also reported by other authors [23]. It is clear that any molar ratio of methanol to oil above 1.5:1 could lead to the deactivation of the bound lipase. Owing to the low solubility of methanol in the oil, excessive methanol might decrease the lipase activity by the contact with insoluble methanol which exists as drops in the oil [24]. However, from the viewpoint of chemical equilibrium, an increase in the methanol-feeding amount will result in higher methyl ester yield and methanol in excess of the stoichiometric molar ratio of 3:1 is required for the complete conversion of the oil to methyl esters. Considering this adverse effect of methanol, Watanabe et al. reported that a three-stepwise addition of methanol, by which methanol concentration in medium was kept low and hence reducing the negative effect of methanol on the lipase activity to some
methanol/oil=0.5:1 methanol/oil=1:1 methanol/oil=1.5:1 methanol/oil=2:1 methanol/oil=3:1
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Reaction time(h) Fig. 8 – Effect of methanol/oil molar ratios on the transesterification of soybean oil. Reaction conditions: immobilized lipase, 40% (w/w oil); reaction temperature, 45 8C.
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biomass and bioenergy 34 (2010) 890–896
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extent, could result in a high conversion of 90% in the enzymatic transesterification of soybean oil [23,25]. Therefore, in the following study, the three-step addition of methanol, with an oil/methanol molar ratio of 1:1 at each step, was used for the transesterification reaction. The effect of lipase dosages on the transesterification of soybean oil is presented in Fig. 9. During the enzymatic transesterification for biodiesel production, the soybean oil conversion was enhanced with an increase in the bound lipase loading, as illustrated in Fig. 9. The conversion of 94% was obtained when 40% immobilized lipase was used (based on oil weight). It is desirable, for economic reason, to find solid catalysts for handling unrefined and waste oils that commonly contain high amounts of free fatty acid (FFA). Therefore, in this work, experiments were performed to examine the effect of FFAs in reactants on the catalytic activity of the bound lipase towards the transesterification reaction. Reactions were carried out by the addition of different amounts of oleic acid into the reactants. When the FFA amount was 1%, 3%, 5%, 7%, and 9% (based on refined soybean oil weight), the conversions to methyl esters on the optimal reaction conditions were 92.5%, 92.3%, 90.7%, 91.4% and 87.5%, respectively. Obviously, within the range studied, the FFA has no significant effect on the conversion to methyl esters, suggesting that the FFA present in the feedstock could not affect clearly the activity of the bound lipase in the transesterification reaction. The operational stability of the immobilized lipase was assessed in successive batch transesterification processes. The results in Fig. 10 indicated that the spent immobilized lipase exhibited a slight decreased activity in the transesterification reaction upon repeated uses, and more than 70% of its initial activity was still retained even after 3 times of recycling. This indicated that the resultant bound lipase had a better reusability, which was desirable for applications in biotechnology. The loss of activity may be ascribed to conformational changes of enzyme or to blocking of lipase active sites or to the gradual loss of the bound lipase during the reaction procedures.
80 70 60 50 40 30 20 10 0 1
2
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Number of runs Fig. 10 – Cyclic use of the immobilized lipase on the transesterification of soybean oil. Reaction conditions: immobilized lipase, 40% (w/w oil); three-stepwise addition of methanol, methanol/oil 1:1 in each step; reaction temperature, 45 8C. The initial activity was defined as 100%.
4.
Conclusions
Lipase was successfully immobilized to magnetic Fe3O4 nanoparticles using EDAC activation and the immobilized lipase so prepared was then used as a biocatalyst for soybean oil transesterification with methanol to produce biodiesel fuel. The TEM and XRD analyses showed that the size and structure of magnetic Fe3O4 nano-particles had no significant change after lipase was bound to support. The immobilized lipase retained most of its activity in wide ranges of pH and temperature than that of the free lipase. The maximal conversion to methyl esters of 94% was attained by using the bound lipase as catalysts. The stepwise addition of methanol has proven to be effective in the transesterification of vegetable oils. In the enzymatic process, the immobilized lipase on magnetic particles lost little activity when it was subjected to repeated uses, thus allowing for its recovery and reuse.
Acknowledgement
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This work was sponsored by Program for Science & Technology Innovation Talents in Universities of Henan Province in China (HASTIT) and Program for Talents in Henan University of Technology.
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Reaction time (h) Fig. 9 – Effect of different amounts of immobilized lipase on the transesterification of soybean oil. Reaction conditions: three-stepwise addition of menthol, methanol/oil 1:1 in each step; reaction temperature, 45 8C.
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Available at www.sciencedirect.com
http://www.elsevier.com/locate/biombioe
Enzymatic transesterification of soybean oil by using immobilized lipase on magnetic nano-particles Wenlei Xie*, Ning Ma School of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou 450001, PR China
article info
abstract
Article history:
Lipase was covalently immobilized onto magnetic Fe3O4 nano-particles by using 1-ethyl-3-
Received 21 December 2009
(3-dimethylaminopropyl) carbodiimide (EDAC) as an activating agent, and the bound lipase
Received in revised form
was used to catalyze the transesterification of vegetable oils with methanol to produce
18 January 2010
fatty acid methyl esters. The binding of lipase to magnetic particles was confirmed by
Accepted 19 January 2010
enzyme assays, transmission electron microscopy (TEM) and Fourier transform infrared
Available online 6 February 2010
(FT-IR) spectra. It was determined that the immobilized lipase exhibited better resistance to temperature and pH inactivation in comparison to free lipase. Using the immobilized
Keywords:
lipase, the major parameters affecting the transesterification reaction, such as the alcohol/
Transesterification
oil molar ratio, enzyme loading and free fatty acid present in reactants were investigated to
Biodiesel
obtain the optimum reaction condition. The conversion of soybean oil to methyl esters
Immobilization
reached over 90% in the three-step transesterification when 40% immobilized lipase was
Magnetic nano-particles
used. Moreover, the lipase catalyst could be used for 3 times without significant decrease of
Thermomyces lanuginosa
the activity. ª 2010 Elsevier Ltd. All rights reserved.
Vegetable oil Glycine max
1.
Introduction
The search for alternative fuels has gained much attention in the recent past because of the potential exhausting and increasing price of petroleum together with environment issues caused by the combustion of fossil fuels. Biodiesel fuel, consisting methyl esters of long chain fatty acids produced by transesterification of vegetable oils or animal fats with methanol, is a promising alternative fuel to mineral-based fossil 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 transesterification of vegetable oils with methanol to produce fatty acid methyl esters. However, the homogeneous processes have major drawbacks due to the difficulties in
catalyst recovery and wastewater treatment, thereby increasing biodiesel production cost [3,4]. To overcome these challenges, heterogeneous transesterification techniques using solid catalysts have been developed and provide clean technology with a simplified downstream process [5–7]. During recent years, the immobilized lipase-mediated transesterification reaction for biodiesel production has been extensively investigated because of its easy recovery from the reaction mixture facilitating its repeated use [8–11]. The supports used for the lipase immobilization are quite important since their interaction with enzyme molecules may influence the activity and stability of immobilized lipase [12]. Considering the facile and fast separation of immobilized enzymes from the reaction mixture, magnetic nano-particles are employed as carriers for enzyme immobilization [13–15]. Moreover, the
* Corresponding author. Tel.: þ86 371 67756302; fax: þ86 371 67756718. E-mail address: [email protected] (W. Xie). 0961-9534/$ – see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biombioe.2010.01.034
biomass and bioenergy 34 (2010) 890–896
covalent binding methods have been recently adopted to immobilize enzyme onto different supports. In this way, an enzyme molecule having an amino acid residue can be sitedirectly immobilized by forming a covalent bond between the amino acid residue and an active group on the support [16–18]. In the present work, the covalent binding of lipase onto the magnetic nano-particles Fe3O4 via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) activation was investigated. The immobilization was confirmed by Fourier transform infrared (FT-IR) spectrum measurement. The size and structure of the resultant particles before and after binding to lipase were characterized by transmission electron microscopy (TEM) and X-ray diffractometer (XRD). The factor affecting the activity recovery and immobilization efficiency was investigated to obtain the optimum condition for lipase immobilization. Dependence of pH and temperature on the hydrolytic activity of the bound lipase was studied. Moreover, the immobilized lipase was tested as a catalyst for the transesterification reaction to produce biodiesel fuels from soybean oil. The effects of various reaction parameters on the transesterification of soybean oil were estimated regarding the conversion of soybean oil to methyl esters.
2.
Experimental
2.1.
Materials
Lipase (Lipozyme-TL) from Thermomyces lanuginosa was a generous gift from Novozymes. Soybean oil was purchased from a local firm having the following composition in fatty acids (wt.%): 12.3% palmitic, 5.8% stearic, 26.5% oleic, 49.4% linoleic and 5.9% linolenic, with 874 g mol1 average molecular weight. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) was purchased from Sigma and used as received. All other chemicals were of analytical grade and used without further purification.
2.2.
Preparation of magnetic nano-particles
Magnetic Fe3O4 nano-particles used for immobilization of lipase were prepared by co-precipitation of Fe2þ (FeSO4$7H2O) and Fe3þ (FeCl3$6H2O) ions in an ammonia solution and treating under hydrothermal conditions [13,18]. Briefly, a 2.78 g portion of FeSO4$7H2O and 5.4 g of FeCl3$6H2O (molar ratio 1:2) were dissolved in 100 cm3 distilled water at a final concentration of 0.3 mol dm3 iron ions. Chemical precipitation was achieved at 25 C by adding NH4OH solution under vigorous stirring. During the reaction process, the pH was maintained at about 10. After incubation for 30 min at 80 C, the mixture was cooled to room temperature with stirring, and the resulting magnetic Fe3O4 nano-particles were separated magnetically and washed three times with distilled water. The product thus obtained was dried in a vacuum oven at 60 C and stored for future use.
2.3.
Lipase immobilization
The use of carbodiimide activation has been an effective method for enzyme immobilization [13]. For preparation of
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lipase-bound magnetic nano-particles, 200 mg of Fe3O4 nano-particles was first dispersed in 2 cm3 of phosphate buffer (0.003 mol dm3 phosphate, pH 6, 0.1 mol dm3 NaCl), and then a solution of carbodiimide (0.5 cm3, 2.5 mg cm3) was added. Afterward, the suspension was treated by sonication at a frequency of 40 kHz for 10 min. Next, 2.5 cm3 of lipase solution (2 mg cm3) was added, and the reaction mixture was sonicated sufficiently for 30 min. The immobilization process was carried out at a constant temperature of 4 C [12,13]. The resulting lipase-bound nano-particles were subsequently recovered from the mixture by magnetic decantation, washed with phosphate buffer (0.003 mol dm3 phosphate, pH 6, 0.1 mol dm3 NaCl) for several times until no free lipase was detected in the supernatant. Finally, the bound lipase was freeze–dried and stored at 4 C for future use. The supernatant was used for enzyme determination according to the method of Bradford by using bovine serum albumin (BSA) as a standard [19]. The immobilized amount of enzyme was calculated by subtracting the amount of unimmobilized enzyme from the total amount of the lipase used for immobilization. The immobilization efficiency of lipase onto the magnetic supports was determined from the following equation. q ¼ Ci Cf V1 =Ci V2 ð%Þ
(1)
where q represents the immobilization efficiency (%), Ci and Cf are the amount-of-substance concentration of the initial soluble enzyme and the final amount-of-substance lipase concentration in the supernatant after immobilization, respectively (mg cm3), and V1 and V2 are the solution volume (cm3). All data in this formula are averages of at least duplicated experiments.
2.4.
Enzyme activity assays
The hydrolytic activity of free and immobilized lipase was tested by olive oil hydrolysis [20]. To 4 cm3 of olive oil emulsion and 5 cm3 of the phosphate buffer (0.025 mol dm3, pH 7.5), a predetermined amount of the free or immobilized lipase was added and the hydrolysis reaction was carried out at 40 C for 15 min. The resultant fatty acids were measured by titration with 0.05 mol dm3 NaOH solution using a phenolphthalein indicator. One unit of lipase activity was defined as the amount of lipase which liberates 1 mmol fatty acid per minute under the assay conditions [21]. The activity recovery (%) remaining after immobilization was the ratio between the activity of bound lipase and the total activity of lipase added in the initial immobilization solution.
2.5.
Characterizations
The size and morphology of magnetic nano-particles were observed by transmission electron microscopy (TEM) using a JEOL model JEM-1200EX at 80 kV. The sample for TEM analysis was obtained by placing a drop of well-dispersed nanoparticles solution onto a copper grid coated with a Formval film, followed by drying the sample at ambient temperature. Once evaporated, the sample was loaded into the microscope for imaging. The accelerating voltage used was 100 kV.
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Enzymatic transesterification reaction
Transesterification reactions were carried out at 45 C in a 50 cm3 capped flask on a shaking incubator. A typical reaction mixture consisted of 9.65 g soybean oil, a weighed amount of the immobilized lipase, and a three-step addition of methanol with 0.35 g methanol in each step. Once the transesterification reaction had completed, the residual methanol was separated with the help of a rotary evaporator under reduced pressure. 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 [22].
2.7.
Reusability assay
To test the stability of immobilized lipase, the immobilized lipase was separated magnetically, washed with phosphate buffer (0.003 mol dm3 phosphate, pH 6, 0.1 mol dm3 NaCl) and tert-butanol, and finally freeze–dried. The recovered immobilized lipase was used in the next batch transesterification with fresh substrates. The assay condition was same as described above.
3.
Results and discussion
3.1. Variables affecting immobilization efficiency and activity recovery for lipase immobilization
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2.6.
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Activity recovery
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Amounts of lipase(mg) Fig. 1 – Effect of lipase amounts on the immobilization efficiency and activity recovery. Immobilization conditions: immobilization time, 45 min; immobilization temperature, 4 8C.
a constant lipase amount of 5 mg, the immobilization efficiency increased from 48.2% to 90.4%. However, the immobilization efficiency on the magnetic nano-particles almost kept constant when the immobilization time prolonged beyond 45 min. It is explained that at the initial stage, immobilization is rapid; and with further increase in immobilization time, the surface of magnetic particles is saturated with lipase. Besides, as shown in Fig. 2, the activity recovery of the immobilized lipase increased with increasing immobilization time and the highest activity recovery was obtained at immobilization time of 45 min. However, the activity recovery started to decrease as the immobilization time was longer than 45 min. Therefore, the optimal immobilization time was considered to be 45 min for the immobilization of lipase on the magnetic Fe3O4 particles.
3.2.
Characteristics of magnetic particles
Immobilization efficiency(%)
Immobilization is normally considered to be an important method to improve the stability of enzyme. In this work, the lipase from T. lanuginosa was covalently bound to the Fe3O4 100
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Immobilization efficiency Activity recovery
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Activity recovery (%)
The lipase was immobilized onto the magnetic Fe3O4 nanoparticles using EDAC as an activating agent. This method is notable owing to its simplicity and high efficiency. In this work, the immobilization was carried out under different variables to determine the optimum condition for the lipase immobilization. In order to determine the proper lipase amount for the immobilization, different lipase weights ranging from 1 to 9 mg were used for immobilization on 200 mg Fe3O4 nanoparticles. The experimental results are given in Fig. 1. It was shown that the immobilization efficiency decreased slightly from 96.2% to 90.3% with the increase of lipase amount from 1 to 5 mg, and then decreased sharply with the further increase of lipase amount from 5 to 9 mg. Although a higher amount of lipase binding occurred as the low lipase amount was used for immobilization, there was substantial loss of enzyme activity. For the activity recovery, it reached its maximal value of 70.8% at a lipase amount of 5 mg. After that, it remained nearly constant with further increasing the lipase amount to 9 mg. By drawing on the results, the proper lipase amount for the immobilization was 5 mg. The immobilization efficiency and activity recovery as a function of time are indicated in Fig. 2. It was found that, with increasing the immobilization time from 10 to 45 min at
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Immobilization efficiency
30
Activity recovery(%)
XRD measurement was performed on a Rigaku D/max-3B X-ray diffractometer using Cu Ka radiation (l ¼ 0.1542 nm). The KBr pellet technique was used for determining the FT-IR spectra of magnetic nano-particles, free lipase, and lipasebound nano-particles. Spectra were recorded on a Shimadzu IR-Prestige-21 spectrometer with 4 cm1 resolution.
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Time(min) Fig. 2 – Effect of immobilization time on the immobilization efficiency and activity recovery. Immobilization conditions: lipase amount, 5 mg; immobilization temperature, 4 8C.
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nano-particles by use of EDAC activation. The binding proof of lipase on the Fe3O4 nano-particles was confirmed by FT-IR spectrum measurement and TEM observation. Besides, the immobilized lipase exhibited excellent susceptibility to applied magnetic field, and thus could be readily separated by magnetic decantation. Fig. 3 shows the FT-IR spectra of the pure lipase (a), the naked Fe3O4 (b), and lipase-bound magnetic particles (c). For the naked Fe3O4, the characteristic absorption peak at 578 cm1 was ascribed to the Fe–O stretching vibrations of Fe3O4, while the hydroxyl group absorption peak was observed at 3441 cm1. For the pure lipase, the IR absorption peaks of 1657 cm1 and 1542 cm1 were characteristic peaks of lipase (spectrum a in Fig. 3) [14]. In the FT-IR spectrum of lipase-bound magnetic particles (spectrum c in Fig. 3), the sample showed the characteristic peaks of both lipase and Fe3O4. As a result, the lipase was successfully bound to the magnetic Fe3O4 nano-particles. Moreover, it is worth noting that the weak characteristic bands of lipase for the enzymebound particles should be owing to the low lipase loading. The TEM micrographs for the magnetic nano-particles without and with bound lipase are illustrated in Fig. 4. According to the TEM pattern, the magnetic Fe3O4 nanoparticles were almost spherical or ellipsoidal with a mean particle size of 11.2 nm, and some aggregation occurred due to interactions between magnetic particles. The particles with bound lipase remained discrete and had the average diameter of 12.7 nm which was similar to that of unbound ones. Therefore, the immobilization process did not significantly result in agglomeration and change in the size of the particles after lipase binding, suggesting that the reaction occurred only on the particle surface. The XRD patterns for naked Fe3O4 nano-particles and lipase-bound Fe3O4 nano-particles are shown in Fig. 5. It can be observed from this figure that the lipase-bound magnetic particles exhibited the same XRD patterns as the magnetic Fe3O4. For both samples, six characteristic peaks for Fe3O4 occurred at 2q of 30.1 , 35.5 , 43.1 , 53.4 , 57.0 , and 62.6 , which are marked by their corresponding indices (220), (311), (400), (422), (511) and (440), respectively, implying that both the magnetic particles before and after binding lipase were pure Fe3O4 [13,14]. Given the results, it can be inferred that the immobilization of lipase did not cause the phase change of Fe3O4 particles.
Fig. 3 – FT-IR spectrum of the magnetic nano-particles with (c) and without (b) bound lipase and the pure lipase (a).
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Fig. 4 – TEM images of magnetic nano-particles without (a) and with (b) bound lipase.
3.3.
Properties of immobilized lipase
The hydrolytic activities of free and immobilized lipase were measured at various temperatures, ranging from 35 C to 65 C, and the results are shown in Fig. 6. As indicated in this
Fig. 5 – XRD patterns for magnetic nano-particles with (a) and without (b) bound lipase.
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immobilized lipase retained higher activity when the pH was far from the optimal pH in comparison to its soluble counterpart, suggesting that the immobilization improved the pH stability of the enzyme, in accordance with the results reported [12].
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3.4. Enzymatic transesterification of soybean oil for biodiesel production 60
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Temperature (ºC) Fig. 6 – Effect of temperature on hydrolytic activities of the free and immobilized lipases. The maximum was defined as 100% activity.
figure, the activity was shown to be strongly dependent on the temperature for both free and bound lipase. Meanwhile, the free and bound lipase displayed the highest activity at approximately 45 C. Besides, the immobilized lipase was less sensitive to the change of temperature than the free enzyme as the temperature ranged from 35 C to 65 C. For example, at a temperature of 55 C the immobilized enzyme exhibited 82% of relative activity, while the free enzyme just showed 51%. Thus, the immobilized lipase exhibited a better thermoresistance compared to its free one. The pH dependence of hydrolytic activity for free and immobilized lipase at pH range between 6 and 9 was also investigated. As illustrated in Fig. 7, it can be seen that the maximum hydrolytic activity was observed at pH 7.0 for both free and immobilized lipase. This indicated that the optimal pH value for enzyme activity had no evident changes after the lipase was immobilized to the support. Furthermore, the
One of the important parameters affecting the enzymatic transesterification reaction is the molar ratio of methanol to oil. Fig. 8 graphically illustrates the change of the conversion to methyl esters in solvent-free medium under the assay conditions as a function of the molar ratio of methanol/oil. The enzymatic transesterification reaction was carried out at 45 C for 12 h, with the methanol/oil molar ratio of 1:0.5, 1:1, 1.5:1, 2:1 and 3:1. As indicated in Fig. 7, the conversion to methyl esters increased by an increase in the molar ratio of methanol/oil from 1:0.5 and reached the maximum value at the molar ratio of 1.5:1. However, the further increase in the molar ratio from 1.5:1 to 3:1 resulted in the decreased conversion to some degree. A similar trend, that an increase in the molar ratio of methanol/oil beyond 1.5:1 decreased the oil conversion to methyl esters catalyzed by lipases, was also reported by other authors [23]. It is clear that any molar ratio of methanol to oil above 1.5:1 could lead to the deactivation of the bound lipase. Owing to the low solubility of methanol in the oil, excessive methanol might decrease the lipase activity by the contact with insoluble methanol which exists as drops in the oil [24]. However, from the viewpoint of chemical equilibrium, an increase in the methanol-feeding amount will result in higher methyl ester yield and methanol in excess of the stoichiometric molar ratio of 3:1 is required for the complete conversion of the oil to methyl esters. Considering this adverse effect of methanol, Watanabe et al. reported that a three-stepwise addition of methanol, by which methanol concentration in medium was kept low and hence reducing the negative effect of methanol on the lipase activity to some
methanol/oil=0.5:1 methanol/oil=1:1 methanol/oil=1.5:1 methanol/oil=2:1 methanol/oil=3:1
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pH Fig. 7 – Effect of medium pH on hydrolytic activities of the free and immobilized lipases. The maximum was defined as 100% activity.
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Reaction time(h) Fig. 8 – Effect of methanol/oil molar ratios on the transesterification of soybean oil. Reaction conditions: immobilized lipase, 40% (w/w oil); reaction temperature, 45 8C.
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extent, could result in a high conversion of 90% in the enzymatic transesterification of soybean oil [23,25]. Therefore, in the following study, the three-step addition of methanol, with an oil/methanol molar ratio of 1:1 at each step, was used for the transesterification reaction. The effect of lipase dosages on the transesterification of soybean oil is presented in Fig. 9. During the enzymatic transesterification for biodiesel production, the soybean oil conversion was enhanced with an increase in the bound lipase loading, as illustrated in Fig. 9. The conversion of 94% was obtained when 40% immobilized lipase was used (based on oil weight). It is desirable, for economic reason, to find solid catalysts for handling unrefined and waste oils that commonly contain high amounts of free fatty acid (FFA). Therefore, in this work, experiments were performed to examine the effect of FFAs in reactants on the catalytic activity of the bound lipase towards the transesterification reaction. Reactions were carried out by the addition of different amounts of oleic acid into the reactants. When the FFA amount was 1%, 3%, 5%, 7%, and 9% (based on refined soybean oil weight), the conversions to methyl esters on the optimal reaction conditions were 92.5%, 92.3%, 90.7%, 91.4% and 87.5%, respectively. Obviously, within the range studied, the FFA has no significant effect on the conversion to methyl esters, suggesting that the FFA present in the feedstock could not affect clearly the activity of the bound lipase in the transesterification reaction. The operational stability of the immobilized lipase was assessed in successive batch transesterification processes. The results in Fig. 10 indicated that the spent immobilized lipase exhibited a slight decreased activity in the transesterification reaction upon repeated uses, and more than 70% of its initial activity was still retained even after 3 times of recycling. This indicated that the resultant bound lipase had a better reusability, which was desirable for applications in biotechnology. The loss of activity may be ascribed to conformational changes of enzyme or to blocking of lipase active sites or to the gradual loss of the bound lipase during the reaction procedures.
80 70 60 50 40 30 20 10 0 1
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Number of runs Fig. 10 – Cyclic use of the immobilized lipase on the transesterification of soybean oil. Reaction conditions: immobilized lipase, 40% (w/w oil); three-stepwise addition of methanol, methanol/oil 1:1 in each step; reaction temperature, 45 8C. The initial activity was defined as 100%.
4.
Conclusions
Lipase was successfully immobilized to magnetic Fe3O4 nanoparticles using EDAC activation and the immobilized lipase so prepared was then used as a biocatalyst for soybean oil transesterification with methanol to produce biodiesel fuel. The TEM and XRD analyses showed that the size and structure of magnetic Fe3O4 nano-particles had no significant change after lipase was bound to support. The immobilized lipase retained most of its activity in wide ranges of pH and temperature than that of the free lipase. The maximal conversion to methyl esters of 94% was attained by using the bound lipase as catalysts. The stepwise addition of methanol has proven to be effective in the transesterification of vegetable oils. In the enzymatic process, the immobilized lipase on magnetic particles lost little activity when it was subjected to repeated uses, thus allowing for its recovery and reuse.
Acknowledgement
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This work was sponsored by Program for Science & Technology Innovation Talents in Universities of Henan Province in China (HASTIT) and Program for Talents in Henan University of Technology.
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references 20 0
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Reaction time (h) Fig. 9 – Effect of different amounts of immobilized lipase on the transesterification of soybean oil. Reaction conditions: three-stepwise addition of menthol, methanol/oil 1:1 in each step; reaction temperature, 45 8C.
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