Enzymatic Production of Biodiesel from Soybean Oil by Using

27 Mar 2014 - School of Chemistry and Chemical Engineering, Henan University of ...... Gulay Bayramoglu , Aydin Akbulut , V. Cengiz Ozalp , M. Yakup A...
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Enzymatic Production of Biodiesel from Soybean Oil by Using Immobilized Lipase on Fe3O4/Poly(styrene-methacrylic acid) Magnetic Microsphere as a Biocatalyst Wenlei Xie* and Jianlong Wang School of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou 450052, People’s Republic of China ABSTRACT: A magnetic composite poly(styrene-methacrylic acid) microsphere, was prepared using oleic acid-coated magnetic nanoparticles as seeds by microemulsion copolymerization of styrene (St) and methacrylic acid (MAA). The lipase from Candida rugosa was then covalently bound to the magnetic polymer-coated microspheres by using 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDAC) as an activation reagent. The immobilization of lipase could enhance the thermal and pH stability of lipase activity when compared to free lipase. The immobilized lipase microspheres were characterized by lipase activity assays, Fourier transform infrared spectroscopy, powder X-ray diffraction, transmission electron microscopy, and vibratingsample magnetometer techniques. The bound lipase showed high activities to soybean oil transesterification with methanol to produce biodiesel. It was found that the oil conversion of 86% was attained at a reaction temperature of 35 °C for 24 h. The immobilized lipase is stable with repeated use for four cycles without severe loss of its activity.

1. INTRODUCTION Seriously diminishing fossil energy resources and rising global environmental concerns have led to a search for an alternative fuel from renewable energy sources such as plant biomass. Renewable fuel such as biodiesel is accepted as a biodegradable, nontoxic, and low air pollutant emitting fuel. Biodiesel, consisting of methyl esters of long-chain fatty acids, is produced from vegetable oils.1,2 Commercially, biodiesel fuel can be produced through the process of transesterification of vegetable oils with methanol in the presence of basic catalysts.3,4 However, these basic catalysts cannot be employed for biodiesel preparation from low-quality vegetable oils, since these oils frequently contain significant quantities of free fatty acids and water.5,6 Recently, the use of lipase for the transesterification of vegetable oils has attracted growing attention for biodiesel production, since it often operates under milder reaction conditions and avoids the use of hazardous toxic chemicals. Apart from this, the lipase might catalyze the esterification of free fatty acids and transesterification of triglycerides simultaneously under the appropriate reaction conditions, thus allowing it to be more competitive as far as a low-cost oil is concerned.7,8 However, the utilization of lipase for the transesterification reaction has barriers to its industrial process because of its high cost, the lack of long-term stability under reaction conditions, the impossibility of multiple reuses, and the difficulty in its separation, recycling, and reuse. For the sake of overcoming the aforementioned limitations, the immobilization of lipase onto a suitable support is a plausible solution and thus is becoming a challenging area of research.9 The immobilized lipase can provide many advantages over native lipase to the transesterification reaction for biodiesel production, such as the ease in catalyst removal from the product, feasible continuous operations, and simple product purification.10−12 Until now, a variety of immobilized lipases have been used for biodiesel production. Liu et al. studied the immobilization of © 2014 American Chemical Society

Burkholderia lipase onto hydrophobic magnetic particles for biodiesel production, and the conversion to biodiesel reached nearly 70% within 12 h by using the immobilized lipase at room temperature.13 The transesterification of Jatropha curcas oil catalyzed by the immobilized Rhizopus oryzae lipase was investigated, and the best conversion to biodiesel of 87.1% was obtained at 40 °C after 17 h when a methanol to oil molar ratio of 5:1 was employed.14 The preparation of biodiesel by enzymatic transesterification of vegetable oils with ethanol was investigated with the immobilized lipase from Candida antarctica (C. antarctica), in which the highest biodiesel yield of 87% was achieved after 24 h of reaction at 30 °C. 15 Biodiesel can also be produced by enzymatic esterification of long-chain fatty acids using a biocatalyst. The immobilized C. antarctica lipase was found to be efficient for the esterification of oleic acid with aliphatic alcohols, providing a yield of biodiesel above 90% after 24 h of reaction.16 There are different types of support materials used for the immobilization of lipase, such as natural macromolecules, inorganic materials, and some synthetic polymers.17,18 The choice of appropriate support materials and efficient attachment methods seems to be important for the immobilization of lipase. Covalent coupling methods are widely employed to immobilize lipase on different supports. In this way, the presence of reactive groups allows enzymes to covalently bind directly to the surface of the support through the condensation reaction between the lipase and the support to facilitate the attachment of lipase .19,20 Specifically, there is a trend to adopt nanostructured solid materials as carriers for the immobilization of lipase. Among the different kinds of nanoparticles, magnetic nanoparticles have recently received particular attention, especially in the fields of enzyme immobilization, mostly due Received: January 15, 2014 Revised: March 27, 2014 Published: March 27, 2014 2624

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activating reagent has been a common method for the immobilization of enzyme, achieving relatively high immobilization efficiency.23,24 In a typical assay, 2 g of magnetic copolymer microsphere was treated with 20 mL of EDAC solution in a shaking water bath at 30 °C for 5 h to activate the carboxylic group prior to lipase immobilization. After magnetic separation, the EDAC-activated microsphere was treated with 30 mL of lipase solution (0.1 mol/L phosphate buffer, pH 7.0), and the resulting solution was then placed in a shaking incubator at 35 °C for 18 h to finish the covalent attachment of lipase. Afterward, the bound lipase was recovered by magnetic decantation, washed thoroughly with phosphate buffer (0.1 mol/L phosphate buffer, pH 7.0), and then employed for the lipase determinations. Finally, the lipase-loaded microspheres were freeze-dried at 4 °C for future use, and the washing solutions were collected to measure the amount of residual lipase. The quantity of lipase in the lipase solution and the washing solution was determined by the Bradford method using BSA as a standard.25 A calibration curve constructed with a BSA solution of known concentration was used for the calculation of the lipase concentration. The immobilization efficiency of lipase on the magnetite copolymer support was estimated from the following equation:

to the rapid separation of the bound lipase by using a magnetic filed.20−22 In the present contribution, for the purpose of providing environmentally friendly procedures for biodiesel preparation, magnetic composite poly(styrene-methacrylic acid) microspheres were first prepared by copolymerization of styrene (St) and methacrylic acid (MAA) in the presence of oleic acid-coated magnetite. In a further step, the lipase from Candida rugosa (C. rugosa) was then immobilized on the magnetic copolymer microspheres using 1-ethyl-3-(3(dimethylamino)propyl)carbodiimide hydrochloride (EDAC) as an activation reagent. The successful copolymer coating and lipase binding were confirmed by enzyme activity assays, and Fourier transform infrared (FT-IR) spectroscopy. The morphology, structure, and magnetic properties of the magnetic copolymer nanoparticles before and after lipase immobilization were characterized by vibrating-sample magnetometer (VSM), transmission electron microscopy (TEM), and powder X-ray diffraction (XRD) techniques. By using the bound lipase, the heterogeneous transesterification of soybean oil with methanol to produce biodiesel was carried out in a batch process under different reaction conditions. The reusability of the bound lipase was also investigated in the present study.

q/% = (C1 − C2)V1/C1V2

(1)

where q is the immobilization efficiency (%) and C1 and C2 are the initial protein concentrations and the final protein concentrations in the supernatant after the immobilization, respectively (mg/mL). V1 is the volume of the initial solution (mL), and V2 is the final solution volume of the supernatant (mL). All data used in this formula are the average of duplicated experiments. 2.4. Lipase Activity Assays. The hydrolytic activities of the native lipase and bound lipase were assayed using olive oil emulsion hydrolysis.26 A known amount of the native or bound lipase was added to 4 mL of olive oil emulsion and 5 mL of buffer solution (0.025 mol/ L phosphate buffer, pH 7.5), and hydrolysis reactions were performed in a shaking incubator at 35 °C for 15 min. The amount of the liberated fatty acid was determined by titration methods with 0.05 mol/L sodium hydroxide solution using phenolphthalein indicator. One unit of lipase activity was expressed as the quantity of the lipase that releases 1 μmol of fatty acids/min under the assay conditions. The activity recovery (%) was determined as the ratio between the activity of bound lipase and the total activity of lipase in the initial solution.27 2.5. Characterizations. The morphologies of the samples were acquired by TEM using a JEOL model JEM-1200EX at 80 kV. The powder X-ray diffraction measurements were carried out on a Rigaku D/max-3B X-ray powder diffractometer (Tokyo, Japan) using filtered Cu Kα radiation (λ = 0.154 nm). The potassium bromide disc technique was employed for recording the FT-IR spectra of the magnetite copolymer support and the native lipase and the bound lipase nanoparticles on a Shimadzu IR-Prestige-21 spectrometer. The magnetization curves of the samples were assessed with a vibratingsample magnetometer using a LakeShore model 7304 at room temperature. The available carboxylic group content of the magnetite copolymer carriers was measured according to the titration method. 2.6. Enzymatic Transesterification Processes. The enzymatic transesterification reactions were conducted in a shaking incubator at 35 °C in a 100 mL conical flask. In a typical assay, a mixture of 48.5 g of soybean oil and a weighed amount of the bound lipase was charged into the reactor, followed by a three-step addition of 5.4 g of methanol to the reaction mixture as the reaction temperature was raised to35 °C. At the end of the transesterification reaction, the immobilized lipase was carefully filtered by magnetic separation and the residual methanol was separated completely from the liquid phase at 60 °C under reduced pressure with a rotary evaporator vacuum. The oil conversion to fatty acid methyl esters could be determined by measuring the hydroxyl content on the transesterified oil as previously described in the literature.28

2. EXPERIMENTAL SECTION 2.1. Materials. Lipase from C. rugosa, EDAC, olive oil, and bovine serum albumin (BSA) were obtained from Sigma-Aldrich. Styrene and divinylbenzene were obtained from Tianjin Chemical Factory (Tianjin, China), distilled under reduced pressure, and stored at 4 °C until use. Commercial soybean oil, with an average molecular weight of 874 g mol −1 , was purchased from a local oil company. Azobis(isobutyronitrile) (AIBN) and other materials were obtained commercially from Tianjin Chemical and of analytical grade. 2.2. Preparation of Oleic Acid-Coated Magnetite Nanoparticles. The oleic acid-coated magnetite nanoparticle was prepared by chemical coprecipitation of Fe2+ and Fe3+ cations by addition of NH3·H2O solution for the covalent immobilization of lipase.19,23 A 4.73 g amount of FeSO4·7H2O and 8.11 g of FeCl3·6H2O were first dissolved in 100 mL of deionized water. After the solution had been heated to 60 °C, the ammonia solution was then added dropwise under nitrogen atmosphere with vigorous stirring, and the resulting slurry was aged in the mother solution at 80 °C for 30 min. The solid precipitates were formed by adding 70 mL of NH3·H2O solution. After this, 10 mL of oleic acid and 5 mL of ethanol were also added dropwise into the suspension solution, and subsequently the solution was heated at 60 °C for 30 min. Thereafter, the precipitates were magnetically decanted and washed thoroughly with water and ethanol. Finally, the solid precipitate was dried in a vacuum oven at room temperature and stored for future use. The oleic acid-coated magnetic nanoparticle was black in color and showed a strong response to an external magnetic filed. To prepare magnetic copolymer microspheres, 2.5 g of sodium dodecyl sulfonate (SDS) was dissolved in 80 mL of ethanol and 20 mL of deionized water, and later, 0.5 g of AIBN, 25 mL of styrene (St), 20 g of the oleic acid-coated magnetic nanoparticles, and 1 mL of divinylbenzene (DVB) were added to this solution. Thereafter, the mixture was stirred vigorously at 70 °C for 1 h. Next, 5 mL of MAA and 1 mL of DVB were introduced into the reaction system, and subsequently the reaction system underwent copolymerization with stirring at 70 °C for another 7 h. The resulting magnetic copolymer microspheres were recovered by magnetic separation and washed with water and ethanol. The magnetic copolymer supports thus obtained had functional groups that could react with the enzyme with the formation of covalent bonds. 2.3. Lipase Immobilization. Owing to carboxylic groups in the magnetite copolymer carriers, the immobilization of lipase was conducted by treatment of lipase solution with the magnetite copolymer carriers. The coupling reaction involving EDAC as an 2625

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3. RESULTS AND DISCUSSION 3.1. Characteristics of the Immobilized Lipase. In this current study, the magnetite copolymer microspheres with carboxyl groups were employed for the immobilization of lipase using a coupling reaction between the terminal amino group of lipase and the carboxyl groups of the microspheres under mild reaction conditions. In the first step, the carboxylic groups of magnetic copolymer microspheres were activated with EDAC. After this, the condensation of amino groups of lipase with EDAC-activated magnetic copolymer microsphere was carried out. Thus, lipase can be bound onto the magnetite copolymer nanoparticles by forming a covalent bound between the carboxylic group of magnetic copolymer microspheres and the terminal amino group of the enzyme.23,24 The comparative FT-IR spectra of the bound lipase particle, naked magnetic copolymer particle, and native lipase are presented in Figure 1. For the native lipase, the IR absorption

magnetic copolymer particles, the strong IR absorption peak at 547 cm−1 was ascribed to the Fe−O bond vibration of magnetic particles (Figure 1, spectrum a). Meanwhile, the characteristic IR peaks at 696, 758, and 1602 cm−1, were originated from the vibrations of the styrene skeleton, and the IR bands at 1697 and 1452 cm−1 were tentatively assigned to the carboxylic groups.29 The appearance of these characteristic bands confirmed that the copolymer was incorporated into and coated the solid magnetic particles. After immobilization of the lipase onto the magnetite copolymer microsphere, the IR bands responsible for the lipase that were chemically covalent-bonded to the magnetic copolymer microspheres were observed at 1647 cm−1 for amide I and at 1539 cm−1 for amide II, suggesting that during the immobilization process the amide bonds are formed between the terminal amino group of the lipase and the carboxyl groups of the magnetic support.30,31 Given the IR results, the lipase is successfully attached onto the surface of magnetite copolymer nanoparticles. The TEM images for the Fe3O4 magnetite, the magnetic copolymer nanoparticle, and the bound lipase microsphere are displayed in Figure 2. As illustrated in this figure, the naked Fe3O4 particles appeared to be essentially fine and nearly spherical in shape, with the mean size of 10.8 nm (Figure 2a). Besides, the Fe3O4 nanoparticles tended to aggregate due to the magnetic dipole−dipole interactions between the particles, since some aggregations formed with tight structure could be observed. Moreover, after the copolymer coating onto the Fe3O4 magnetite, the magnetic copolymer nanoparticles showed their good monodispersity, and the composite nanoparticles had the average diameter of 13.2 nm (Figure 2b). In the current study, the Fe3O4 nanoparticles were coated with the copolymer layer to prevent the aggregation of the magnetite. The copolymer coating layer could effectively separate the magnetic nanoparticles, and this distance reduces the dipole−dipole interactions between the magnetic nanoparticles and thus could decrease the aggregation of nanoparticles. Further, the morphology and size of magnetic copolymer particles with bound lipase were similar to those of unbound ones (Figure 2c), which suggested that the

Figure 1. FT-IR spectra of magnetic P(St-MAA) microspheres (a), bound lipase (b), and the native lipase (c).

bands of 1654 cm−1 and 1541 cm−1 were characteristic bands of lipase (Figure 1, spectrum c).21 In the case of the naked

Figure 2. TEM images of Fe3O4 (a) and the magnetic P(St-MAA) microspheres without (b) and with (c) bound lipase. 2626

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morphology and size of the magnetic copolymer particles remained unchanged after the lipase binding. Therefore, the immobilization process does not cause agglomeration and variation in the size of particles, revealing that the coupling reaction occurs only on the surface of magnetite copolymer particles. The copolymer layer not only does protect the magnetite but also can be employed for further immobilization of the lipase. The XRD patterns for the Fe3O4 magnetite nanoparticle, the magnetic copolymer nanoparticle, and the bound lipase microsphere are shown in Figure 3. For the Fe3O4 magnetite

Figure 4. Room-temperature magnetization curves for (a) Fe3O4 and (b) magnetic P(St-MAA) microspheres with bound lipase.

magnetically from the reaction mixture easily by applying a magnetic field. 3.2. Factors Affecting the Immobilization Efficiency and Activity Recovery for Lipase Immobilization. For the covalent immobilization of lipase, the carboxyl groupcontaining magnetite copolymer microspheres were prepared from St, MMA, and DVB using oleic acid-coated magnetite nanoparticles as seeds by microemulsion polymerization. The content of available carboxyl groups of the magnetic copolymer microsphere was calculated on the basis of the titration method and amounted to 0.093 mmol/g. In the present study, different concentrations of lipase between 1.2 and 2.8 mg/mL (30 mL) were tested for immobilization on a 2 g of EDAC-activated magnetic copolymer microsphere. The influence of lipase concentration on the activity recovery and immobilization efficiency is illustrated in Figure 5. As observed from Figure 5, the amount

Figure 3. XRD patterns for Fe3O4 (a) and the magnetic P(St-MAA) microspheres with (c) and without (b) bound lipase.

nanoparticles, six diffraction peaks of Fe3O4 recorded at 2θ of 30.1°, 35.4°, 43.2°, 53.5°, 57.1°, and 62.5° could be attributed to the (220), (311), (400), (422), (511), and (440) planes, respectively, which indicated that the magnetic particles were the cubic spinel crystal structure of pure Fe3O4 magnetite (JCPDS database file, No. 85-1436).21,23 For magnetite copolymer particles, the same six XRD peaks were also recorded. When the lipase was immobilized onto the magnetite copolymer particles, the sample exhibited XRD patterns identical to that of Fe3O4 particles. These results show that the incorporation of copolymer and the immobilization of lipase could not vary the cubic spinel structure of Fe3O4 that is essential for retention of magnetic properties. As a consequence, the immobilized lipase could preserve the magnetic behaviors, which is beneficial for application in a separation process. The magnetic properties of the Fe3O4 magnetite particles and the immobilized lipase microspheres were evaluated by VSM techniques. The typical magnetization curves are shown in Figure 4. As can be seen, the two magnetization curves showed an S-shape over the magnetic filed and exhibited typical superparamagnetic behavior. The saturation magnetization of the bound lipase microsphere was determined to be 15.16 emu g−1, and for pure Fe3O4 particles the saturation magnetization was shown to be 65.07 emu g−1. Clearly, the magnetization moment of the magnetite nanoparticle was decreased after the nonmagnetic copolymer coating and the immobilization of lipase. The weak hysteresis in the magnetization indicated that the magnetic material was nearly superparamagnetic, which is a significant property for the magnetic supports.32 Accordingly, the obtained magnetic copolymer microspheres with bound lipase can respond quickly to the external magnetic field owing to the superparamagnetic characteristics and high magnetization value, thus suggesting that they can be isolated

Figure 5. Influence of lipase concentrations on the immobilization efficiency and activity recovery. Immobilization conditions: immobilization time, 4 h; reaction temperature, 35 °C; EDAC concentration, 0.25%.

of lipase covalently attached to the support was gradually decreased when the lipase concentration rose in the concentration range studied. However, there was a maximum lipase concentration to be found at 2.0 mg/mL for the activity recovery. In this case, on increasing the lipase concentration from 1.2 to 2.0 mg/mL, the activity recovery was increased from 38% to 63%; but a lipase concentration higher than 2.0 mg/mL led the activity recovery to decrease slightly. With the 2627

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excess of lipase loading, the surface of the magnetic support was saturated, which could result in a steric hindrance among the lipase molecules thereby blocking the active sites on the support. Based on the results, the best lipase concentration of 2.0 mg/mL could be employed for the immobilization of lipase. The effects of immobilizing time on the immobilization efficiency and the activity recovery were studied, and the obtained results are presented in Figure 6. As can be seen, an

Figure 7. Influence of EDAC concentration on the immobilization efficiency and activity recovery. Immobilization conditions: lipase concentration, 2 mg/mL; immobilization time, 4 h; immobilization temperature, 35 °C.

Figure 6. Influence of immobilization time on the immobilization efficiency and activity recovery. Immobilization conditions: lipase concentration, 2 mg/mL; immobilization temperature, 35 °C; EDAC concentration, 0.25%.

increase in the immobilization duration from 1 to 4 h resulted in an increase in the immobilization efficiency from 72% to 84%, and then this relation seemed to level off after 4 h. Besides, according to the results presented in Figure 6, the activity recovery was shown to increase with increasing immobilizing time, and the highest activity recovery was achieved as 63% when the immobilization time was 4 h. However, the activity recovery was found to decrease if the immobilizing time was longer than 4 h. Long immobilization time can increase the number of covalent bonds between lipase and carrier; however a further increase in the immobilization time beyond 4 h results in the decreased activity recovery. Therefore, it can be inferred that the best immobilization time is 4 h. Figure 7 shows the influence of EDAC concentration on the immobilization efficiency and the activity recovery. It was shown that the highest activity recovery and immobilization efficiency were achieved at the EDAC concentration of 0.25 mg/mL. Increasing the EDAC concentration from 0.05 to 0.25 mg/mL could result in the decrease of immobilization efficiency from 57% to 84%. Similarly, the activity recovery was increased from 42% to 63% with the EDAC concentration increasing from 0.05 to 0.25 mg/mL. However, higher EDAC concentrations than 0.25 mg/mL led to a decline in the activity recovery as well as immobilization efficiency as given in Figure 7. Therefore, the best EDAC concentration is considered to be 0.25 mg/mL. The immobilization efficiency and activity recovery for varied temperatures between 25 and 45 °C were determined, and the results are indicated in Figure 8. It was shown that the best immobilization temperature was 35 °C. As shown in Figure 8, the immobilization efficiency was increased with an increase in the temperature from 25 to 35 °C. The highest immobilization efficiency was obtained as 83% at a temperature of 35 °C, and

Figure 8. Influence of temperature on the immobilization efficiency and activity recovery. Immobilization conditions: lipase concentration, 2 mg/mL; immobilization time, 4 h; EDAC concentration, 0.25%.

the extent of immobilization was observed to decrease as the temperature increased beyond 35 °C. Besides, there was the same trend for the activity recovery with the temperature. The thermal deactivation of enzyme is a well-known phenomenon during the enzyme immobilization processes, and the enzymatic activity is decreased as the temperature is higher or lower than the best temperature. Accordingly, the best temperature for the immobilization of lipase is 35 °C. 3.3. Properties of the Bound Lipase. A comparative investigation between the native and immobilized lipase is provided in terms of pH and temperature. The influence of temperature on the hydrolytic activity of native and bound lipases was determined in the buffer solution (0.025 mol/L phosphate buffer, pH 7.5) by using olive oil emulsion as substrates. As shown in Figure 9, the two temperature profiles for the free lipase and immobilized lipase exhibited similar patterns. With the rise in temperature, both types of lipase became increasingly active until the temperature reached 35 °C, and after this temperature the denaturation of the enzyme would take place. Moreover, according to the results presented in Figure 9, the bound lipase showed it was less sensitive to the variation of temperature than the free lipase when the temperature ranged from 25 to 45 °C. Evidently, the immobilization of lipase could enhance the thermal stability 2628

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immobilization of lipase onto magnetic copolymer microspheres, these formed biocatalysts displayed high transesterification activities. Stoichiometrically, the transesterification reaction needs a methanol-to-oil molar ratio of 3:1 to afford more methyl esters as products. Since the transesterification is a reversible reaction, an excess methanol can enhance the conversion to methyl esters by shifting the equilibrium favorably toward the forward direction. However, a high methanol loading might have significant inhibitory effects on the lipase activity due to the low solubility of methanol in oils, which can cause the inactivation of the immobilized lipase. When a portion of methanol has remained insoluble, it forms droplets which can coat the lipase leading to the deactivation of the enzyme.9,33 For this adverse effect of methanol, the stepwise addition of methanol, by which methanol concentration in the reaction mixture remains at a low level and in this case the enzyme activity inhibition is weaker, has been usually employed in the transesterification reaction. The stepwise successive addition manner of methanol can afford a higher conversion to methyl esters, even when an excess of methanol is employed in the enzymatic transesterification process.20 Accordingly, in this present study, a three-step addition of methanol was adopted for the enzymatic transesterification of soybean oil. The influence of the methanol-to-oil ratio on the conversion was investigated using a three-step addition procedure with this immobilized lipase. The obtained results showed that when the methanol-to-oil molar ratios were 1:1, 2:1, 3:1, 4:1, 5:1, and 7:1 with the three stepwise additions of methanol, the oil conversion was found to reach 48%, 59%, 78%, 86%, 88%, and 70%, respectively. Obviously, the oil conversion of 86% was attained at the methanol-to-oil ratio of 4:1; and a further increase in the methanol-to-oil ratio to 5:1 could lead to a slight increase in the conversion. When the methanol-to-oil molar ratio increased to 7:1, the oil conversion was decreased remarkably to 70% due to the methanol inhibition effect. Such a result indicates that the appropriate amount of methanol-to-oil molar ratio of is 4:1. The Influence of bound lipase dosages on the transesterification reaction was also investigated. The change of the oil conversion when different loadings of this bound lipase were used is illustrated in Figure 11. The amount of this biocatalyst was based on the oil mass. Here, 1 g of the

Figure 9. Influence of temperature on hydrolytic activities of the free and immobilized lipases. The maximum was defined as 100% activity.

of enzyme activity as compared to the free lipase. As a consequence, the covalent bond formation might have resulted in a more stable enzyme conformation and thus a higher resistance against the variation of temperature when compared to the free lipase. The pH profile on the hydrolytic activity of the native and bound lipase was investigated in the pH value ranging from 1 to 10. The plot of hydrolytic activity versus the pH value is presented in Figure 10. As evident from the obtained results,

Figure 10. Influence of medium pH on hydrolytic activities of the free and immobilized lipases. The maximum was defined as 100% activity.

the optimum pH was 7.5 for the native lipase as well as for the bound lipase. Moreover, the activity of the bound lipase was found to be higher than that of native lipase at the pH value between 1 and 10. As a result, the immobilization could, to some extent, improve the stability of lipase against the variation of pH. In view of these results, the lipase is indeed attached by covalent bonds on the surface of the support, thereby limiting the transition of enzyme conformation and resulting in the stabilization of the bound lipase. Therefore, the bound lipase has better adaptability than the free lipase. 3.4. Immobilized Lipase-Catalyzed Transesterification of Soybean Oil. The catalytic activity of magnetic copolymer nanoparticles and the bound lipase microspheres in the transesterification reaction was evaluated. As expected, the magnetic copolymer microspheres did not exhibit particular catalytic activities in the transesterification reaction. But after

Figure 11. Influence of various amounts of bound lipase on the enzymatic transesterification of soybean oil. Reaction conditions: methanol/oil, 5:1; three-step addition of methanol; reaction temperature, 35 °C. 2629

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with the immobilized lipase is necessary for the maintenance of catalytically active conformation of lipase in the reaction medium. However, the higher water amount might make the lipase aggregating, thus resulting in a loss of lipase activity. 3.5. Reusability of the Bound Lipase. The reusability of the immobilized lipase is of great importance from the practical application point of view. The immobilized lipase was recovered by magnetic separation, washed thoroughly with phosphate buffer (0.1 mol L−1 phosphate buffer, pH 7.0), rinsed with tert-butanol, and then used again for successive batch reaction at 35 °C. Each batch reaction time was 24 h, and the transesterification conditions remained the same as those previously described. The obtained results showed that the oil conversion was 84%, 82%, 81%, and 73% when the bound lipase was recycled for 1, 2, 3, and 4 times. Clearly, about 85% residual activity could be retained after four cycles. However, the oil conversion to methyl esters was significantly decreased to 47% after five cycles of reuse, mostly owing to protein denaturation and byproduct inhibition. In view of the results, it is evident that the bound lipase has a better durability and can be employed up to four cycles. The bound lipase studied in this work is considered as an effective biocatalyst for biodiesel production. The oil conversion of 86% is achieved using the bound lipase. Several other bound lipases are also developed in the literature for biodiesel preparation. For instance, immobilized lipase from Rhizomucor miehei (Lipozyme RM IM) gave a high conversion (>80%) after 24 h in the transesterification reaction for biodiesel production.36 Using the immobilized lipase PS from Pseudomonas cepacia, a 67% conversion to methyl esters was obtained.37 Ribeiro et al. reported the enzymatic transesterification converted 80.5% of coconut oil to biodiesel using the immobilized lipase.38 Although the bound lipase thus prepared shows a high activity under the transesterification conditions, the biocatalyst loading seems to be so high for obtaining a better conversion to methyl esters. Nevertheless, the bound lipase is meaningful because of its magnetic properties and the easy separation of the biocatalyst from the reaction mixture. Besides, some other researchers also employed the immobilized lipase for the transesterification reaction, where >40% loading of the biocatalyst was used for a good yield of biodiesel.17,39

immobilized lipase corresponded to 24.1 mg of free lipase. As can be seen, the conversion to methyl esters was increased with increasing catalyst loading; and there was no significant increase in the conversion beyond the loading amount of 50%. Thus, the suitable amount of the bound lipase chosen to conduct the transesterification reaction is 50% (based on the oil mass). In the previous investigation,34 more than 50% of the amount of immobilized lipase was also reported to be employed for the enzymatic conversion of vegetable oils to biodiesel. On the other hand, it could also be observed from Figure 11 that there was gradual increase in the oil conversion as the reaction duration increased from 3 to 24 h, and thereafter the conversion seemed to level off. Therefore, in the process of the three-step transesterification, the highest conversion is 86% when 24 h of reaction time is employed. The amount of water present in the reactant plays a very important role in biocatalytic transesterification reaction, which is reported to influence the lipase activity in nonaqueous media.20,35 In the present study, the transesterification activity of the bound lipase was low (a oil conversion of 78%) without water, implying that a minimum amount of water is needed for the bound lipase to catalyze the transesterification reaction. The influence of the initial water amount in the range from 0.1% (w/w) to 0.5% (w/w) on the transesterification activity of the bound lipase was examined. The results depicted in Figure 12

Figure 12. Influence of water amount on the enzymatic transesterification of soybean oil. Reaction conditions: methanol/oil, 5:1; three-step addition of methanol; catalyst amount, 50%; reaction temperature, 35 °C.

4. CONCLUSION The covalent immobilization of lipase from Candida rugosa onto magnetic copolymer nanoparticles was achieved using EDAC as an activating reagent. The TEM and XRD results showed that the size and structure of magnetite copolymer nanoparticles had no significant change after lipase immobilization. The maximum immobilization efficiency was obtained as 84% with the activity recovery of 63%. The bound lipase on the magnetic copolymer microspheres possessed good magnetic behaviors and can be easily separated by applying a magnetic filed. After immobilization of lipase onto the support, the bound lipase showed better pH and thermal stability than its free counterpart. Biodiesel production using this bound lipase was achieved by a three-step addition of methanol to avoid the methanol inhibition of lipase. By using this bound lipase for the transesterification of soybean oil, the biodiesel conversion of 86% was achieved at 35 °C for 24 h with a methanol-to-oil molar ratio of 4:1. The bound lipase is shown to be a potential environmental friendly biocatalyst for biodiesel production.

showed that there was an increase in the oil conversion on increasing the water amount to 0.1% (w/w). However, the subsequent increment in the water amount beyond 0.1% (w/w) could result in a reverse effect on the activity of the bound lipase. With an increasing water amount from 0.1% (w/w) to 0.5% (w/w), the soybean oil conversion suffered a drop from 86% to 69%. After an optimum water amount, the transesterification activity went down, since too much water can facilitate lipase aggregation which ultimately results in the decrease in enzyme activity. Similar results were also observed in the production of biodiesel by using other immobilized lipases.9,12,20 In this work, the addition of water up to 0.1% (w/ w) is necessary to improve the catalytic activity of the bound lipase. Water can boost the surface area as the available oil− water interface allows the lipase to preserve the activity of the enzyme.35 Therefore, the proper amount of water associated 2630

dx.doi.org/10.1021/ef500131s | Energy Fuels 2014, 28, 2624−2631

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Article

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-371-67756302. Fax: +86-371-67756718. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the support from the National Natural Science Foundation of China (Project No. 21276066), the Plan for Scientific Innovation Talent of Henan Province (Grant 144200510006), and the Program for the Innovative Research Team in the Universities of Henan province in China (Grant 2012IRTSTHN009).

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