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Ultrasonic irradiation with vibration for biodiesel production from soybean oil by Novozym 435
Process Biochemistry 45 (2010) 519–525
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Ultrasonic irradiation with vibration for biodiesel production from soybean oil by Novozym 435 Dahai Yu *, Li Tian, Hao Wu, Song Wang, Ye Wang, Dongxiao Ma, Xuexun Fang * Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, 2519 Jiefang Road, Changchun 130021, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 October 2009 Accepted 16 November 2009
The production of biodiesel with soybean oil and methanol through transesterification by Novozym 435 (Candida antarctica lipase B immobilized on polyacrylic resin) were conducted under two different conditions—ultrasonic irradiation and vibration to compare their overall effects. Compared with vibration, ultrasonic irradiation significantly enhanced the activity of Novozym 435. The reaction rate was further increased under the condition of ultrasonic irradiation with vibration (UIV). Effects of reaction conditions, such as ultrasonic power, water content, organic solvents, ratio of solvent/oil, ratio of methanol/oil, enzyme dosage and temperature on the activity of Novozym 435 were investigated under UIV. Under the optimum conditions (50% of ultrasonic power, 50 rpm vibration, water content of 0.5%, tert-amyl alcohol/oil volume ratio of 1:1, methanol/oil molar ratio of 6:1, 6% Novozym 435 and 40 8C), 96% yield of fatty acid methyl ester (FAME) could be achieved in 4 h. Furthermore, repeated use of Novozym 435 after five cycles showed no obvious loss in enzyme activity, which suggested this enzyme was stable under the UIV condition. These results indicated that UIV was a fast and efficient method for biodiesel production. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Ultrasonic irradiation Vibration Biodiesel Soybean oil Novozym 435
1. Introduction Biodiesel (monoalkyl esters of fatty acids) produced from vegetable oils, animal fats and microalgal oils by transesterification or esterification with short chain alcohols has been viewed as promising renewable sources of fuel because of its biodegradability, low toxicity, renewability, and less dependence on petroleum products [1,2]. Acid and base catalysts were adopted in the conventional method for biodiesel production to form fatty acid alkyl esters [3]. Alkaline transesterification is most often used in the industrial production of biodiesel today due to its high activity under mild reaction conditions and relatively low cost [4,5]. However, these homogeneous alkaline catalysts could easily react with free fatty acids in the feed oil to form the unwanted soap and water by-product, which would adversely affect the quality of biodiesel, and an expensive separation would be required to purify the biodiesel [6]. Acid catalysts are rarely used because they are corrosive and slower in catalyzing reactions which would result in a lower yield of biodiesel [7]. Recently, enzymatic production of biodiesel has attracted considerable interest, since it is more efficient and highly selective, involves less energy consumption, and produces less side products
* Corresponding authors. Tel.: +86 431 88498113; fax: +86 431 88980440. E-mail addresses: [email protected] (D. Yu), [email protected] (X. Fang). 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.11.012
or waste [8,9]. Furthermore, no complex operation is needed for the recovery of glycerol and also for eliminating the catalyst and salt in comparison with the chemical methods [10]. It has been reported that some lipases can effectively catalyze the methanolysis of vegetable oils and fats to produce biodiesel [11]. Most of the recent researches focus on determining the best enzyme source and optimizing the reaction conditions (substrate molar ratio, solvent or no solvent, temperature, water content, free fatty acid level, percent conversion, acyl migration, and substrate flow rate in packed bed bioreactors) to improve the yield of biodiesel for possible industrial scale-ups and use [12,13]. However, from an economic point of view, the enzyme activity is low in lipasecatalyzed biodiesel production, compared with chemical catalyst [3]; therefore it is necessary to find a proper method to increase the reaction rate to promote the application of enzyme catalytic biodiesel production. Ultrasound has been used to accelerate the rates of numerous chemical reactions [14–16], and the rate enhancements, mediated by cavitation, are believed to be originated from the build-up of high local pressures (up to 1000 atm) and temperatures (up to 5000 K), as well as increased catalytic surface areas. Ultrasound assisted lipase-catalyzed reaction method witnesses a fast development. It has been reported that ultrasonic mixing has a significant effect on enzymatic transesterification. The enhancement of enzyme activity of Novozym 435 by ultrasound irradiation have been presented in many literature [17,18]. Ultrasound irradiation has also been proved to offer a fast and easy route to
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Scheme 1. Transesterification of soybean oil and methanol with Novozym 435 under UIV.
biodiesel production in the use of acid or base catalysts [19,20]. The higher reaction rates have been typically attributed to the overcome of the mass transfer limitations (increase in interfacial area and activity of the microscopic and macroscopic bubbles formed) when ultrasonic waves applied to a two-phase reaction system. However, little research has been done to date on the applications of ultrasound irradiation in lipase-catalyzed biodiesel production, and the demonstration of its utility remains a pressing concern. Herein, a low-frequency ultrasonic (40 kHz) waves is used in biodiesel production from soybean oil and methanol through transesterification with Novozym 435 as the catalyst (Scheme 1). A comparison between vibration and ultrasound irradiation on the biodiesel production is conducted. Our study for the first time showed that ultrasonic irradiation with vibration (UIV) could be used to facilitate biodiesel production. Under UIV, the effects of reaction conditions on the enzyme activity have also been investigated. 2. Materials and methods 2.1. Materials Refined soybean oil was obtained locally, Novozym 435 (EC 3.1.1.3) from Candida antarctica was a gift from Novo-Nordisk (Bagsværd, Denmark). The reference substances (methyl oleate, methyl linoleate, methyl stearate and methyl palmitate), N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), glycerol and methyl p-hydroxybenzoate (MP) were purchased from Sigma and were chromatographically pure. Other chemicals were obtained commercially and were of analytical grade. All reactants were dehydrated by 4 A˚ molecular sieve before use. 2.2. Ultrasound equipment A transsonic digital ultrasonic bath (KQ 500DV, Kunshan Corporation, power rating of 500 W and frequency of irradiation of 40 kHz) was used as the source of ultrasonic irradiation to perform the transesterification reaction. The power for the reaction could be adjusted from 40 to 100%. The temperature of the reaction mixture was monitored and kept constant (2 8C) during the reaction. 2.3. Methods 2.3.1. Enzymatic transesterification of soybean oil The transesterification reactions were performed in an Erlenmeyer flask, which was fastened on a speed controlled oscillator (vibrator). The flask was placed above
the ultrasonic bath and was partly emerged into the water. The vibration reactions were performed by using a digitally controlled velocity-variation oscillator (Changchou Guohua Electrical Instrument Factory, HY-4). A standard reaction mixture was consisted of 40% ultrasonic power, 50 rpm vibration, water content of 0.5%, tert-amyl alcohol/oil volume ratio of 1:1, methanol/oil molar ratio of 6:1, 6% immobilized lipase based on the oil weight at 40 8C. One unit (U) of enzyme specific activity was defined as the amount of enzyme that was necessary to produce 1 mmol fatty acid methyl esters per minute in the first 2 h. 2.3.2. Analytical procedure Gas chromatographic (GC) procedure was used to determine the trimethylsilylatd glycerol, mono- and diglycerides in a single run [21]. Briefly, samples were withdrawn from the reaction mixture at specified time intervals and centrifuged at 8000 rpm for 3 min to remove the enzyme. 100 ml methyl p-hydroxybenzoate stock solution (5 mg/ml) in pyridine was added as internal standards to 100 ml reaction mixture in a clean, dry reaction vial, then 100 ml MSTFA was added and incubated at room temperature for 15 min. After cooling to room temperature, the silylated mixtures were quantified by using a gas chromatograph (GC 2014, Shimadzu, Kyoto, Japan) equipped with an Rtx-1 capillary column (0.25 mm 30 m; Hewlett– Packard, U.S.A.). The column temperature was kept at 50 8C for 1 min, and then raised to 330 8C at 10 8C/min and maintained at this temperature until the triglycerides was eluted. The temperatures of the injector and detector were maintained at 280 and 350 8C, respectively. The yield of products was identified by comparing the peak areas of standard methyl p-hydroxybenzoate at particular retention times. Quantification of the final product (FAME) was done according to the calibration curves of pure reference substrate. All the experiments were performed in triplicate and the results were reported as the mean standard deviation.
3. Results 3.1. Ultrasonic irradiation versus vibration for biodiesel production The transesterification of soybean oil and methanol by Novozym 435 was carried out under ultrasonic irradiation and vibration conditions (with and without vibration). An oscillator, instead of a stirrer, was used in the present experiment, as a stirrer can cause a visible breakage of Novozym 435, which might result in the loss of enzyme activity and recyclability. The speed of vibration had a significant impact on the reaction as shown in Fig. 1A, the reaction rate improved up to 1.5 folds under 200 rpm vibration compared with that under 50 rpm vibration. No FAME was
Fig. 1. (A) Effect of vibration speed on biodiesel production. (*) 0 rpm, (&) 50 rpm, (~) 100 rpm, (&) 150 rpm, and (^) 200 rpm. (B) Comparison of reaction process without enzyme under ultrasonic irradiation (*), with enzyme under 50 rpm vibration (&), with enzyme under ultrasonic irradiation (~) and with enzyme under 50 rpm stirring and ultrasonic irradiation (^). Conditions: Reactions were carried out in tert-amyl alcohol (40 ml), soybean oil (4 mmol), methanol (24 mmol), 50% ultrasonic power, Novozym 435 (6% based on the oil weight), water content of 0.5% at 40 8C, respectively.
D. Yu et al. / Process Biochemistry 45 (2010) 519–525
produced when the mixture was not vibrated, which indicated that without contact between enzyme and substrate the reaction could not be initiated. However, compared with ultrasonic irradiation, the reaction rate was much lower under vibration. As shown in Fig. 1B, 93% yield of FAME was achieved in 12 h under ultrasonic irradiation, whereas only 62% yield was achieved under vibration at the maximum speed. The results also showed that a shorter time was needed under ultrasonic radiation compared to vibration with the same yield of FAME. The reaction rate was under the influence of mass transfer limitations [22]. The lower rates of the reactions by vibration might due to insufficient liquid circulation currents generated that facilitate uniform mixing at microlevel. While under ultrasonic irradiation, ultrasonic cavitation could destroy the two-tier structure of the interface of methanol and triolein, increase the interfacial area, emulsification speed and activity of the microscopic and macroscopic bubbles, which might increase the reaction rate [23]. It was also possible that compared to vibration, the enzyme behaved more active under ultrasonic irradiation, as the movement increase of the liquid molecules might facilitate the substrate to approach the active site. However, the mechanism of the increased enzymatic activity was still unclear, since only irreversible effects could be measured by comparing the properties of the enzyme before and after ultrasonic irradiation and the real time detection of the effect of ultrasonic radiation on proteins could hardly be done due to the limitations of the measurement technology [24]. Ultrasonic alone without the enzyme could not catalyze the reaction as shown in Fig. 1B. Although ultrasonic irradiation could generate a faster reaction rate, the contact between the substrate and the enzyme was still insufficient, as using an ultrasonic irradiation with vibration further increased the reaction rate (Fig. 1B). Considering that the decrease of the vibration speed from 200 to 50 rpm did not decrease the reaction rate to a great extent and the low energy consumption, a low vibration speed (50 rpm) was used. By combining the ultrasonic irradiation and vibration, the reaction reached equilibrium after 4 h, and the yield of FAME was 96 wt%, while the reaction did not reach equilibrium and comparable yield until 12 h by using either ultrasonic or vibration. This indicated that ultrasonic irradiation in combination with vibration could facilitate biodiesel production. The rapid emulsification resulted from ultrasonic irradiation and the sufficient contact between enzyme and substrate resulted from vibration might cause accelerated transportation of reactants or products, and thus improve the reaction rate. Because higher enzyme activity and yields of biodiesel formation were observed under UIV, all further experiments were performed in the presence of UIV to optimize the biodiesel formation.
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Fig. 2. Effect of ultrasonic power on biodiesel production. Conditions: Reactions were carried out in tert-amyl alcohol (4 ml), soybean oil (4 mmol), methanol (24 mmol), 50% ultrasonic power, Novozym 435 (6% based on the oil weight) at 40 8C and 50 rpm vibration under ultrasonic irradiation in 2 h. The ultrasonic power varied from 40 to 100%.
3.3. Effect of water content on biodiesel production Enzyme hydration was one of the critical parameters affecting enzyme activity in low-water environments. Water played a crucial role in minimizing solvent-induced conformational rigidity, which was one of the causes for the reduced catalytic activities observed in nonaqueous media [25]. In the present study, the water content varied in the range of 0–4%. As shown in Fig. 3, the enzyme activities exhibited a bell shaped curve with the changing of water content. When water content was <0.5%, the enzyme activity increased with the increase in water content. Novozym 435 exhibited highest activity when water content was 0.5%. With a further increase of water content, the enzyme activities decreased dramatically. No transesterification was detected with dried enzymes. In nonaqueous media, a certain amount of water was necessary for the enzyme to maintain its proper conformation
3.2. Effect of ultrasonic power on biodiesel production The activity of Novozym 435 in biodiesel production under UIV was evaluated by changing the ultrasound power, water content, solvents, solvent ratio (solvent/oil), substrate ratio (methanol/oil), enzyme dosage and temperature of the reaction. To obtain the maximum rates of biodiesel formation and yield at the possible minimum energy consumption, the ultrasonic power was set in the range of 40–100% to investigate its effect on biodiesel production. Fig. 2 shows the increase of ultrasonic power resulted in no obvious difference in enzyme activity, which was inconsistent with the previous studies showing that ultrasonic power had a positive impact on the enzyme activity before a point of diminishing return was reached. Based on our experimental results, 50% ultrasonic power was chosen considering both the higher enzyme activity and lower energy consumption.
Fig. 3. Effect of water content on biodiesel production. Conditions: Reactions were carried out in tert-amyl alcohol (4 ml), soybean oil (4 mmol), methanol (24 mmol), 50% ultrasonic power, Novozym 435 (6% based on the oil weight) at 40 8C and 50 rpm vibration under ultrasonic irradiation in 2 h. The water content varied from 0 to 4%.
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Table 1 Effect of solvents on the enzyme activity in biodiesel production. Solvent n-Hexane n-Pentanol Tert-amyl alcohol 2-Pentanol Cyclohexanol n-Butanol Tert-butanol 2-propanol Acetone 1,4-Dioxane
Log P 3.50 1.51 1.50 1.42 1.20 0.80 0.80 0.42 0.23 1.10
Viscosity
Dielectric constant
Solubility parameter
Enzyme activity (mmol/min g)
0.30 3.31 3.70 3.86 4.60 2.95 4.31 2.37 0.31 1.30
1.89 13.92 15.44 14.71 15.00 17.70 12.47 17.94 20.71 2.21
7.22 11.10 10.43 10.91 11.40 11.46 10.65 11.63 9.67 10.11
98.40 189.60 672.20 399.20 312.00 336.80 572.80 427.20 148.80 18.96
Conditions: Reactions were carried out in 4 ml solvent, soybean oil (4 mmol), methanol (24 mmol), 50% ultrasonic power, Novozym 435 (6% based on the oil weight), water content of 0.5% at 40 8C and 50 rpm vibration under ultrasonic irradiation in 2 h. Data source of solvent parameters: Refs. [32,33].
to keep its catalytic activity. At low-water content, the conformation of Novozym 435 was excessively rigid which disturbed the ‘‘induced-fit’’ process of the enzyme and decreased the enzyme activity [26]. At higher water content, the decrease in enzyme activity could be attributed to the observed enzyme particle aggregation that might consequently lead to limited access of the substrate to the enzyme active site [27]. An alternative possibility was that the conformation of Novozym 435 was more flexible at higher water content, and the water in the reaction mixture might act as competing nucleophile for acyl-enzyme, thus suppressing the expected acyl transfer and causing unfavorable equilibrium position in reversed hydrolysis. Overall, these results suggested that water content strongly influenced the hydration level of the enzyme that consequently affected the transesterification activity for biodiesel production.
Other small alcohols, such as 2-propanol, n-butanol, n-pentanol and 2-pentanol were inappropriate to use, because they served not only as solvents, but also as acyl acceptors in the transesterification reaction with triglyceride and some by-product was formed. Particularly, some solvents with very similar values of viscosity (npentanol and tert-amyl alcohol), dielectric constant (n-pentanol and 2-pentanol), and solubility parameter (tert-amyl alcohol and 1,4-dioxane) exhibited quite different effects to the enzyme activity. Therefore, viscosity, dielectric constant, solubility parameter, or even log P by themselves did not appear to be reliable parameters to assess the solvent effect on enzyme activity and a comprehensive consideration of all solvent parameters should be taken when choosing an appropriate solvent. Tert-amyl alcohol, instead of widely used tert-butanol, was chosen as the solvent considering the higher enzyme activity in this solvent.
3.4. Effect of solvents on biodiesel production
3.5. Effect of tert-amyl alcohol concentration on biodiesel production
It was well described in the literature that enzyme activity were strongly affected by organic solvents [28]. In the present study, the effects of several organic solvents with various log P (the partition coefficient of the solvent for the standard octanol/water two-phase system), dielectric constant, solubility parameter and other parameters were investigated for the transesterification of soybean oil. As shown in Table 1, there was an increase in enzyme activity as the solvent hydrophobicity increased in the range of 1.1 to 0.42. Klibanov pointed out that a minimum of water was required to preserve the conformation of the enzyme [29]. In hydrophilic solvent, such as acetone and 1,4-dioxane, water had higher affinity in hydrophilic solvent rather than bounding to the enzyme. As a consequence, the enzyme might lose its flexibility conformation due to the lack of bound water and thus losing its activity [30]. The explanation of the increasing enzyme activity with the increasing hydrophobicity might be that solvents with higher log P had better miscibility with methanol and glycerol, and stronger hydrophobicity protected the enzyme from deactivation by these alcohols [31]. No apparent correlation between log P and enzyme activity were observed when log P 0.8. The enzyme activity was greatly influenced by different functional groups and molecular structure of the solvents. Enzyme activity was improved by adding the solvents with hydroxyl group, because they solubilized both methanol and glycerol. Alcohols with side-chain molecular structures had higher enzyme activity compared to the linear ones, especially with tertiary alcohols, like tert-butanol and tertamyl alcohol (Table 1). This phenomenon might also be attributed to the differences in miscibility with triglyceride. As an indication of solubility parameter, for alcohol molecules with the same carbon numbers, the branched ones had better miscibility with triglyceride (solubility parameter of 7.43) than the linear isomers.
Biodiesel synthesis was greatly influenced by addition of tert-amyl alcohol to the reaction mixture. Different amounts of tert-amyl alcohol were added to the reaction mixture in order to observe the effect of tert-amyl alcohol concentration on the transesterification reaction. The enzyme activity was very low in solvent-free system due to the toxicity of excessive methanol on enzyme activity as shown in Fig. 4. The enzyme activity increased
Fig. 4. Effect of tert-amyl alcohol concentration on biodiesel production. Conditions: Reactions were carried out in soybean oil (4 mmol), methanol (24 mmol), 50% ultrasonic power, Novozym 435 (6% based on the oil weight) and water content of 0.5% at 40 8C and 50 rpm vibration under ultrasonic irradiation in 2 h. The tert-amyl alcohol/oil volume ratio varied from 0 to 4.
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Fig. 5. Effect of substrate ratio on biodiesel production. Conditions: Reactions were carried out in tert-amyl alcohol (4 ml), soybean oil (4 mmol), 50% ultrasonic power, Novozym 435 (6% based on the oil weight) and water content of 0.5% at 40 8C and 50 rpm vibration under ultrasonic irradiation in 2 h. The molar ratio of methanol/oil varied from 1 to 36.
greatly with the increase in tert-amyl alcohol/oil volume ratio with a maximum value at a tert-amyl alcohol/oil volume ratio of 1:1. Further increased in tert-amyl alcohol concentration resulted no increment of enzyme activity. The presence of tert-amyl alcohol in the system could improve the solubility of methanol in the reaction mixture, as a result, the inhibition effects of methanol in biodiesel production were eliminated, and lipase maintained high activity even with high amount of methanol presented in the system. 3.6. Effect of substrate ratio on biodiesel production The rate of an enzyme catalytic reaction depends on the concentrations of enzyme and substrate. Variation of substrate ratio might have significant effect on the rate of the reaction. In the present experiments, the substrate molar ratio (methanol/oil) was changed from 1 to 36 while keeping the amount of soybean oil constant at 4 mmol. The amount of the enzyme was kept constant. The enzymatic activities increased with the increase of substrate ratio from 1:1 to 6:1, and reached the maximum of 680 mmol/ min g when the substrate ratio was 6:1 (Fig. 5). As the amount of methanol increases, more cavitation bubbles might be generated because the cavitation events were easier in methanol than in oil, and thus improve the reaction. After this point, with the further increasing of substrate ratio, the enzyme activity decreased gradually. It was probably due to the inhibitory effect of methanol on enzyme become more significant with higher concentrations of methanol. Hence, the molar ratio of 6:1 was considered as the optimum substrate ratio.
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Fig. 6. Effect of enzyme dosage on biodiesel production. Conditions: Reactions were carried out in tert-amyl alcohol (4 ml), soybean oil (4 mmol), methanol (24 mmol), 50% ultrasonic power, water content of 0.5% at 40 8C and 50 rpm vibration under ultrasonic irradiation in 2 h. The amount of enzyme varied from 0 to 10% based on the oil weight.
yield of methyl ester witnessed a steep increase, while after the point, the yield increase was much slower), and was adopted in the experiments. 3.8. Effect of temperature on biodiesel production Theoretically elevated temperature can help the substrate molecules to obtain adequate energy to pass over the energy barrier and enhance the reaction rate. In contrast, enzymes were temperature-depended and easily deactivated at high temperature. The effect of temperature on the activity of Novozym 435 was examined in the range of 30–70 8C. The results in Fig. 7 shows that the enzyme activity increased as temperature increased from 30 to 40 8C, followed by a decrease at higher temperature. As the reaction temperature elevated, the collision chance between enzyme and substrate molecules increased,
3.7. Effect of enzyme dosage on biodiesel production Our ultimate aim was to develop a process that could consume the least amount of enzyme and time to produce the possible maximum amount of biodiesel. The effect of Novozym 435 dosage on the biodiesel production was studied under UIV. The mole ratios of the reactants were kept constant while the amount of enzyme was changed from 0 to 10% based on the oil weight. As the loading of Novozym 435 increased, the yield of FAME was also increased (Fig. 6). Considering the cost of the enzyme, 6% of Novozym 435 was proved to be the most efficient amount (before this point, the
Fig. 7. Effect of temperature on biodiesel production. Conditions: Reactions were carried out in tert-amyl alcohol (4 ml), soybean oil (4 mmol), methanol (24 mmol), 50% ultrasonic power, Novozym 435 (6% based on the oil weight) and water content of 0.5% and 50 rpm vibration under ultrasonic irradiation in 2 h. Temperature varied from 30 to 70 8C.
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in 4 h under UIV. The enzyme was ultrasonic stable as no obvious loss in lipase activity was observed after repeated use for five cycles under UIV. Acknowledgment Financial support for this work was by the Program for New Century Excellent Talents from the Chinese Ministry of Education (No. NCET-08-0244). References
Fig. 8. Reusability of enzyme on biodiesel production. Conditions: Reactions were carried out in tert-amyl alcohol (4 ml), soybean oil (4 mmol), methanol (24 mmol), 50% ultrasonic power, Novozym 435 (6% based on the oil weight) and water content of 0.5% at 40 8C and 50 rpm vibration under ultrasonic irradiation in 2 h.
which might help to form enzyme–substrate complexes and then led to the increase of enzyme activity. The increased of reaction temperature also decreased of viscosity of soybean oil, increased of cavitation events and the rate of emulsion formation, thus consequently increased the biodiesel formation. In addition, the molecules of the proteins could fluctuate to relieve the steric repulsion by increasing the temperature, and such a fluctuation could contribute to the rate acceleration at elevated temperatures [34]. As for the decrease of enzyme activity with further increase of the temperature above 40 8C, it was mostly likely due to the denaturation (alteration) of protein structure resulted from heat-induced destruction of noncovalent interactions (the breakdown of the weak ionic and hydrogen bonding that stabilized the three dimensional structure of the enzyme) [35]. 3.9. Reusability of Novozym 435 under UIV Since the enzyme was costly, the reusability of enzyme was an essential factor for the production of biodiesel in industry. The catalyst reusability studies were carried out to investigate the stability of the enzyme under UIV. As shown in Fig. 8, Novozym 435 was not deactivated or denatured under UIV, only a slightly decrease (4%) in enzyme activity was observed after five uses. These results suggested that Novozym 435 was suitable for catalytic usage in UIV conditions. 4. Conclusions In the present study, ultrasonic irradiation with vibration was proved to be an efficient method for enzymatic biodiesel production. Under ultrasonic irradiation, enzyme activity of Novozym 435 was enhanced in transesterification reaction of soybean oil and methanol. The ultrasonic assisted reaction generated equivalent yield of FAME in relatively shorter time compared with that of vibration. When ultrasonic irradiation and vibration were used together, biodiesel production rate was further enhanced. Under the optimum conditions (50% of ultrasonic power, 50 rpm vibration, water content of 0.5%, tert-amyl alcohol/oil volume ratio of 1:1, methanol/oil molar ratio of 6:1, 6% Novozym 435 and 40 8C), a 96% yield of fatty acid methyl ester could be achieved
[1] Demirbas A. Progress and recent trends in biofuels. Prog Energy Combust Sci 2007;33(1):1–18. [2] Meher LC, Sagar DV, Naik SN. Technical aspects of biodiesel production by transesterification: a review. Renew Sustain Energy Rev 2006;10(3): 248–68. [3] Akoh CC, Chang SW, Lee GC, Shaw JF. Enzymatic approach to biodiesel production. J Agric Food Chem 2007;55(22):8995–9005. [4] Freedman B, Pryde EH, Mounts TL. Variables affecting the yields of fatty esters from transesterified vegetable oils. J Am Oil Chem Soc 1984;61(10): 1638–43. [5] Akoh CC, Swanson BG. Base-catalyzed transesterification of vegetable oils. J Food Process Preserv 1988;12(2):139–49. [6] Ma F, Hanna MA. Biodiesel production: a review. Bioresour Technol 1999;70(1):1–15. [7] Canakci M, Van Gerpen JH. Biodiesel production via acid catalysis. Trans ASAE 1999;42(5):1203–10. [8] Fjerbaek L, Christensen KV, Norddahl B. A review of the current state of biodiesel production using enzymatic transesterification. Biotechnol Bioeng 2009;102(5):1298–315. [9] Shah S, Sharma S, Gupta MN. Enzymatic transesterification for biodiesel production. Indian J Biochem Biophys 2003;40(6):392–9. [10] Marchetti JM, Miguel VU, Errazu AF. Possible methods for biodiesel production. Renew Sust Energy Rev 2007;11(6):1300–11. [11] Nelson L, Foglia T, Marmer W. Lipase-catalyzed production of biodiesel. J Am Oil Chem Soc 1996;73(9):1191–5. [12] Nie K, Xie F, Wang F, Tan T. Lipase catalyzed methanolysis to produce biodiesel: optimization of the biodiesel production. J Mol Catal B Enzym 2006;43(1–4):142–7. [13] Shaw JF, Chang SW, Lin SC, Wu TT, Ju HY, Akoh CC, et al. Continuous enzymatic synthesis of biodiesel with Novozym 435. Energy Fuels 2008;22(2):840–4. [14] Miethchen R. Selected applications of sonochemistry in organic chemistry. Ultrasonics 1992;30(33):173–9. [15] Mason TJ. Ultrasound in synthetic organic chemistry. Chem Soc Rev 1997;26(6): 443–51. [16] Zhou SJ, Hawley MC. A study of ultrasonic reaction rate enhancement effect in adhesive bonding of polymers and composites. Compos Struct 2003;61(4): 303–9. [17] Sinisterra JV. Application of ultrasound to biotechnology: an overview. Ultrasonics 1992;30(3):180–5. [18] Lin G, Liu H. Ultrasound promoted lipase catalyzed reactions. Tetrahedron Lett 1995;36(34):6067–8. [19] Mahamuni NN, Adewuyi YG. Optimization of the synthesis of biodiesel via ultrasound-enhanced base-catalyzed transesterification of soybean oil using a multifrequency ultrasonic reactor. Energy Fuels 2009;23:2757–66. [20] Deshmane VG, Gogate PR, Pandit AB. Ultrasound-assisted synthesis of biodiesel from palm fatty acid distillate. Ind Eng Chem Res 2009;48(17): 7923–7. [21] Plank C, Lorbeer E. Simultaneous determination of glycerol and mono-, di and triglycerides in vegetable oil methyl esters by capillary gas chromatography. J Chromatogr A 1995;697(1–2):461–8. [22] Xiu GH, Jiang L, Li P. Mass-transfer limitations for immobilized enzymecatalyzed kinetic resolution of racemate in a batch reactor. Ind Eng Chem Res 2000;39(11):4054–62. [23] Bradley M, Ashokkumar M, Grieser F. Sonochemical production of fluorescent and phosphorescent latex particles. J Am Chem Soc 2003;125(2):525–9. [24] Shah S, Gupta MN. The effect of ultrasonic pre-treatment on the catalytic activity of lipases in aqueous and non-aqueous media. Chem Cent J 2008;2(1):1–9. [25] Ma L, Persson M, Adlercreutz P. Water content dependence of lipase catalysis in organic media explains successful transesterification reactions. Enzyme Microb Technol 2002;31(7):1024–9. [26] Halling PJ. Enzymatic conversion in organic and other low-water media. In: Drauz K, Waldmann H, editors. 2nd ed., Enzyme catalysis in organic synthesis a comprehensive handbook, vol. I, 2nd ed. Weinheim, Germany: Wiley–VCH Verlag GmbH; 2002. p. 259–86. [27] Karboune S, Archelas A, Baratti J. Properties of epoxide hydrolase from Aspergillus niger for the hydrolytic kinetic resolution of epoxides in pure organic media. Enzyme Microb Technol 2006;39(2):318–24. [28] Laane C, Boeren S, Vos K, Veeger C. Rules for optimization of biocatalysis in organic solvents. Biotechnol Bioeng 1987;30(1):81–7.
D. Yu et al. / Process Biochemistry 45 (2010) 519–525 [29] Klibanov AM. Improving enzymes by using them in organic solvents. Nature 2001;409(6817):241–6. [30] Perraud R, Laboret F. Optimization of methyl propionate production by Mucor miehei lipase. Appl Microbiol Biotechnol 1995;44(3–4):321–6. [31] Dossat V, Combes D, Marty A. Continuous enzymatic transesterification of high oleic sunflower oil in a packed bed reactor: influence of the glycerol production. Enzyme Microb Technol 1999;25(3–5):194–200. [32] Lide DR. CRC handbook of chemistry and physics, 87th ed., Boca Raton, FL: CRC Press/Taylor and Francis; 2006.
525
[33] Secundo F, Riva S, Carrea G. Effects of medium and of reaction conditions on the enantioselectivity of lipases in organic solvents and possible rationales. Tetrahedron Asym 1992;3(2):267–80. [34] Yu HM, Chen ST, Suree P, Nuansri R, Wang KT. Effect of microwave irradiation on acid-catalyzed hydrolysis of starch. J Org Chem 1996;61(26):9608–9. [35] Yadav GD, Lathi PS. Intensification of enzymatic synthesis of propylene glycol monolaurate from 1,2-propanediol and lauric acid under microwave irradiation: kinetics of forward and reverse reactions. Enzyme Microb Technol 2006;38(6):814–20.
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Ultrasonic irradiation with vibration for biodiesel production from soybean oil by Novozym 435 Dahai Yu *, Li Tian, Hao Wu, Song Wang, Ye Wang, Dongxiao Ma, Xuexun Fang * Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, 2519 Jiefang Road, Changchun 130021, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 October 2009 Accepted 16 November 2009
The production of biodiesel with soybean oil and methanol through transesterification by Novozym 435 (Candida antarctica lipase B immobilized on polyacrylic resin) were conducted under two different conditions—ultrasonic irradiation and vibration to compare their overall effects. Compared with vibration, ultrasonic irradiation significantly enhanced the activity of Novozym 435. The reaction rate was further increased under the condition of ultrasonic irradiation with vibration (UIV). Effects of reaction conditions, such as ultrasonic power, water content, organic solvents, ratio of solvent/oil, ratio of methanol/oil, enzyme dosage and temperature on the activity of Novozym 435 were investigated under UIV. Under the optimum conditions (50% of ultrasonic power, 50 rpm vibration, water content of 0.5%, tert-amyl alcohol/oil volume ratio of 1:1, methanol/oil molar ratio of 6:1, 6% Novozym 435 and 40 8C), 96% yield of fatty acid methyl ester (FAME) could be achieved in 4 h. Furthermore, repeated use of Novozym 435 after five cycles showed no obvious loss in enzyme activity, which suggested this enzyme was stable under the UIV condition. These results indicated that UIV was a fast and efficient method for biodiesel production. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Ultrasonic irradiation Vibration Biodiesel Soybean oil Novozym 435
1. Introduction Biodiesel (monoalkyl esters of fatty acids) produced from vegetable oils, animal fats and microalgal oils by transesterification or esterification with short chain alcohols has been viewed as promising renewable sources of fuel because of its biodegradability, low toxicity, renewability, and less dependence on petroleum products [1,2]. Acid and base catalysts were adopted in the conventional method for biodiesel production to form fatty acid alkyl esters [3]. Alkaline transesterification is most often used in the industrial production of biodiesel today due to its high activity under mild reaction conditions and relatively low cost [4,5]. However, these homogeneous alkaline catalysts could easily react with free fatty acids in the feed oil to form the unwanted soap and water by-product, which would adversely affect the quality of biodiesel, and an expensive separation would be required to purify the biodiesel [6]. Acid catalysts are rarely used because they are corrosive and slower in catalyzing reactions which would result in a lower yield of biodiesel [7]. Recently, enzymatic production of biodiesel has attracted considerable interest, since it is more efficient and highly selective, involves less energy consumption, and produces less side products
* Corresponding authors. Tel.: +86 431 88498113; fax: +86 431 88980440. E-mail addresses: [email protected] (D. Yu), [email protected] (X. Fang). 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.11.012
or waste [8,9]. Furthermore, no complex operation is needed for the recovery of glycerol and also for eliminating the catalyst and salt in comparison with the chemical methods [10]. It has been reported that some lipases can effectively catalyze the methanolysis of vegetable oils and fats to produce biodiesel [11]. Most of the recent researches focus on determining the best enzyme source and optimizing the reaction conditions (substrate molar ratio, solvent or no solvent, temperature, water content, free fatty acid level, percent conversion, acyl migration, and substrate flow rate in packed bed bioreactors) to improve the yield of biodiesel for possible industrial scale-ups and use [12,13]. However, from an economic point of view, the enzyme activity is low in lipasecatalyzed biodiesel production, compared with chemical catalyst [3]; therefore it is necessary to find a proper method to increase the reaction rate to promote the application of enzyme catalytic biodiesel production. Ultrasound has been used to accelerate the rates of numerous chemical reactions [14–16], and the rate enhancements, mediated by cavitation, are believed to be originated from the build-up of high local pressures (up to 1000 atm) and temperatures (up to 5000 K), as well as increased catalytic surface areas. Ultrasound assisted lipase-catalyzed reaction method witnesses a fast development. It has been reported that ultrasonic mixing has a significant effect on enzymatic transesterification. The enhancement of enzyme activity of Novozym 435 by ultrasound irradiation have been presented in many literature [17,18]. Ultrasound irradiation has also been proved to offer a fast and easy route to
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Scheme 1. Transesterification of soybean oil and methanol with Novozym 435 under UIV.
biodiesel production in the use of acid or base catalysts [19,20]. The higher reaction rates have been typically attributed to the overcome of the mass transfer limitations (increase in interfacial area and activity of the microscopic and macroscopic bubbles formed) when ultrasonic waves applied to a two-phase reaction system. However, little research has been done to date on the applications of ultrasound irradiation in lipase-catalyzed biodiesel production, and the demonstration of its utility remains a pressing concern. Herein, a low-frequency ultrasonic (40 kHz) waves is used in biodiesel production from soybean oil and methanol through transesterification with Novozym 435 as the catalyst (Scheme 1). A comparison between vibration and ultrasound irradiation on the biodiesel production is conducted. Our study for the first time showed that ultrasonic irradiation with vibration (UIV) could be used to facilitate biodiesel production. Under UIV, the effects of reaction conditions on the enzyme activity have also been investigated. 2. Materials and methods 2.1. Materials Refined soybean oil was obtained locally, Novozym 435 (EC 3.1.1.3) from Candida antarctica was a gift from Novo-Nordisk (Bagsværd, Denmark). The reference substances (methyl oleate, methyl linoleate, methyl stearate and methyl palmitate), N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), glycerol and methyl p-hydroxybenzoate (MP) were purchased from Sigma and were chromatographically pure. Other chemicals were obtained commercially and were of analytical grade. All reactants were dehydrated by 4 A˚ molecular sieve before use. 2.2. Ultrasound equipment A transsonic digital ultrasonic bath (KQ 500DV, Kunshan Corporation, power rating of 500 W and frequency of irradiation of 40 kHz) was used as the source of ultrasonic irradiation to perform the transesterification reaction. The power for the reaction could be adjusted from 40 to 100%. The temperature of the reaction mixture was monitored and kept constant (2 8C) during the reaction. 2.3. Methods 2.3.1. Enzymatic transesterification of soybean oil The transesterification reactions were performed in an Erlenmeyer flask, which was fastened on a speed controlled oscillator (vibrator). The flask was placed above
the ultrasonic bath and was partly emerged into the water. The vibration reactions were performed by using a digitally controlled velocity-variation oscillator (Changchou Guohua Electrical Instrument Factory, HY-4). A standard reaction mixture was consisted of 40% ultrasonic power, 50 rpm vibration, water content of 0.5%, tert-amyl alcohol/oil volume ratio of 1:1, methanol/oil molar ratio of 6:1, 6% immobilized lipase based on the oil weight at 40 8C. One unit (U) of enzyme specific activity was defined as the amount of enzyme that was necessary to produce 1 mmol fatty acid methyl esters per minute in the first 2 h. 2.3.2. Analytical procedure Gas chromatographic (GC) procedure was used to determine the trimethylsilylatd glycerol, mono- and diglycerides in a single run [21]. Briefly, samples were withdrawn from the reaction mixture at specified time intervals and centrifuged at 8000 rpm for 3 min to remove the enzyme. 100 ml methyl p-hydroxybenzoate stock solution (5 mg/ml) in pyridine was added as internal standards to 100 ml reaction mixture in a clean, dry reaction vial, then 100 ml MSTFA was added and incubated at room temperature for 15 min. After cooling to room temperature, the silylated mixtures were quantified by using a gas chromatograph (GC 2014, Shimadzu, Kyoto, Japan) equipped with an Rtx-1 capillary column (0.25 mm 30 m; Hewlett– Packard, U.S.A.). The column temperature was kept at 50 8C for 1 min, and then raised to 330 8C at 10 8C/min and maintained at this temperature until the triglycerides was eluted. The temperatures of the injector and detector were maintained at 280 and 350 8C, respectively. The yield of products was identified by comparing the peak areas of standard methyl p-hydroxybenzoate at particular retention times. Quantification of the final product (FAME) was done according to the calibration curves of pure reference substrate. All the experiments were performed in triplicate and the results were reported as the mean standard deviation.
3. Results 3.1. Ultrasonic irradiation versus vibration for biodiesel production The transesterification of soybean oil and methanol by Novozym 435 was carried out under ultrasonic irradiation and vibration conditions (with and without vibration). An oscillator, instead of a stirrer, was used in the present experiment, as a stirrer can cause a visible breakage of Novozym 435, which might result in the loss of enzyme activity and recyclability. The speed of vibration had a significant impact on the reaction as shown in Fig. 1A, the reaction rate improved up to 1.5 folds under 200 rpm vibration compared with that under 50 rpm vibration. No FAME was
Fig. 1. (A) Effect of vibration speed on biodiesel production. (*) 0 rpm, (&) 50 rpm, (~) 100 rpm, (&) 150 rpm, and (^) 200 rpm. (B) Comparison of reaction process without enzyme under ultrasonic irradiation (*), with enzyme under 50 rpm vibration (&), with enzyme under ultrasonic irradiation (~) and with enzyme under 50 rpm stirring and ultrasonic irradiation (^). Conditions: Reactions were carried out in tert-amyl alcohol (40 ml), soybean oil (4 mmol), methanol (24 mmol), 50% ultrasonic power, Novozym 435 (6% based on the oil weight), water content of 0.5% at 40 8C, respectively.
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produced when the mixture was not vibrated, which indicated that without contact between enzyme and substrate the reaction could not be initiated. However, compared with ultrasonic irradiation, the reaction rate was much lower under vibration. As shown in Fig. 1B, 93% yield of FAME was achieved in 12 h under ultrasonic irradiation, whereas only 62% yield was achieved under vibration at the maximum speed. The results also showed that a shorter time was needed under ultrasonic radiation compared to vibration with the same yield of FAME. The reaction rate was under the influence of mass transfer limitations [22]. The lower rates of the reactions by vibration might due to insufficient liquid circulation currents generated that facilitate uniform mixing at microlevel. While under ultrasonic irradiation, ultrasonic cavitation could destroy the two-tier structure of the interface of methanol and triolein, increase the interfacial area, emulsification speed and activity of the microscopic and macroscopic bubbles, which might increase the reaction rate [23]. It was also possible that compared to vibration, the enzyme behaved more active under ultrasonic irradiation, as the movement increase of the liquid molecules might facilitate the substrate to approach the active site. However, the mechanism of the increased enzymatic activity was still unclear, since only irreversible effects could be measured by comparing the properties of the enzyme before and after ultrasonic irradiation and the real time detection of the effect of ultrasonic radiation on proteins could hardly be done due to the limitations of the measurement technology [24]. Ultrasonic alone without the enzyme could not catalyze the reaction as shown in Fig. 1B. Although ultrasonic irradiation could generate a faster reaction rate, the contact between the substrate and the enzyme was still insufficient, as using an ultrasonic irradiation with vibration further increased the reaction rate (Fig. 1B). Considering that the decrease of the vibration speed from 200 to 50 rpm did not decrease the reaction rate to a great extent and the low energy consumption, a low vibration speed (50 rpm) was used. By combining the ultrasonic irradiation and vibration, the reaction reached equilibrium after 4 h, and the yield of FAME was 96 wt%, while the reaction did not reach equilibrium and comparable yield until 12 h by using either ultrasonic or vibration. This indicated that ultrasonic irradiation in combination with vibration could facilitate biodiesel production. The rapid emulsification resulted from ultrasonic irradiation and the sufficient contact between enzyme and substrate resulted from vibration might cause accelerated transportation of reactants or products, and thus improve the reaction rate. Because higher enzyme activity and yields of biodiesel formation were observed under UIV, all further experiments were performed in the presence of UIV to optimize the biodiesel formation.
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Fig. 2. Effect of ultrasonic power on biodiesel production. Conditions: Reactions were carried out in tert-amyl alcohol (4 ml), soybean oil (4 mmol), methanol (24 mmol), 50% ultrasonic power, Novozym 435 (6% based on the oil weight) at 40 8C and 50 rpm vibration under ultrasonic irradiation in 2 h. The ultrasonic power varied from 40 to 100%.
3.3. Effect of water content on biodiesel production Enzyme hydration was one of the critical parameters affecting enzyme activity in low-water environments. Water played a crucial role in minimizing solvent-induced conformational rigidity, which was one of the causes for the reduced catalytic activities observed in nonaqueous media [25]. In the present study, the water content varied in the range of 0–4%. As shown in Fig. 3, the enzyme activities exhibited a bell shaped curve with the changing of water content. When water content was <0.5%, the enzyme activity increased with the increase in water content. Novozym 435 exhibited highest activity when water content was 0.5%. With a further increase of water content, the enzyme activities decreased dramatically. No transesterification was detected with dried enzymes. In nonaqueous media, a certain amount of water was necessary for the enzyme to maintain its proper conformation
3.2. Effect of ultrasonic power on biodiesel production The activity of Novozym 435 in biodiesel production under UIV was evaluated by changing the ultrasound power, water content, solvents, solvent ratio (solvent/oil), substrate ratio (methanol/oil), enzyme dosage and temperature of the reaction. To obtain the maximum rates of biodiesel formation and yield at the possible minimum energy consumption, the ultrasonic power was set in the range of 40–100% to investigate its effect on biodiesel production. Fig. 2 shows the increase of ultrasonic power resulted in no obvious difference in enzyme activity, which was inconsistent with the previous studies showing that ultrasonic power had a positive impact on the enzyme activity before a point of diminishing return was reached. Based on our experimental results, 50% ultrasonic power was chosen considering both the higher enzyme activity and lower energy consumption.
Fig. 3. Effect of water content on biodiesel production. Conditions: Reactions were carried out in tert-amyl alcohol (4 ml), soybean oil (4 mmol), methanol (24 mmol), 50% ultrasonic power, Novozym 435 (6% based on the oil weight) at 40 8C and 50 rpm vibration under ultrasonic irradiation in 2 h. The water content varied from 0 to 4%.
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Table 1 Effect of solvents on the enzyme activity in biodiesel production. Solvent n-Hexane n-Pentanol Tert-amyl alcohol 2-Pentanol Cyclohexanol n-Butanol Tert-butanol 2-propanol Acetone 1,4-Dioxane
Log P 3.50 1.51 1.50 1.42 1.20 0.80 0.80 0.42 0.23 1.10
Viscosity
Dielectric constant
Solubility parameter
Enzyme activity (mmol/min g)
0.30 3.31 3.70 3.86 4.60 2.95 4.31 2.37 0.31 1.30
1.89 13.92 15.44 14.71 15.00 17.70 12.47 17.94 20.71 2.21
7.22 11.10 10.43 10.91 11.40 11.46 10.65 11.63 9.67 10.11
98.40 189.60 672.20 399.20 312.00 336.80 572.80 427.20 148.80 18.96
Conditions: Reactions were carried out in 4 ml solvent, soybean oil (4 mmol), methanol (24 mmol), 50% ultrasonic power, Novozym 435 (6% based on the oil weight), water content of 0.5% at 40 8C and 50 rpm vibration under ultrasonic irradiation in 2 h. Data source of solvent parameters: Refs. [32,33].
to keep its catalytic activity. At low-water content, the conformation of Novozym 435 was excessively rigid which disturbed the ‘‘induced-fit’’ process of the enzyme and decreased the enzyme activity [26]. At higher water content, the decrease in enzyme activity could be attributed to the observed enzyme particle aggregation that might consequently lead to limited access of the substrate to the enzyme active site [27]. An alternative possibility was that the conformation of Novozym 435 was more flexible at higher water content, and the water in the reaction mixture might act as competing nucleophile for acyl-enzyme, thus suppressing the expected acyl transfer and causing unfavorable equilibrium position in reversed hydrolysis. Overall, these results suggested that water content strongly influenced the hydration level of the enzyme that consequently affected the transesterification activity for biodiesel production.
Other small alcohols, such as 2-propanol, n-butanol, n-pentanol and 2-pentanol were inappropriate to use, because they served not only as solvents, but also as acyl acceptors in the transesterification reaction with triglyceride and some by-product was formed. Particularly, some solvents with very similar values of viscosity (npentanol and tert-amyl alcohol), dielectric constant (n-pentanol and 2-pentanol), and solubility parameter (tert-amyl alcohol and 1,4-dioxane) exhibited quite different effects to the enzyme activity. Therefore, viscosity, dielectric constant, solubility parameter, or even log P by themselves did not appear to be reliable parameters to assess the solvent effect on enzyme activity and a comprehensive consideration of all solvent parameters should be taken when choosing an appropriate solvent. Tert-amyl alcohol, instead of widely used tert-butanol, was chosen as the solvent considering the higher enzyme activity in this solvent.
3.4. Effect of solvents on biodiesel production
3.5. Effect of tert-amyl alcohol concentration on biodiesel production
It was well described in the literature that enzyme activity were strongly affected by organic solvents [28]. In the present study, the effects of several organic solvents with various log P (the partition coefficient of the solvent for the standard octanol/water two-phase system), dielectric constant, solubility parameter and other parameters were investigated for the transesterification of soybean oil. As shown in Table 1, there was an increase in enzyme activity as the solvent hydrophobicity increased in the range of 1.1 to 0.42. Klibanov pointed out that a minimum of water was required to preserve the conformation of the enzyme [29]. In hydrophilic solvent, such as acetone and 1,4-dioxane, water had higher affinity in hydrophilic solvent rather than bounding to the enzyme. As a consequence, the enzyme might lose its flexibility conformation due to the lack of bound water and thus losing its activity [30]. The explanation of the increasing enzyme activity with the increasing hydrophobicity might be that solvents with higher log P had better miscibility with methanol and glycerol, and stronger hydrophobicity protected the enzyme from deactivation by these alcohols [31]. No apparent correlation between log P and enzyme activity were observed when log P 0.8. The enzyme activity was greatly influenced by different functional groups and molecular structure of the solvents. Enzyme activity was improved by adding the solvents with hydroxyl group, because they solubilized both methanol and glycerol. Alcohols with side-chain molecular structures had higher enzyme activity compared to the linear ones, especially with tertiary alcohols, like tert-butanol and tertamyl alcohol (Table 1). This phenomenon might also be attributed to the differences in miscibility with triglyceride. As an indication of solubility parameter, for alcohol molecules with the same carbon numbers, the branched ones had better miscibility with triglyceride (solubility parameter of 7.43) than the linear isomers.
Biodiesel synthesis was greatly influenced by addition of tert-amyl alcohol to the reaction mixture. Different amounts of tert-amyl alcohol were added to the reaction mixture in order to observe the effect of tert-amyl alcohol concentration on the transesterification reaction. The enzyme activity was very low in solvent-free system due to the toxicity of excessive methanol on enzyme activity as shown in Fig. 4. The enzyme activity increased
Fig. 4. Effect of tert-amyl alcohol concentration on biodiesel production. Conditions: Reactions were carried out in soybean oil (4 mmol), methanol (24 mmol), 50% ultrasonic power, Novozym 435 (6% based on the oil weight) and water content of 0.5% at 40 8C and 50 rpm vibration under ultrasonic irradiation in 2 h. The tert-amyl alcohol/oil volume ratio varied from 0 to 4.
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Fig. 5. Effect of substrate ratio on biodiesel production. Conditions: Reactions were carried out in tert-amyl alcohol (4 ml), soybean oil (4 mmol), 50% ultrasonic power, Novozym 435 (6% based on the oil weight) and water content of 0.5% at 40 8C and 50 rpm vibration under ultrasonic irradiation in 2 h. The molar ratio of methanol/oil varied from 1 to 36.
greatly with the increase in tert-amyl alcohol/oil volume ratio with a maximum value at a tert-amyl alcohol/oil volume ratio of 1:1. Further increased in tert-amyl alcohol concentration resulted no increment of enzyme activity. The presence of tert-amyl alcohol in the system could improve the solubility of methanol in the reaction mixture, as a result, the inhibition effects of methanol in biodiesel production were eliminated, and lipase maintained high activity even with high amount of methanol presented in the system. 3.6. Effect of substrate ratio on biodiesel production The rate of an enzyme catalytic reaction depends on the concentrations of enzyme and substrate. Variation of substrate ratio might have significant effect on the rate of the reaction. In the present experiments, the substrate molar ratio (methanol/oil) was changed from 1 to 36 while keeping the amount of soybean oil constant at 4 mmol. The amount of the enzyme was kept constant. The enzymatic activities increased with the increase of substrate ratio from 1:1 to 6:1, and reached the maximum of 680 mmol/ min g when the substrate ratio was 6:1 (Fig. 5). As the amount of methanol increases, more cavitation bubbles might be generated because the cavitation events were easier in methanol than in oil, and thus improve the reaction. After this point, with the further increasing of substrate ratio, the enzyme activity decreased gradually. It was probably due to the inhibitory effect of methanol on enzyme become more significant with higher concentrations of methanol. Hence, the molar ratio of 6:1 was considered as the optimum substrate ratio.
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Fig. 6. Effect of enzyme dosage on biodiesel production. Conditions: Reactions were carried out in tert-amyl alcohol (4 ml), soybean oil (4 mmol), methanol (24 mmol), 50% ultrasonic power, water content of 0.5% at 40 8C and 50 rpm vibration under ultrasonic irradiation in 2 h. The amount of enzyme varied from 0 to 10% based on the oil weight.
yield of methyl ester witnessed a steep increase, while after the point, the yield increase was much slower), and was adopted in the experiments. 3.8. Effect of temperature on biodiesel production Theoretically elevated temperature can help the substrate molecules to obtain adequate energy to pass over the energy barrier and enhance the reaction rate. In contrast, enzymes were temperature-depended and easily deactivated at high temperature. The effect of temperature on the activity of Novozym 435 was examined in the range of 30–70 8C. The results in Fig. 7 shows that the enzyme activity increased as temperature increased from 30 to 40 8C, followed by a decrease at higher temperature. As the reaction temperature elevated, the collision chance between enzyme and substrate molecules increased,
3.7. Effect of enzyme dosage on biodiesel production Our ultimate aim was to develop a process that could consume the least amount of enzyme and time to produce the possible maximum amount of biodiesel. The effect of Novozym 435 dosage on the biodiesel production was studied under UIV. The mole ratios of the reactants were kept constant while the amount of enzyme was changed from 0 to 10% based on the oil weight. As the loading of Novozym 435 increased, the yield of FAME was also increased (Fig. 6). Considering the cost of the enzyme, 6% of Novozym 435 was proved to be the most efficient amount (before this point, the
Fig. 7. Effect of temperature on biodiesel production. Conditions: Reactions were carried out in tert-amyl alcohol (4 ml), soybean oil (4 mmol), methanol (24 mmol), 50% ultrasonic power, Novozym 435 (6% based on the oil weight) and water content of 0.5% and 50 rpm vibration under ultrasonic irradiation in 2 h. Temperature varied from 30 to 70 8C.
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in 4 h under UIV. The enzyme was ultrasonic stable as no obvious loss in lipase activity was observed after repeated use for five cycles under UIV. Acknowledgment Financial support for this work was by the Program for New Century Excellent Talents from the Chinese Ministry of Education (No. NCET-08-0244). References
Fig. 8. Reusability of enzyme on biodiesel production. Conditions: Reactions were carried out in tert-amyl alcohol (4 ml), soybean oil (4 mmol), methanol (24 mmol), 50% ultrasonic power, Novozym 435 (6% based on the oil weight) and water content of 0.5% at 40 8C and 50 rpm vibration under ultrasonic irradiation in 2 h.
which might help to form enzyme–substrate complexes and then led to the increase of enzyme activity. The increased of reaction temperature also decreased of viscosity of soybean oil, increased of cavitation events and the rate of emulsion formation, thus consequently increased the biodiesel formation. In addition, the molecules of the proteins could fluctuate to relieve the steric repulsion by increasing the temperature, and such a fluctuation could contribute to the rate acceleration at elevated temperatures [34]. As for the decrease of enzyme activity with further increase of the temperature above 40 8C, it was mostly likely due to the denaturation (alteration) of protein structure resulted from heat-induced destruction of noncovalent interactions (the breakdown of the weak ionic and hydrogen bonding that stabilized the three dimensional structure of the enzyme) [35]. 3.9. Reusability of Novozym 435 under UIV Since the enzyme was costly, the reusability of enzyme was an essential factor for the production of biodiesel in industry. The catalyst reusability studies were carried out to investigate the stability of the enzyme under UIV. As shown in Fig. 8, Novozym 435 was not deactivated or denatured under UIV, only a slightly decrease (4%) in enzyme activity was observed after five uses. These results suggested that Novozym 435 was suitable for catalytic usage in UIV conditions. 4. Conclusions In the present study, ultrasonic irradiation with vibration was proved to be an efficient method for enzymatic biodiesel production. Under ultrasonic irradiation, enzyme activity of Novozym 435 was enhanced in transesterification reaction of soybean oil and methanol. The ultrasonic assisted reaction generated equivalent yield of FAME in relatively shorter time compared with that of vibration. When ultrasonic irradiation and vibration were used together, biodiesel production rate was further enhanced. Under the optimum conditions (50% of ultrasonic power, 50 rpm vibration, water content of 0.5%, tert-amyl alcohol/oil volume ratio of 1:1, methanol/oil molar ratio of 6:1, 6% Novozym 435 and 40 8C), a 96% yield of fatty acid methyl ester could be achieved
[1] Demirbas A. Progress and recent trends in biofuels. Prog Energy Combust Sci 2007;33(1):1–18. [2] Meher LC, Sagar DV, Naik SN. Technical aspects of biodiesel production by transesterification: a review. Renew Sustain Energy Rev 2006;10(3): 248–68. [3] Akoh CC, Chang SW, Lee GC, Shaw JF. Enzymatic approach to biodiesel production. J Agric Food Chem 2007;55(22):8995–9005. [4] Freedman B, Pryde EH, Mounts TL. Variables affecting the yields of fatty esters from transesterified vegetable oils. J Am Oil Chem Soc 1984;61(10): 1638–43. [5] Akoh CC, Swanson BG. Base-catalyzed transesterification of vegetable oils. J Food Process Preserv 1988;12(2):139–49. [6] Ma F, Hanna MA. Biodiesel production: a review. Bioresour Technol 1999;70(1):1–15. [7] Canakci M, Van Gerpen JH. Biodiesel production via acid catalysis. Trans ASAE 1999;42(5):1203–10. [8] Fjerbaek L, Christensen KV, Norddahl B. A review of the current state of biodiesel production using enzymatic transesterification. Biotechnol Bioeng 2009;102(5):1298–315. [9] Shah S, Sharma S, Gupta MN. Enzymatic transesterification for biodiesel production. Indian J Biochem Biophys 2003;40(6):392–9. [10] Marchetti JM, Miguel VU, Errazu AF. Possible methods for biodiesel production. Renew Sust Energy Rev 2007;11(6):1300–11. [11] Nelson L, Foglia T, Marmer W. Lipase-catalyzed production of biodiesel. J Am Oil Chem Soc 1996;73(9):1191–5. [12] Nie K, Xie F, Wang F, Tan T. Lipase catalyzed methanolysis to produce biodiesel: optimization of the biodiesel production. J Mol Catal B Enzym 2006;43(1–4):142–7. [13] Shaw JF, Chang SW, Lin SC, Wu TT, Ju HY, Akoh CC, et al. Continuous enzymatic synthesis of biodiesel with Novozym 435. Energy Fuels 2008;22(2):840–4. [14] Miethchen R. Selected applications of sonochemistry in organic chemistry. Ultrasonics 1992;30(33):173–9. [15] Mason TJ. Ultrasound in synthetic organic chemistry. Chem Soc Rev 1997;26(6): 443–51. [16] Zhou SJ, Hawley MC. A study of ultrasonic reaction rate enhancement effect in adhesive bonding of polymers and composites. Compos Struct 2003;61(4): 303–9. [17] Sinisterra JV. Application of ultrasound to biotechnology: an overview. Ultrasonics 1992;30(3):180–5. [18] Lin G, Liu H. Ultrasound promoted lipase catalyzed reactions. Tetrahedron Lett 1995;36(34):6067–8. [19] Mahamuni NN, Adewuyi YG. Optimization of the synthesis of biodiesel via ultrasound-enhanced base-catalyzed transesterification of soybean oil using a multifrequency ultrasonic reactor. Energy Fuels 2009;23:2757–66. [20] Deshmane VG, Gogate PR, Pandit AB. Ultrasound-assisted synthesis of biodiesel from palm fatty acid distillate. Ind Eng Chem Res 2009;48(17): 7923–7. [21] Plank C, Lorbeer E. Simultaneous determination of glycerol and mono-, di and triglycerides in vegetable oil methyl esters by capillary gas chromatography. J Chromatogr A 1995;697(1–2):461–8. [22] Xiu GH, Jiang L, Li P. Mass-transfer limitations for immobilized enzymecatalyzed kinetic resolution of racemate in a batch reactor. Ind Eng Chem Res 2000;39(11):4054–62. [23] Bradley M, Ashokkumar M, Grieser F. Sonochemical production of fluorescent and phosphorescent latex particles. J Am Chem Soc 2003;125(2):525–9. [24] Shah S, Gupta MN. The effect of ultrasonic pre-treatment on the catalytic activity of lipases in aqueous and non-aqueous media. Chem Cent J 2008;2(1):1–9. [25] Ma L, Persson M, Adlercreutz P. Water content dependence of lipase catalysis in organic media explains successful transesterification reactions. Enzyme Microb Technol 2002;31(7):1024–9. [26] Halling PJ. Enzymatic conversion in organic and other low-water media. In: Drauz K, Waldmann H, editors. 2nd ed., Enzyme catalysis in organic synthesis a comprehensive handbook, vol. I, 2nd ed. Weinheim, Germany: Wiley–VCH Verlag GmbH; 2002. p. 259–86. [27] Karboune S, Archelas A, Baratti J. Properties of epoxide hydrolase from Aspergillus niger for the hydrolytic kinetic resolution of epoxides in pure organic media. Enzyme Microb Technol 2006;39(2):318–24. [28] Laane C, Boeren S, Vos K, Veeger C. Rules for optimization of biocatalysis in organic solvents. Biotechnol Bioeng 1987;30(1):81–7.
D. Yu et al. / Process Biochemistry 45 (2010) 519–525 [29] Klibanov AM. Improving enzymes by using them in organic solvents. Nature 2001;409(6817):241–6. [30] Perraud R, Laboret F. Optimization of methyl propionate production by Mucor miehei lipase. Appl Microbiol Biotechnol 1995;44(3–4):321–6. [31] Dossat V, Combes D, Marty A. Continuous enzymatic transesterification of high oleic sunflower oil in a packed bed reactor: influence of the glycerol production. Enzyme Microb Technol 1999;25(3–5):194–200. [32] Lide DR. CRC handbook of chemistry and physics, 87th ed., Boca Raton, FL: CRC Press/Taylor and Francis; 2006.
525
[33] Secundo F, Riva S, Carrea G. Effects of medium and of reaction conditions on the enantioselectivity of lipases in organic solvents and possible rationales. Tetrahedron Asym 1992;3(2):267–80. [34] Yu HM, Chen ST, Suree P, Nuansri R, Wang KT. Effect of microwave irradiation on acid-catalyzed hydrolysis of starch. J Org Chem 1996;61(26):9608–9. [35] Yadav GD, Lathi PS. Intensification of enzymatic synthesis of propylene glycol monolaurate from 1,2-propanediol and lauric acid under microwave irradiation: kinetics of forward and reverse reactions. Enzyme Microb Technol 2006;38(6):814–20.