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Articles Continuous Enzymatic Synthesis of Biodiesel with Novozym 435 J.-F. Shaw,†,‡,§ S.-W. Chang,‡,⊥ S.-C. Lin,| T.-T. Wu,| H.-Y. Ju,‡ C. C. Akoh,# R.-H. Chang,∆ and C.-J. Shieh*,‡,O Department of Nutrition and Health Science, Chung-Chou UniVersity of Technology, Number 6, Lane 2, Section 3, Shanjiao Road, Yuan-Lin, Chang-Hua, 503, Taiwan, Department of Food Science and Biotechnology, Department of Chemical Engineering, and Biotechnology Center, National Chung Hsing UniVersity, Taichung, 402, Taiwan, Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei, 115, Taiwan, Department of Bioindustry Technology, Dayeh UniVersity, Chang-Hua, 515, Taiwan, Department of Food Science and Technology, UniVersity of Georgia, Athens, Georgia 30602, and Econergy Taiwan Co. Ltd., Da-an, Taipei, 106, Taiwan ReceiVed August 21, 2007. ReVised Manuscript ReceiVed October 29, 2007
A continuous process for the enzymatic synthesis of biodiesel, fatty acid methyl esters (FAMEs) from soybean oil, catalyzed by immobilized lipase from Candida antarctica (Novozym 435) was investigated. Novozym 435 was packed in a packed-bed reactor and used to catalyze the alcoholysis of methanol and soybean oil to produce FAMEs in a cosolvent system. Response surface methodology (RSM) and 3-factor-3-level fractional factorial design were employed to evaluate the effects of synthesis parameters, such as reaction temperature (45-65 °C), flow rate (0.1-0.5 mL/min), and substrate molar ratio of methanol to soybean oil (3:1-5:1) on percentage molar conversion of FAMEs by alcoholysis. Reaction temperature and flow rate had significant effects on the percent molar conversion. On the basis of ridge max analysis, the optimum conditions for synthesis were as follows: reaction temperature 52 °C, flow rate 0.1 mL/min, and substrate molar ratio 4.3:1. The predicted value was 74.2%, and the actual experimental value was 75.2% molar conversion.
Introduction Biodiesel (fatty acid methyl esters, FAMEs), an alternative fuel resource for diesel engines, is currently of great interest because of its environmental benefits, such as its renewable, biodegradable, and nontoxic properties. Commercially, biodiesel has been industrially produced by chemical processes that are efficient in terms of reaction time and high yields but has drawbacks in terms of the recovery of glycerol, removal requirement of salt residues, large amount of wastewater, and high energy cost. To overcome these drawbacks, the utilization of biocatalysts (lipases) to synthesize biodiesel by alcoholysis of triacylglycerol and short-chain alcohol under mild conditions has attracted considerable attention in recent years. Several research works have been carried out using enzymatic methods.1–6 * To whom all correspondence should be addressed. Biotechnology Center, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 40227, Taiwan. Tel.: +886-4-2284-0450-2 ext. 5121. Fax: +886-4-22861905. E-mail:
[email protected]. † Department of Food Science and Biotechnology, National Chung Hsing University. ‡ Dayeh University. § Academia Sinica. ⊥ Chung-Chou University of Technology. # University of Georgia. | Department of Chemical Engineering, National Chung Hsing University. ∆ Econergy Taiwan Co. Ltd. O Biotechnology Center, National Chung Hsing University. (1) Nelson, L. A.; Foglia, T. A.; Marmer, W. N. Lipase-catalyzed production of biodiesel. J. Am. Oil Chem. Soc. 1996, 73, 1191–1195.
Nelson et al.1 reported that the Candida antarctica lipase was efficient for the transesterification of triacylglycerols with secondary alcohols to give branched alkyl esters. Shimada et al.2 found that immobilized C. antarctica (Novozym 435) lipase was inactivated in mixtures containing greater than 1.5 mol equiv of methanol in oil and that stepwise addition of methanol prevented the inactivation of lipase. Kose et al.7 investigated alcoholysis of refined cottonseed oil in the presence of immobilized C. antarctica lipase and suggested optimum conditions with a maximum methyl esters content of 91.5%. Previously, we have utilized the immobilized C. antarctica lipase (Novozym 435) to synthesize biodiesel via alcoholysis (2) Shimada, Y.; Watanabe, Y.; Samukawa, T.; Sugihara, A.; Noda, H.; Fukuda, H.; Tominaga, Y. Conversion of vegetable oil to biodiesel using immobilized Candida antarctica lipase. J. Am. Oil Chem. Soc. 1999, 76, 789–793. (3) Iso, M.; Chen, B.; Eguchi, M.; Kudo, T.; Shrestha, S. Production of biodiesel fuel from triglycerides and alcohol using immobilized lipase. J. Mol. Catal. B: Enzym. 2001, 16, 53–58. (4) Shieh, C. J.; Liao, H. F.; Lee, C. C. Optimization of lipase-catalyzed biodiesel by response surface methodology. Bioresour. Technol. 2003, 88, 103–106. (5) Chen, J. W.; Wu, W. T. Regeneration of immobilized Candida antarctica lipase for transesterfication. J. Biosci. Bioeng. 2003, 95, 466– 469. (6) Chang, H. M.; Liao, H. F.; Lee, C. C.; Shieh, C. J. Optimized synthesis of lipase-catalyed biodiesel by Novozyme 435. J. Chem. Technol. Biot. 2005, 80, 307–312. (7) Kose, O.; Tuter, M.; Aksov, H. A. Immobilized Candida antarctica lipase catalyzed alcoholysis of cotton seed oil in a solvent-free medium. Bioresour. Technol. 2002, 82, 125–129.
10.1021/ef7005047 CCC: $40.75 2008 American Chemical Society Published on Web 02/21/2008
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Table 1. Box-Behnken Design and Experimental Data for 3-Level-3-Factor Response Surface Analysis factors treatment no.
temperature (°C) X1
flow rate (mL/min) X2
molar ratio (methanol/soybean oil) X3
molar conversion (%) Y1
production rate (µmol/min) Y2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1(65)a 1(65) -1(45) -1(45) 1(65) 1(65) -1(45) -1(45) 0(55) 0(55) 0(55) 0(55) 0(55) 0(55) 0(55)
1(0.5) -1(0.1) 1(0.5) -1(0.1) 0(0.3) 0(0.3) 0(0.3) 0(0.3) 1(0.5) 1(0.5) -1(0.1) -1(0.1) 0(0.3) 0(0.3) 0(0.3)
0(4:1) 0(4:1) 0(4:1) 0(4:1) 1(5:1) -1(3:1) 1(5:1) -1(3:1) 1(5:1) -1(3:1) 1(5:1) -1(3:1) 0(4:1) 0(4:1) 0(4:1)
36.06 ( 0.90 38.63 ( 0.55 17.37 ( 0.06 73.74 ( 0.10 37.37 ( 0.12 28.61 ( 0.06 31.92 ( 0.01 48.74 ( 0.61 36.93 ( 0.01 44.36 ( 0.06 74.08 ( 0.09 46.93 ( 0.03 60.36 ( 0.28 59.23 ( 0.39 59.64 ( 0.22
54.09 ( 1.35 11.58 ( 0.17 26.06 ( 0.09 22.12 ( 0.03 33.63 ( 0.10 25.75 ( 0.05 28.72 ( 0.01 43.87 ( 0.55 55.40 ( 0.01 66.55 ( 0.08 22.22 ( 0.03 14.08 ( 0.01 54.02 ( 0.67 53.60 ( 0.77 53.68 ( 0.20
a
Numbers in parenthesis represent actual experimental amounts.
of canola oil and methanol in a batch system containing n-hexane. An optimal weight conversion of 99.4% at a reaction time of 12.4 h, temperature of 38.0 °C, substrate molar ratio of 3.5:1, enzyme concentration of 42.3%, and 7.2% of added water by using central composite rotatable design (CCRD) and response surface methodology (RSM) analysis was obtained.6 This indicated that the Novozym 435 could be a good choice for high yield enzymatic biodiesel production. However, there is still limited investigation with regards to the large-scale enzymatic synthesis of biodiesel. In this study, we have developed a packed-bed reactor system which possesses advantages of continuity, ease of operation, high stability, and lower byproduct formation to allow largescale biodiesel production in a cosolvent system. The present work focused on the reaction parameters that affected lipasecatalyzed continuous synthesis of biodiesel in a packed-bed reactor. Our aims were to better understand the relationship between the reaction variables (reaction temperature, flow rate, and substrate molar ratio) and response (molar conversion in percent) and to establish the optimum condition for continuous enzymatic synthesis of biodiesel by using statistical experimental design and RSM analysis. Experimental Section Experimental Materials. Immobilized lipase (triacylglycerol hydrolase, EC 3.1.1.3; Novozym 435) from C. antarctica supported on acrylic resin beads was purchased from Novo Nordisk Bioindustrials, Inc. (Bagsvaerd, Denmark). The catalytic activity of Novozym 435 was 7000 PLU/g (propyl laurate units) containing 1–2% (w/w) water. Soybean oil was purchased from Taiwan Sugar Corp. (Taipei, Taiwan). Methanol (99.5% pure) and tert-butanol (95% pure) were purchased from Katayama Chemical Co. (Tokyo, Japan). Glyceryl tributyrate was purchased from Sigma Chemical Co. (St. Louis, MO) and used as an internal standard. Molecular sieve 4 Å was purchased from Davison Chemical (Baltimore, MD), and n-hexane was obtained from Merck Chemical Co. (Darmstadt, Germany). All other chemicals were analytical reagent grade. Experimental Design. A 3-level-3-factor Box-Behnken design was employed in this study, requiring 15 experiments. To avoid bias, 15 runs were performed in a totally random order. The variables and their levels selected for the study of biodiesel synthesis were reaction temperature (45–65 °C), flow rate (0.1–0.5 mL/min), and substrate molar ratio (3:1–5:1; methanol:soybean oil). All experiments were performed in a packed-bed reactor containing cosolvent (n-hexane:tert-butanol ) 9:1 v/v) system to prevent
enzyme inactivation by residual glycerol during the continuous reaction period. Enzymatic Synthesis of FAMEs in a Continuous Reactor. All materials were dehydrated by molecular sieve 4 Å for 24 h before use. Soybean oil (100 mM), different molar ratios of methanol, and cosolvent (n-hexane:tert-butanol ) 9:1 v/v) were mixed well in a feeding flask. The alcoholysis reaction was carried out in a packed-bed reactor consisting of a stainless steel tube 25 cm in length with a 0.25 cm inner diameter. The mixture was pumped through a continuous reactor (packed with 1.0 g Novozym 435) at the designed conditions. All products were collected at the outlet of the reaction tube after 30 min of reaction time for further FAMEs analysis. The whole system was placed in the temperature controlled chamber to prevent any possible temperature variation. FAMEs Confirmation and Analysis. The biodiesel formation was determined by injecting a 1 µL aliquot into a gas chromatograph (Hewlett-Packard 6890, Avondale, PA) equipped with a flameionization detector (FID) and a MXT-65TG fused silica capillary column (30 m × 0.25 mm i.d.; film thickness 1 µm; Restek, Bellefonte, PA) and operated in a splitless mode. Injector and FID temperatures were set at 300 and 320 °C, respectively. The isothermal run was maintained at 195 °C of oven temperature for 11 min. Nitrogen was used as the carrier gas. The percentage molar conversion was defined as (millimoles of FAMEs)/(3 × millimoles of initial soybean oil) × 100% and was estimated using peak area integrated by online software Hewlett-Packard 6890 Series II ChemStation. Among which, the millimoles of FAMEs were quantified by the equations derived from a standard curve. Statistical Analysis. The experimental data (Table 1) were analyzed by the response surface regression (RSREG) procedure of SAS software to fit the following second-order polynomial equation:8 3
Y ) βk0 +
∑βx
ki i
i)1
3
+
∑β
kiixi
i)1
2
+
3
∑ ∑
βkijxixj (1)
i ) 1 j ) i+ 1
Where, Y is response (percent of molar conversion); βk0, βki, βkii, and βkij were constant coefficients; and xi is the uncoded independent variable. The option of RIDGE MAX was employed to compute the estimated ridge of maximum response for increasing radii from the center of the original design.
Results and Discussion Effect of Flow Rate. Figure 1 shows the effect of various flow rates on the alcoholysis of soybean oil and methanol (8) SAS user guide; SAS Institute Inc.: Cary, NC, 1990. (9) Ott, L. An Introduction to Statistical Methods and Data Analysis; PWS-Kent Publishing: Boston, 1988. (10) Watanabe, Y.; Shimada, Y.; Sugihara, A. Conversion of degummed soybean oil to biodiesel fuel with immobilized Candida antarctica lipase. J. Mol. Catal. B: Enzym. 2002, 17, 151–155.
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Figure 1. Effect of flow rate at 50 °C and substrate molar ratio 4:1 (methanol:soybean oil) in a cosolvent system. The reaction was carried out in a 4.3 mL stainless steel column packed with 1.0 g of Novozym 435. Table 2. Analysis of Variance for Synthetic Variables Pertaining to Response Percent Molar Conversion degree of freedom sum of squares prob > f
source model
9 3 3 3 3 2 5
linear quadratic crossproduct lack of fit pure error total error 0.963 R2
3602.118557 1335.926950 1080.513007 1185.678600 137.633750 1.281667 138.915417
0.0045 0.0054 0.0086 0.0070 0.0138
Table 3. Analysis of Variance for Joint Test factor
degree of freedom
sum of squares
Prob > fa
temperature (X1) flow rate (X2) molar ratio (X3)
4 4 4
1944.784860 2251.093476 665.150991
0.0038 0.0027 0.0381b
a
Prob > f ) level of significance. b Not significant at p < 0.01.
(substrate molar ratio 4:1; methanol:soybean oil) catalyzed by Novozym 435 in a cosolvent system at 50 °C. The percentage molar conversion of biodiesel increased up to 75% at a 0.1 mL/ min flow rate. Therefore, the range of flow rate from 0.1 to 0.5
mL/min was chosen in this study. The selection of flow rate range needs to be extremely precise in Box-Behnken design; otherwise, the optimal condition of synthesis cannot be found inside the experimental region through statistical analysis and contour plots. Model Fitting. The major objective of this work is the development and evaluation of a statistical approach to better understand the relationship between the variables of a continuous enzymatic biodiesel synthesis reaction and the response (percent molar conversion of FAMEs). Based on this concept, a largescale process can be optimized to obtain alternative diesel with lower costs in terms of time and manpower requirements. In comparison with one-factor-at-a-time design, RSM as employed in this study is more efficient in reducing the experimental runs and time for optimal biodiesel production. The RSREG procedure was employed to fit the secondorder polynomial eq 1 to the experimental dataspercent molar conversion (Table 1). Among the various treatments, the highest molar conversion (74.08%) was treatment no. 11 (55 °C, flow rate 0.1 mL/min, and substrate molar ratio 5:1), and the lowest conversion (17.37%) was treatment no. 3 (45 °C, flow rate 0.5 mL/min, and substrate molar ratio 4:1). From the SAS output of RSREG, the second-order polynomial eq 2 is given below: Y ) - 313.960 + 12.742x1 - 228.550x2 + 36.974x3 0.160x1 + 6.740x2x1 - 50.521x22 + 0.630x3x1 43.325x3x2 - 7.136x32 (2) Analysis of variance (Table 2) indicated that the second-order polynomial model was highly significant and adequate to represent the actual relationship between the response (percent molar conversion) and the significant variables with very small p-value (0.0045) and a satisfactory coefficient of determination (R2 ) 0.963). Furthermore, the overall effect of the three synthesis variables on the percent molar conversion of soybean oil to biodiesel was further analyzed by a joint test (Table 3). The results revealed that the reaction temperature (x1) and flow rate (x2) were the most important parameters and exerted a statistically significant overall effect (p < 0.01) on the percent molar conversion to form biodiesel. Mutual Effect of Parameters. The relationships between reaction factors and response can be better understood by examining the planned series of contour plots generated from
Figure 2. Contour plots of molar conversion in the continuous enzymatic synthesis of FAMEs showing the effect of synthesis temperature at 45 (A), 55 (B), and 65 °C (C). Enzyme load in all experiments was constant at 1.0 g. Numbers inside the contour plots indicate the molar conversion of FAMEs at given reaction conditions.
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Figure 3. Contour plots of molar conversion in the continuous enzymatic synthesis of FAMEs showing the effect of flow rate at 0.1 (A), 0.3 (B), and 0.5 mL/min (C). Enzyme load in all experiments was constant at 1.0 g. Numbers inside the contour plots indicate the molar conversion of FAMEs at given reaction conditions.
Figure 4. Contour plots of molar conversion in the continuous enzymatic synthesis of FAMEs showing the effect of substrate molar ratio at 3:1 (A), 4:1 (B), and 5:1 (C). Enzyme load in all experiments was constant at 1.0 g. Numbers inside the contour plots indicate the molar conversion of FAMEs at given reaction conditions. Table 4. Estimated Ridge of Maximum Response for Variable Percent Molar Conversion uncoded factor values coded estimated response standard radius (% conversion) error 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
59.77 61.07 62.40 63.77 65.16 66.59 68.04 69.54 71.06 72.62 74.21
3.04 3.03 3.01 2.97 2.92 2.87 2.84 2.83 2.88 2.98 3.17
X1 (°C) 55.00 54.70 54.40 54.09 53.78 53.48 53.18 52.88 52.58 52.28 51.99
X2 X3 (methanol/ (mL/min) soybean oil) 0.30 0.28 0.26 0.24 0.23 0.21 0.19 0.17 0.15 0.13 0.12
4.00 4.02 4.03 4.06 4.08 4.11 4.13 4.16 4.19 4.22 4.25
the predicted model (eq 2) by holding constant the synthesis temperature (Figure 2), flow rate (Figure 3), or substrate molar ratio (Figure 4). As the temperature reached 55 °C, the molar conversion of FAMEs increased up to 70% with 0.15 mL/min of flow rate and higher substrate molar ratio (alcohol:oil 4:1) as shown in Figure 2B. However, as the temperature increased up to 65 °C, the FAMEs conversion was significantly reduced (Figure 2C). This means that the most suitable temperature for Novozym 435-catalyzed alcoholysis reaction under our reaction conditions would be
around 55 °C; higher temperatures (over 55 °C) might cause protein inactivation or decrease the conversion ability of the enzyme for long-term operation time. The FAMEs molar conversions were increased with decreasing flow rate as the reaction temperature e55 °C (Figure 3), indicating that a lower flow rate might extend the contact time between substrate and enzyme and result in higher conversion level to FAMEs. As the flow rate dropped to 1.0 mL/min, higher substrate molar ratio (g3.5) resulted in 70% FAMEs molar conversion in a moderate temperature range from 45 to 55 °C (Figure 3A). Although the substrate molar ratio exhibited no significant effect on the FAMEs molar conversion from 3:1 to 4:1, however, the percentage conversion of FAMEs was significantly affected as the molar ratio was raised from 4:1 to 5:1 as shown in Figure 4B. This result revealed that the optimal substrate molar ratio was g4:1 (methanol to soybean oil), meaning that at least a 4-fold molar excess methanol to soybean oil is required for economical FAMEs production. At any given temperature and substrate molar ratio, the FAMEs molar conversion was increased as the flow rate decreased (Figure 4C) which is consistent with Figures 2 and 3. Overall, the average maximum molar conversion of FAMEs obtained in this study was around 70% which represents lower conversion than that reported in several studies.4,7,9 However, in comparison with 7, 12.4, and 48 h
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reaction times used by Shieh et al.,4 Kose et al.,7 and Watanabe et al.,10 respectively, only 30 min was required in our system demonstrating the high production efficiency of the continuous bioreactor developed in the present work. Attaining Optimum Synthesis Conditions. The optimum point was determined by ridge max analysis.8 The method of ridge analysis computes the estimated ridge of maximum response for increasing radii from the center of the original design. The ridge max analysis (Table 4) indicated that maximum molar conversion was 74 ( 3.2% at 0.1 mL/min, 52 °C, and a 4.3:1 substrate molar ratio. Model Verification. The validity of the predicted model was examined by experiments at the suggested optimum synthesis conditions and three center points of fractional factorial design
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(treatment nos. 13, 14, and 15). The predicted value was 74.2 ( 3.2% obtained by ridge max analysis, and the actual value was 75.2 ( 2.5%. A ξ-square test (p-value ) 0.981, degrees of freedom ) 5) indicated that the observed values were essentially the same as the predicted values and that the generated model adequately predicted the percent molar conversion.10 Thus, the optimization of lipase-catalyzed synthesis of biodiesel from soybean oil, with Novozym 435, was successfully developed by RSM. Acknowledgment. This research was supported by the National Science Council (NSC-96-2317-B-005-002), Taiwan, Republic of China. EF7005047
