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Lipase-immobilized biocatalytic membranes for biodiesel production
Bioresource Technology xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Lipase-immobilized biocatalytic membranes for biodiesel production Chia-Hung Kuo a, Li-Ting Peng b, Shu-Chen Kan b, Yung-Chuan Liu b,c,⇑, Chwen-Jen Shieh a,⇑ a
Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan c Agricultural Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan b
a r t i c l e
i n f o
Article history: Received 16 October 2012 Received in revised form 10 December 2012 Accepted 11 December 2012 Available online xxxx Keywords: Immobilized lipase Biodiesel Polyvinylidene fluoride membrane Response surface methodology
a b s t r a c t Microbial lipase from Candida rugosa (Amano AY-30) has good transesterification activity and can be used for biodiesel production. In this study, polyvinylidene fluoride (PVDF) membrane was grafted with 1,4diaminobutane and activated by glutaraldehyde for C. rugosa lipase immobilization. After immobilization, the biocatalytic membrane was used for producing biodiesel from soybean oil and methanol via transesterification. Response Surface Methodology (RSM) in combination with a 5-level-5-factor central composite rotatable design (CCRD) was employed to evaluate the effects of reaction time, reaction temperature, enzyme amount, substrate molar ratio and water content on the yield of soybean oil methyl ester. By ridge max analysis, the predicted and experimental yields under the optimum synthesis conditions were 97% and 95%, respectively. The lipase-immobilized PVDF membrane showed good reuse ability for biodiesel production, enabling operation for at least 165 h during five reuses of the batch, without significant loss of activity. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Biodiesel, also known as fatty acid methyl esters (FAME), has attracted considerable attention in recent years because of the depletion of fossil fuels, increased crude oil prices and its environmental benefits. Biodiesel can be produced by the transesterification of triacylglycerols with alcohols, such as plant oil, animal fat or edible oil, and is generally carried out with NaOH or KOH as a catalyst. Under alkali conditions, however, an undesirable by-product, soap, is accumulated in the presence of water. The existence of soap leads to emulsification that increases the costs of the separation of biodiesel and glycerol, as well as the amount of water required for purification (Mendow et al., 2011). Alternatively, the use of lipase (triacylglycerol hydrolase, EC 3.1.1.3) in transesterification to synthesize biodiesel does not produce soap and can be carried out in mild conditions, has shown more potential than chemical processes (Hama and Kondo, 2012; Shieh et al., 2003; Szczesna Antczak et al., 2009). The lipase AY, produced from C. rugosa, is one of the most commonly used enzymes in organic solvents owing to its high activity in hydrolysis, esterification, transesterification and aminolysis ⇑ Corresponding authors at: Biotechnology Center and Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan. Tel.: +886 4 2285 3769; fax: +886 4 2285 4734 (Y.-C. Liu), tel.: +886 4 2284 0452x5121; fax: +886 4 2286 1905 (C.-J. Shieh). E-mail addresses: [email protected] (Y.-C. Liu), [email protected] (C.-J. Shieh).
(Villeneuve et al., 2000). It has been widely used in bio-transformations, such as resolution of racemic acids and resolution of secondary alcohols, due to its high enantioselectivity (Sánchez et al., 2000). However, utilization of free lipase for industrial applications has some disadvantages, such as high cost, low stability and nonreusability (Zhang et al., 2012). By means of an appropriate immobilization process, the operational costs of industrial processes involving lipase can be significantly reduced (Chang et al., 2008). Polyvinylidene fluoride (PVDF) is a hydrophobic polymer which is widely used for microfiltration and ultrafiltration membranes because of its excellent process ability, high chemical resistance, high thermal stability and inertness to many corrosive solvents (Han et al., 2011; Ying et al., 2002). Lipase immobilized on membranes offers some advantages over beaded supports, such as no intra-particle diffusion, short axial-diffusion path, low pressure drop, no bed compaction and easier scale up, which are usually limited in the conventional packed-bed systems. Besides, lipases are known to be a more hydrophobic enzyme than other proteins (Hiol et al., 2000). The hydrophobic supports involve hydrophobic interfaces, which make it possible to change the lipase structure from a close to an open conformation to promote lipase hyperactivation after immobilization (Chen et al., 2012; Jin et al., 2011; Shakeri and Kawakami, 2009). In this study, the multipoint covalent attachment of C. rugosa lipase onto PVDF membrane was prepared. The biocatalytic PVDF membrane was employed as a catalyst for the transesterification of soybean oil with methanol in n-hexane. The main objectives of this work were to better understand the effect of the reaction
0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.12.054
Please cite this article in press as: Kuo, C.-H., et al. Lipase-immobilized biocatalytic membranes for biodiesel production. Bioresour. Technol. (2013), http:// dx.doi.org/10.1016/j.biortech.2012.12.054
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C.-H. Kuo et al. / Bioresource Technology xxx (2013) xxx–xxx
variables (time, temperature, enzyme amount, substrate molar ratio and added water content) on the biodiesel yield, and to obtain the optimum conditions for biodiesel production using central composite rotatable design (CCRD) and RSM analysis.
ity is defined as the amount of enzyme which liberates 1 mmol of p-nitrophenol from p-nitrophenyl palmitate per minute.
2. Methods
A 5-level-5-factor CCRD was employed in this study, requiring 29 experiments. The variables and their levels selected for the study of biodiesel synthesis were reaction time (8–40 h), temperature (30–50 °C), enzyme amount (1–5 pieces of membrane), substrate molar ratio (3:1–7:1; methanol: soybean oil) and added water content (0–10% by weight of soybean oil). Table 1 shows the independent factors (xi), levels and experimental design in terms of coded and uncoded.
2.1. Materials The polyvinylidene fluoride (PVDF) membrane (150 mg, d: 47 mm, pore size: 0.45 lm, thickness: 140 lm) was purchased from Pall (Mexico). 1,4-Diaminobutane (DA) and glutaraldehyde (GA) were obtained from Acros Organics (NJ, USA). Lipase from C. rugosa (Amano AY-30) was purchased from Amano International Enzyme Co. (Nagoya, Japan). Soybean oil was purchased from the Taiwan Sugar Corp. (Taipei, Taiwan). Methanol (99.5% pure), nhexane and p-nitrophenyl palmitate (p-NPP) were purchased from Sigma-Aldrich Chemical Co. (MO, USA). The molecular sieve 4 Å was purchased from Ridel-deHaen (MO, USA). All other chemicals were of analytical reagent grade. 2.2. Immobilization of lipase onto PVDF membrane The pre-activated PVDF membrane was prepared for lipase immobilization. Briefly, the PVDF membrane was aminated with 1,4-diaminobutane and activated with glutaraldehyde, as described in the previous study (Kuo et al., 2012). The lipase immobilization was performed by immersion of the pre-activated PVDF membrane in 20 mL of 50 mM, pH 6 phosphate buffer at a lipase concentration of 7 mg mL1, 35 °C for 90 min. After that, the lipase-immobilized PVDF membranes were washed three times with distilled water and preserved at 4 °C until use. The activity of the immobilized lipase was determined to be 60 U g1 membrane (9 U per piece of membrane). One unit (U) of enzyme activ-
2.3. Experimental design
2.4. Synthesis and analysis All materials were dehydrated by a molecular sieve 4 Å for 24 h before reaction. Soybean oil (0.5 g) and different molar ratios of methanol were added to 6 mL of n-hexane, followed by different amounts of water (0–10% by weight of soybean oil) and lipaseimmobilized PVDF membrane (1–5 pieces). The mixtures of soybean oil, methanol and lipase-immobilized membrane were agitated in an orbital shaking water bath (100 rpm) at different reaction temperatures and reaction times (Table 1). Then, the analysis was performed by injecting a 1 lL aliquot in splitless mode into an Agilent Technologies 7890A gas chromatograph (South Taft, Loveland, Colorado, USA) equipped with a flame-ionization detector (FID) and a MXT-65TG fused-silica capillary column (30 m 0.25 mm id; film thickness 0.1 lm; Restek, Bellefonte, PA, USA). Injector and detector temperatures were set at 300 °C. The oven’s initiating temperature was set at 160 °C, elevated to 190 °C at 10 °C min1, then held for 10 min. Pure nitrogen was used as a carrier gas. The percentage of molar conversion was defined as (mmol of biodiesel per 3 mmol of initial soybean oil) 100%.
Table 1 Central-composite rotatable design and experimental data for 5-level-5-factor response surface analysis. Treatment no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Factors Time (h) X1
Temp (°C) X2
Enzyme amount (Pieces of membrane) X3
Substrate molar ratio(MeOH: oil) X4
H2O (%wt of soybean oil) X5
2(40) 1(16) 0(24) 1(16) 1(32) 0(24) 1(16) 0(24) 1(32) 1(16) 0(24) 0(24) 0(24) 0(24) 1(16) 1(32) 1(32) 1(32) 1(32) 0(24) 1(16) 1(32) 1(32) 1(16) 0(24) 0(24) 0(24) 2 (8) 1(16)
0(40) 1(35) 0(40) 1(45) 1(35) 0(40) 1(35) 0(40) 1(45) 1(35) 0(40) 2(30) 0(40) 0(40) 1(45) 1(35) 1(45) 1(45) 1(35) 2(50) 1(35) 1(35) 1(45) 1(45) 0(40) 0(40) 0(40) 0(40) 1(45)
0(3) 1(4) 2(1) 1(4) 1(2) 0(3) 1(2) 0(3) 1(2) 1(2) 0(3) 0(3) 2(5) 0(3) 1(2) 1(4) 1(4) 1(4) 1(2) 0(3) 1(4) 1(4) 1(2) 1(2) 0(3) 0(3) 0(3) 0(3) 1(4)
0(5:1) 1(4:1) 0(5:1) 1(6:1) 1(6:1) 2(7:1) 1(6:1) 0(5:1) 1(4:1) 1(4:1) 0(5:1) 0(5:1) 0(5:1) 0(5:1) 1(4:1) 1(4:1) 1(4:1) 1(6:1) 1(4:1) 0(5:1) 1(6:1) 1(6:1) 1(6:1) 1(6:1) 0(5:1) 0(5:1) 2(3:1) 0(5:1) 1(4:1)
0(5.0) 1(7.5) 0(5.0) 1(7.5) 1(2.5) 0(5.0) 1(7.5) 2(0.0) 1(2.5) 1(2.5) 2(10) 0(5.0) 0(5.0) 0(5.0) 1(7.5) 1(2.5) 1(7.5) 1(2.5) 1(7.5) 0(5.0) 1(2.5) 1(7.5) 1(7.5) 1(2.5) 0(5.0) 0(5.0) 0(5.0) 0(5.0) 1(2.5)
Observed yield (%) Y
82.9 36.5 26.1 45.6 63.2 46.1 14.1 45.8 19.3 15.2 32.1 39.0 89.9 73.8 11.2 70.3 69.8 39.6 26.4 13.3 27.3 42.7 19.5 11.4 81.7 78.9 76.7 40.9 23.6
Please cite this article in press as: Kuo, C.-H., et al. Lipase-immobilized biocatalytic membranes for biodiesel production. Bioresour. Technol. (2013), http:// dx.doi.org/10.1016/j.biortech.2012.12.054
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3.2. Effect of synthesis parameters
Table 2 ANOVA of the response surface model and joint factor. Source
Degree of freedom
Linear Quadratic Interaction Total Model R2 = 0.95 Joint factor Time (x1) Temperature (x2) Enzyme amount (x3) Substrate molar ratio (x4) Added water (x5) a b
Sum of squares
Prob > Fa b
5 5 10 20
7145.12 7276.99 2277.86 16700.00
0.0010 0.0009b 0.1437 0.0027b
6 6 6 6 6
3970.87 7031.31 5947.73 1661.34 4767.92
0.0101b 0.0017b 0.0029b 0.1036 0.0059b
Prob > F = level of significance. Significant at 0.05 level.
3. Results and discussion 3.1. Model fitting The RSREG procedure of SAS software (SAS Institute, Cary, NC, USA) was employed to fit the second-order polynomial equation to the experimental conversions shown in Table 1. The second-order polynomial Eq. (1) is given below:
Y ¼ 1216:64449 þ 11:03731v1 þ 44:13050v2 þ 38:27679v3 þ 86:64257v4 þ 0:23889v5 0:082891v1 v2 þ 0:10039v1 v3 0:25586v1 v4 0:19984v1 v5 þ 0:74188v2 v3 0:083125v2 v4 þ 0:542752v2 v5 5:07187v3 v4 þ 1:79125v3 v5 0:87875v4 v5 0:092563v21 0:59446v22 6:89030v23 6:04905v24 1:86585x25
ð1Þ
The analysis of variance (ANOVA) results, as shown in Table 2, indicated that the second-order polynomial model was statistically significant and adequate to represent the actual relationship between the response (yield) and the significant variables, with a very small p-value (0.0027) and a satisfactory coefficient of determination (R2 = 0.95). The ANOVA results also showed that the linear and quadratic terms had a significant influence (p < 0.05), while the interaction term had less influence (p > 0.05). The overall effect of the five synthesis variables on the biodiesel yield was evaluated by a joint test. The results (Table 2) revealed that the reaction time (x1), temperature (x2), enzyme content (x3) and added water content (x5) were the important factors, exerting a statistically significant overall effect (p < 0.05) on the response, and that the substrate molar ratio (x4) had a less significant effect (p > 0.05) (See Fig. 1).
Yield (%)
100 80
(B)
(A)
Since the interaction term had less influence, the effect of each parameter on the biodiesel yield could be better understood by the predicted plots generated from Eq. (1) by keeping the other four parameters constant at their respective center levels. The effect of reaction time was investigated in the range of 8–40 h. As shown in Fig. 1(A), the biodiesel yield increased as the reaction time increased, and then gradually leveled off after 30 h. The yield gradually leveling off after 30 h was probably due to the synthesis product inhibiting the alcoholysis reaction. The effect of reaction temperature on the biodiesel yield is shown in Fig. 1(B). It can be seen that the yield increased with increases in temperature up to 40 °C and then rapidly decreased. This result indicated that the excessive heat energy inhibited the lipase activity, as lipase can be inactivated by thermal denaturation. It has been reported that for most lipases used for biodiesel synthesis, the optimal temperature is between 30 and 55 °C and is dependent on the operational stability of the lipase and the rate of the transesterification reaction (Lee et al., 2011; Wang et al., 2010). The relationship between the biodiesel yield and enzyme amount is shown in Fig. 1(C). The highest yield (87%) was obtained with four pieces of lipase-immobilized membrane. The biodiesel yield gradually leveled off or slightly decreased at higher enzyme amounts (over four pieces) because all the active sites could not be exposed to the substrates in the present of excess lipase in the reaction. In order to save costs, the amount of enzyme used should be minimized as much as possible, as an excess of enzyme does not effectively increase the yield. Fig. 1(D) shows the effect of the substrate molar ratio on biodiesel synthesis. An increase in biodiesel yield was observed with the increase of the MeOH:oil molar ratio from 3:1 to 5:1, and then the trend was reversed. The biodiesel yield at a seven substrate molar ratio was lower than that at five. This result indicated that more methanol was needed for the transesterification reaction to proceed at a reasonable rate; however, too much of an increase of methanol can inhibit enzyme activity and result in a decreased yield. Shimada et al. observed the inactivation of C. antarctica lipase by methanol and ethanol when the alcohol: oil molar ratio was higher than 3:1, which was overcome by using immobilized enzyme or added methanol stepwise (Shimada et al., 2002). The effect of added water content on biodiesel synthesis was investigated in the range of 0–10%, as shown in Fig. 1(E). Our result showed that the biodiesel yield was increased with water content between 0 and 5.5%. Studies have demonstrated that the addition of a small amount of water to a lipase-catalyzed reaction mixture increased catalytic efficiency and the rate of fatty acid ester synthesis (Kaieda et al., 2001; Tongboriboon et al., 2010). In an organic solvent, a small amount of water is essential to maintaining the specific three-dimensional structure of the enzyme. However, lipases usually catalyze hydrolysis in aqueous media, so excess
(E)
(D)
(C)
60 40 20 10
20 30 Time (h)
40
30
35
40 45 Temp (oC)
50
1
2 3 4 Enzyme amount (Pieces)
5
3
4 5 6 Substrate ratio (MeOH:Oil)
7
0
2
4 6 8 H2O (% wt. of oil)
10
Fig. 1. Relationship between the synthesis parameters: (A) reaction time; (B) temperature; (C) enzyme amount; (D) substrate molar ratio; and (E) added water content and biodiesel yield. Solid lines are model data, and symbols are experimental data.
Please cite this article in press as: Kuo, C.-H., et al. Lipase-immobilized biocatalytic membranes for biodiesel production. Bioresour. Technol. (2013), http:// dx.doi.org/10.1016/j.biortech.2012.12.054
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water may also stimulate the competing hydrolysis reaction and lead to a decrease in transesterification yields. In this study, the highest yield was found with a water content of 4–6%. 3.3. Attaining optimum conditions and reusability of immobilized lipase The optimum synthesis of enzymatic biodiesel was determined by the ridge max analysis, which showed that the maximum yield was 97.2% at 33 h, 40 °C, 4.3 pieces of lipase-immobilized membrane, 4:1 substrate molar ratio and 5.2% added water content. A verification experiment performed at the suggested optimum conditions obtained a yield of 95.3%, which was close to the predicted yield; thus, the predicted model was successfully developed. In this study, the lipase-catalyzed synthesis of biodiesel was carried out in a low-water organic environment. Lipase exposed in n-hexane and methanol for an extended period can cause an inactivation effect. In particular, methanol inhibition is generally observed in biodiesel production via lipase-catalyzed transesterification reactions (Shimada et al., 2002). To examine the enzyme reusability, the ability of immobilized lipase for biodiesel synthesis was investigated under optimum conditions. The immobilized lipase was recovered from the reaction medium after reaction and directly reused in the next batch. After the batch of immobilized lipase was reused five times, the biodiesel yields still remained at about 90%. This result showed that the immobilized lipase remained stable through a long-term hexane and methanol exposure. Thus, the result demonstrated that the lipase immobilized on PVDF membrane could be effectively applied for biodiesel synthesis and that the stability was high enough to permit reuse. Acknowledgements This research was supported by the National Science Council, Taiwan, ROC (Grants no. 101-2313-B-005-040-MY3) and was also supported in part by the Ministry of Education, Taiwan, ROC, under the ATU plan. References Chang, S.W., Shaw, J.F., Yang, K.H., Chang, S.F., Shieh, C.J., 2008. Studies of optimum conditions for covalent immobilization of Candida rugosa lipase on poly (cglutamic acid) by RSM. Bioresour. Technol. 99, 2800–2805.
Chen, G.J., Kuo, C.H., Chen, C.I., Yu, C.C., Shieh, C.J., Liu, Y.C., 2012. Effect of membranes with various hydrophobic/hydrophilic properties on lipase immobilized activity and stability. J. Biosci. Bioeng. 113, 166–172. Hama, S., Kondo, A., 2012. Enzymatic biodiesel production: An overview of potential feedstocks and process development. Bioresour. Technol. (http://dx.doi.org/ 10.1016/j.biortech.2012.08.014). Han, M.J., Baroña, G.N.B., Jung, B., 2011. Effect of surface charge on hydrophilically modified poly(vinylidene fluoride) membrane for microfiltration. Desalination 270, 76–83. Hiol, A., Jonzo, M.D., Rugani, N., Druet, D., Sarda, L., Comeau, L.C., 2000. Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit. Enzyme Microb. Technol. 26, 421–430. Jin, Q., Jia, G., Zhang, Y., Yang, Q., Li, C., 2011. Hydrophobic surface induced activation of Pseudomonas cepacia lipase immobilized into mesoporous silica. Langmuir 27, 12016–12024. Kaieda, M., Samukawa, T., Kondo, A., Fukuda, H., 2001. Effect of methanol and water contents on production of biodiesel fuel from plant oil catalyzed by various lipases in a solvent-free system. J. Biosci. Bioeng. 91, 12–15. Kuo, C.H., Chen, G.J., Twu, Y.K., Liu, Y.C., Shieh, C.J., 2012. Optimum lipase immobilized on diamine-grafted PVDF membrane and its characterization. Ind. Eng. Chem. Res. 51, 5141–5147. Lee, J.H., Kim, S.B., Kang, S.W., Song, Y.S., Park, C., Han, S.O., Kim, S.W., 2011. Biodiesel production by a mixture of Candida rugosa and Rhizopus oryzae lipases using a supercritical carbon dioxide process. Bioresour. Technol. 102, 2105– 2108. Mendow, G., Veizaga, N.S., Querini, C.A., 2011. Ethyl ester production by homogeneous alkaline transesterification: Influence of the catalyst. Bioresour. Technol. 102, 6385–6391. Sánchez, A., del Río, J.L., Valero, F., Lafuente, J., Faus, I., Solà, C., 2000. Continuous enantioselective esterification of trans-2-phenyl-1-cyclohexanol using a new Candida rugosa lipase in a packed bed bioreactor. J. Biotechnol. 84, 1–12. Shakeri, M., Kawakami, K., 2009. Enhancement of Rhizopus oryzae lipase activity immobilized on alkyl-functionalized spherical mesocellular foam: influence of alkyl chain length. Microporous Mesoporous Mater. 118, 115–120. Shieh, C.J., Liao, H.F., Lee, C.C., 2003. Optimization of lipase-catalyzed biodiesel by response surface methodology. Bioresour. Technol. 88, 103–106. Shimada, Y., Watanabe, Y., Sugihara, A., Tominaga, Y., 2002. Enzymatic alcoholysis for biodiesel fuel production and application of the reaction to oil processing. J. Mol. Catal. B: Enzym. 17, 133–142. Szczesna Antczak, M., Kubiak, A., Antczak, T., Bielecki, S., 2009. Enzymatic biodiesel synthesis-key factors affecting efficiency of the process. Renew. Energy 34, 1185–1194. Tongboriboon, K., Cheirsilp, B., H-Kittikun, A., 2010. Mixed lipases for efficient enzymatic synthesis of biodiesel from used palm oil and ethanol in a solventfree system. J. Mol. Catal. B: Enzym. 67, 52–59. Villeneuve, P., Muderhwa, J.M., Graille, J., Haas, M.J., 2000. Customizing lipases for biocatalysis: a survey of chemical, physical and molecular biological approaches. J. Mol. Catal. B: Enzym. 9, 113–148. Wang, Y.D., Shen, X.Y., Li, Z.L., Li, X., Wang, F., Nie, X.A., Jiang, J.C., 2010. Immobilized recombinant Rhizopus oryzae lipase for the production of biodiesel in solvent free system. J. Mol. Catal. B: Enzym. 67, 45–51. Ying, L., Kang, E., Neoh, K., 2002. Covalent immobilization of glucose oxidase on microporous membranes prepared from poly (vinylidene fluoride) with grafted poly (acrylic acid) side chains. J. Membr. Sci. 208, 361–374. Zhang, B., Weng, Y., Xu, H., Mao, Z., 2012. Enzyme immobilization for biodiesel production. Appl. Microbiol. Biotechnol. 93, 61–70.
Please cite this article in press as: Kuo, C.-H., et al. Lipase-immobilized biocatalytic membranes for biodiesel production. Bioresour. Technol. (2013), http:// dx.doi.org/10.1016/j.biortech.2012.12.054
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Lipase-immobilized biocatalytic membranes for biodiesel production Chia-Hung Kuo a, Li-Ting Peng b, Shu-Chen Kan b, Yung-Chuan Liu b,c,⇑, Chwen-Jen Shieh a,⇑ a
Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan c Agricultural Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan b
a r t i c l e
i n f o
Article history: Received 16 October 2012 Received in revised form 10 December 2012 Accepted 11 December 2012 Available online xxxx Keywords: Immobilized lipase Biodiesel Polyvinylidene fluoride membrane Response surface methodology
a b s t r a c t Microbial lipase from Candida rugosa (Amano AY-30) has good transesterification activity and can be used for biodiesel production. In this study, polyvinylidene fluoride (PVDF) membrane was grafted with 1,4diaminobutane and activated by glutaraldehyde for C. rugosa lipase immobilization. After immobilization, the biocatalytic membrane was used for producing biodiesel from soybean oil and methanol via transesterification. Response Surface Methodology (RSM) in combination with a 5-level-5-factor central composite rotatable design (CCRD) was employed to evaluate the effects of reaction time, reaction temperature, enzyme amount, substrate molar ratio and water content on the yield of soybean oil methyl ester. By ridge max analysis, the predicted and experimental yields under the optimum synthesis conditions were 97% and 95%, respectively. The lipase-immobilized PVDF membrane showed good reuse ability for biodiesel production, enabling operation for at least 165 h during five reuses of the batch, without significant loss of activity. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Biodiesel, also known as fatty acid methyl esters (FAME), has attracted considerable attention in recent years because of the depletion of fossil fuels, increased crude oil prices and its environmental benefits. Biodiesel can be produced by the transesterification of triacylglycerols with alcohols, such as plant oil, animal fat or edible oil, and is generally carried out with NaOH or KOH as a catalyst. Under alkali conditions, however, an undesirable by-product, soap, is accumulated in the presence of water. The existence of soap leads to emulsification that increases the costs of the separation of biodiesel and glycerol, as well as the amount of water required for purification (Mendow et al., 2011). Alternatively, the use of lipase (triacylglycerol hydrolase, EC 3.1.1.3) in transesterification to synthesize biodiesel does not produce soap and can be carried out in mild conditions, has shown more potential than chemical processes (Hama and Kondo, 2012; Shieh et al., 2003; Szczesna Antczak et al., 2009). The lipase AY, produced from C. rugosa, is one of the most commonly used enzymes in organic solvents owing to its high activity in hydrolysis, esterification, transesterification and aminolysis ⇑ Corresponding authors at: Biotechnology Center and Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan. Tel.: +886 4 2285 3769; fax: +886 4 2285 4734 (Y.-C. Liu), tel.: +886 4 2284 0452x5121; fax: +886 4 2286 1905 (C.-J. Shieh). E-mail addresses: [email protected] (Y.-C. Liu), [email protected] (C.-J. Shieh).
(Villeneuve et al., 2000). It has been widely used in bio-transformations, such as resolution of racemic acids and resolution of secondary alcohols, due to its high enantioselectivity (Sánchez et al., 2000). However, utilization of free lipase for industrial applications has some disadvantages, such as high cost, low stability and nonreusability (Zhang et al., 2012). By means of an appropriate immobilization process, the operational costs of industrial processes involving lipase can be significantly reduced (Chang et al., 2008). Polyvinylidene fluoride (PVDF) is a hydrophobic polymer which is widely used for microfiltration and ultrafiltration membranes because of its excellent process ability, high chemical resistance, high thermal stability and inertness to many corrosive solvents (Han et al., 2011; Ying et al., 2002). Lipase immobilized on membranes offers some advantages over beaded supports, such as no intra-particle diffusion, short axial-diffusion path, low pressure drop, no bed compaction and easier scale up, which are usually limited in the conventional packed-bed systems. Besides, lipases are known to be a more hydrophobic enzyme than other proteins (Hiol et al., 2000). The hydrophobic supports involve hydrophobic interfaces, which make it possible to change the lipase structure from a close to an open conformation to promote lipase hyperactivation after immobilization (Chen et al., 2012; Jin et al., 2011; Shakeri and Kawakami, 2009). In this study, the multipoint covalent attachment of C. rugosa lipase onto PVDF membrane was prepared. The biocatalytic PVDF membrane was employed as a catalyst for the transesterification of soybean oil with methanol in n-hexane. The main objectives of this work were to better understand the effect of the reaction
0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.12.054
Please cite this article in press as: Kuo, C.-H., et al. Lipase-immobilized biocatalytic membranes for biodiesel production. Bioresour. Technol. (2013), http:// dx.doi.org/10.1016/j.biortech.2012.12.054
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variables (time, temperature, enzyme amount, substrate molar ratio and added water content) on the biodiesel yield, and to obtain the optimum conditions for biodiesel production using central composite rotatable design (CCRD) and RSM analysis.
ity is defined as the amount of enzyme which liberates 1 mmol of p-nitrophenol from p-nitrophenyl palmitate per minute.
2. Methods
A 5-level-5-factor CCRD was employed in this study, requiring 29 experiments. The variables and their levels selected for the study of biodiesel synthesis were reaction time (8–40 h), temperature (30–50 °C), enzyme amount (1–5 pieces of membrane), substrate molar ratio (3:1–7:1; methanol: soybean oil) and added water content (0–10% by weight of soybean oil). Table 1 shows the independent factors (xi), levels and experimental design in terms of coded and uncoded.
2.1. Materials The polyvinylidene fluoride (PVDF) membrane (150 mg, d: 47 mm, pore size: 0.45 lm, thickness: 140 lm) was purchased from Pall (Mexico). 1,4-Diaminobutane (DA) and glutaraldehyde (GA) were obtained from Acros Organics (NJ, USA). Lipase from C. rugosa (Amano AY-30) was purchased from Amano International Enzyme Co. (Nagoya, Japan). Soybean oil was purchased from the Taiwan Sugar Corp. (Taipei, Taiwan). Methanol (99.5% pure), nhexane and p-nitrophenyl palmitate (p-NPP) were purchased from Sigma-Aldrich Chemical Co. (MO, USA). The molecular sieve 4 Å was purchased from Ridel-deHaen (MO, USA). All other chemicals were of analytical reagent grade. 2.2. Immobilization of lipase onto PVDF membrane The pre-activated PVDF membrane was prepared for lipase immobilization. Briefly, the PVDF membrane was aminated with 1,4-diaminobutane and activated with glutaraldehyde, as described in the previous study (Kuo et al., 2012). The lipase immobilization was performed by immersion of the pre-activated PVDF membrane in 20 mL of 50 mM, pH 6 phosphate buffer at a lipase concentration of 7 mg mL1, 35 °C for 90 min. After that, the lipase-immobilized PVDF membranes were washed three times with distilled water and preserved at 4 °C until use. The activity of the immobilized lipase was determined to be 60 U g1 membrane (9 U per piece of membrane). One unit (U) of enzyme activ-
2.3. Experimental design
2.4. Synthesis and analysis All materials were dehydrated by a molecular sieve 4 Å for 24 h before reaction. Soybean oil (0.5 g) and different molar ratios of methanol were added to 6 mL of n-hexane, followed by different amounts of water (0–10% by weight of soybean oil) and lipaseimmobilized PVDF membrane (1–5 pieces). The mixtures of soybean oil, methanol and lipase-immobilized membrane were agitated in an orbital shaking water bath (100 rpm) at different reaction temperatures and reaction times (Table 1). Then, the analysis was performed by injecting a 1 lL aliquot in splitless mode into an Agilent Technologies 7890A gas chromatograph (South Taft, Loveland, Colorado, USA) equipped with a flame-ionization detector (FID) and a MXT-65TG fused-silica capillary column (30 m 0.25 mm id; film thickness 0.1 lm; Restek, Bellefonte, PA, USA). Injector and detector temperatures were set at 300 °C. The oven’s initiating temperature was set at 160 °C, elevated to 190 °C at 10 °C min1, then held for 10 min. Pure nitrogen was used as a carrier gas. The percentage of molar conversion was defined as (mmol of biodiesel per 3 mmol of initial soybean oil) 100%.
Table 1 Central-composite rotatable design and experimental data for 5-level-5-factor response surface analysis. Treatment no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Factors Time (h) X1
Temp (°C) X2
Enzyme amount (Pieces of membrane) X3
Substrate molar ratio(MeOH: oil) X4
H2O (%wt of soybean oil) X5
2(40) 1(16) 0(24) 1(16) 1(32) 0(24) 1(16) 0(24) 1(32) 1(16) 0(24) 0(24) 0(24) 0(24) 1(16) 1(32) 1(32) 1(32) 1(32) 0(24) 1(16) 1(32) 1(32) 1(16) 0(24) 0(24) 0(24) 2 (8) 1(16)
0(40) 1(35) 0(40) 1(45) 1(35) 0(40) 1(35) 0(40) 1(45) 1(35) 0(40) 2(30) 0(40) 0(40) 1(45) 1(35) 1(45) 1(45) 1(35) 2(50) 1(35) 1(35) 1(45) 1(45) 0(40) 0(40) 0(40) 0(40) 1(45)
0(3) 1(4) 2(1) 1(4) 1(2) 0(3) 1(2) 0(3) 1(2) 1(2) 0(3) 0(3) 2(5) 0(3) 1(2) 1(4) 1(4) 1(4) 1(2) 0(3) 1(4) 1(4) 1(2) 1(2) 0(3) 0(3) 0(3) 0(3) 1(4)
0(5:1) 1(4:1) 0(5:1) 1(6:1) 1(6:1) 2(7:1) 1(6:1) 0(5:1) 1(4:1) 1(4:1) 0(5:1) 0(5:1) 0(5:1) 0(5:1) 1(4:1) 1(4:1) 1(4:1) 1(6:1) 1(4:1) 0(5:1) 1(6:1) 1(6:1) 1(6:1) 1(6:1) 0(5:1) 0(5:1) 2(3:1) 0(5:1) 1(4:1)
0(5.0) 1(7.5) 0(5.0) 1(7.5) 1(2.5) 0(5.0) 1(7.5) 2(0.0) 1(2.5) 1(2.5) 2(10) 0(5.0) 0(5.0) 0(5.0) 1(7.5) 1(2.5) 1(7.5) 1(2.5) 1(7.5) 0(5.0) 1(2.5) 1(7.5) 1(7.5) 1(2.5) 0(5.0) 0(5.0) 0(5.0) 0(5.0) 1(2.5)
Observed yield (%) Y
82.9 36.5 26.1 45.6 63.2 46.1 14.1 45.8 19.3 15.2 32.1 39.0 89.9 73.8 11.2 70.3 69.8 39.6 26.4 13.3 27.3 42.7 19.5 11.4 81.7 78.9 76.7 40.9 23.6
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3.2. Effect of synthesis parameters
Table 2 ANOVA of the response surface model and joint factor. Source
Degree of freedom
Linear Quadratic Interaction Total Model R2 = 0.95 Joint factor Time (x1) Temperature (x2) Enzyme amount (x3) Substrate molar ratio (x4) Added water (x5) a b
Sum of squares
Prob > Fa b
5 5 10 20
7145.12 7276.99 2277.86 16700.00
0.0010 0.0009b 0.1437 0.0027b
6 6 6 6 6
3970.87 7031.31 5947.73 1661.34 4767.92
0.0101b 0.0017b 0.0029b 0.1036 0.0059b
Prob > F = level of significance. Significant at 0.05 level.
3. Results and discussion 3.1. Model fitting The RSREG procedure of SAS software (SAS Institute, Cary, NC, USA) was employed to fit the second-order polynomial equation to the experimental conversions shown in Table 1. The second-order polynomial Eq. (1) is given below:
Y ¼ 1216:64449 þ 11:03731v1 þ 44:13050v2 þ 38:27679v3 þ 86:64257v4 þ 0:23889v5 0:082891v1 v2 þ 0:10039v1 v3 0:25586v1 v4 0:19984v1 v5 þ 0:74188v2 v3 0:083125v2 v4 þ 0:542752v2 v5 5:07187v3 v4 þ 1:79125v3 v5 0:87875v4 v5 0:092563v21 0:59446v22 6:89030v23 6:04905v24 1:86585x25
ð1Þ
The analysis of variance (ANOVA) results, as shown in Table 2, indicated that the second-order polynomial model was statistically significant and adequate to represent the actual relationship between the response (yield) and the significant variables, with a very small p-value (0.0027) and a satisfactory coefficient of determination (R2 = 0.95). The ANOVA results also showed that the linear and quadratic terms had a significant influence (p < 0.05), while the interaction term had less influence (p > 0.05). The overall effect of the five synthesis variables on the biodiesel yield was evaluated by a joint test. The results (Table 2) revealed that the reaction time (x1), temperature (x2), enzyme content (x3) and added water content (x5) were the important factors, exerting a statistically significant overall effect (p < 0.05) on the response, and that the substrate molar ratio (x4) had a less significant effect (p > 0.05) (See Fig. 1).
Yield (%)
100 80
(B)
(A)
Since the interaction term had less influence, the effect of each parameter on the biodiesel yield could be better understood by the predicted plots generated from Eq. (1) by keeping the other four parameters constant at their respective center levels. The effect of reaction time was investigated in the range of 8–40 h. As shown in Fig. 1(A), the biodiesel yield increased as the reaction time increased, and then gradually leveled off after 30 h. The yield gradually leveling off after 30 h was probably due to the synthesis product inhibiting the alcoholysis reaction. The effect of reaction temperature on the biodiesel yield is shown in Fig. 1(B). It can be seen that the yield increased with increases in temperature up to 40 °C and then rapidly decreased. This result indicated that the excessive heat energy inhibited the lipase activity, as lipase can be inactivated by thermal denaturation. It has been reported that for most lipases used for biodiesel synthesis, the optimal temperature is between 30 and 55 °C and is dependent on the operational stability of the lipase and the rate of the transesterification reaction (Lee et al., 2011; Wang et al., 2010). The relationship between the biodiesel yield and enzyme amount is shown in Fig. 1(C). The highest yield (87%) was obtained with four pieces of lipase-immobilized membrane. The biodiesel yield gradually leveled off or slightly decreased at higher enzyme amounts (over four pieces) because all the active sites could not be exposed to the substrates in the present of excess lipase in the reaction. In order to save costs, the amount of enzyme used should be minimized as much as possible, as an excess of enzyme does not effectively increase the yield. Fig. 1(D) shows the effect of the substrate molar ratio on biodiesel synthesis. An increase in biodiesel yield was observed with the increase of the MeOH:oil molar ratio from 3:1 to 5:1, and then the trend was reversed. The biodiesel yield at a seven substrate molar ratio was lower than that at five. This result indicated that more methanol was needed for the transesterification reaction to proceed at a reasonable rate; however, too much of an increase of methanol can inhibit enzyme activity and result in a decreased yield. Shimada et al. observed the inactivation of C. antarctica lipase by methanol and ethanol when the alcohol: oil molar ratio was higher than 3:1, which was overcome by using immobilized enzyme or added methanol stepwise (Shimada et al., 2002). The effect of added water content on biodiesel synthesis was investigated in the range of 0–10%, as shown in Fig. 1(E). Our result showed that the biodiesel yield was increased with water content between 0 and 5.5%. Studies have demonstrated that the addition of a small amount of water to a lipase-catalyzed reaction mixture increased catalytic efficiency and the rate of fatty acid ester synthesis (Kaieda et al., 2001; Tongboriboon et al., 2010). In an organic solvent, a small amount of water is essential to maintaining the specific three-dimensional structure of the enzyme. However, lipases usually catalyze hydrolysis in aqueous media, so excess
(E)
(D)
(C)
60 40 20 10
20 30 Time (h)
40
30
35
40 45 Temp (oC)
50
1
2 3 4 Enzyme amount (Pieces)
5
3
4 5 6 Substrate ratio (MeOH:Oil)
7
0
2
4 6 8 H2O (% wt. of oil)
10
Fig. 1. Relationship between the synthesis parameters: (A) reaction time; (B) temperature; (C) enzyme amount; (D) substrate molar ratio; and (E) added water content and biodiesel yield. Solid lines are model data, and symbols are experimental data.
Please cite this article in press as: Kuo, C.-H., et al. Lipase-immobilized biocatalytic membranes for biodiesel production. Bioresour. Technol. (2013), http:// dx.doi.org/10.1016/j.biortech.2012.12.054
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water may also stimulate the competing hydrolysis reaction and lead to a decrease in transesterification yields. In this study, the highest yield was found with a water content of 4–6%. 3.3. Attaining optimum conditions and reusability of immobilized lipase The optimum synthesis of enzymatic biodiesel was determined by the ridge max analysis, which showed that the maximum yield was 97.2% at 33 h, 40 °C, 4.3 pieces of lipase-immobilized membrane, 4:1 substrate molar ratio and 5.2% added water content. A verification experiment performed at the suggested optimum conditions obtained a yield of 95.3%, which was close to the predicted yield; thus, the predicted model was successfully developed. In this study, the lipase-catalyzed synthesis of biodiesel was carried out in a low-water organic environment. Lipase exposed in n-hexane and methanol for an extended period can cause an inactivation effect. In particular, methanol inhibition is generally observed in biodiesel production via lipase-catalyzed transesterification reactions (Shimada et al., 2002). To examine the enzyme reusability, the ability of immobilized lipase for biodiesel synthesis was investigated under optimum conditions. The immobilized lipase was recovered from the reaction medium after reaction and directly reused in the next batch. After the batch of immobilized lipase was reused five times, the biodiesel yields still remained at about 90%. This result showed that the immobilized lipase remained stable through a long-term hexane and methanol exposure. Thus, the result demonstrated that the lipase immobilized on PVDF membrane could be effectively applied for biodiesel synthesis and that the stability was high enough to permit reuse. Acknowledgements This research was supported by the National Science Council, Taiwan, ROC (Grants no. 101-2313-B-005-040-MY3) and was also supported in part by the Ministry of Education, Taiwan, ROC, under the ATU plan. References Chang, S.W., Shaw, J.F., Yang, K.H., Chang, S.F., Shieh, C.J., 2008. Studies of optimum conditions for covalent immobilization of Candida rugosa lipase on poly (cglutamic acid) by RSM. Bioresour. Technol. 99, 2800–2805.
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Please cite this article in press as: Kuo, C.-H., et al. Lipase-immobilized biocatalytic membranes for biodiesel production. Bioresour. Technol. (2013), http:// dx.doi.org/10.1016/j.biortech.2012.12.054