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Highly efficient transformation of waste oil to biodiesel by immobilized lipase from Penicillium expansum
Process Biochemistry 44 (2009) 685–688
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Highly efficient transformation of waste oil to biodiesel by immobilized lipase from Penicillium expansum Nan-Wei Li a,b, Min-Hua Zong a,*, Hong Wu a a b
Lab of Applied Biocatalysis, South China University of Technology, Guangzhou 510640, China College of Light Industry and Food, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 June 2008 Received in revised form 2 December 2008 Accepted 18 February 2009
An inexpensive self-made immobilized lipase from Penicillium expansum was shown to be an efficient biocatalyst for biodiesel production from waste oil with high acid value in organic solvent. It was revealed that water from the esterification of free fatty acids and methanol prohibited a high methyl ester yield. Adsorbents could effectively control the concentration of water in the reaction system, resulting in an improved methyl ester yield. Silica gel was proved to be the optimal adsorbent, affording a ME yield of 92.8% after 7 h. Moreover, the enzyme preparation displayed a higher stability in waste oil than in corn oil, with 68.4% of the original enzymatic activity retained after being reused for 10 batches. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Biodiesel Waste oil High acid value Immobilized lipase Penicillium expansum Adsorbent
1. Introduction Recently, the price of fossil oil has been soaring up and this important resource will eventually be exhausted if its large-scale use continues [1]. Biodiesel, defined as monoalkyl esters of long chain fatty acids, is one of the alternative fuels derived from renewable lipid feedstock, such as vegetable oils or animal fats [2]. It has attracted much attention due to its renewable nature, improved exhaust emissions, and biodegradability [3]. However, one of the major obstacles to its wide application is its high cost as compared to fossil diesel [4]. It has been reported that the cost of raw materials amounts to about 75% of the total biodiesel production cost [5]. Therefore, waste oil was used as the feedstock in the present work in order to reduce the cost of biodiesel production. Enzymatic approaches serve as a promising technology for biodiesel production due to the mild reaction conditions, easy recovery of product, being environmentally friendly and low demanding on raw materials compared with chemical methods [6–8]. However, the high cost of commercially available enzymes limits the enzymatic production of biodiesel on an industrial scale [8,9]. On the other hand, poor solubility of methanol in oil and adsorption of glycerol onto the lipase lead to the accumulation of
* Corresponding author. Fax: +86 20 22236669. E-mail address: [email protected] (M.-H. Zong). 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.02.012
methanol around the enzyme, resulting in the inactivation of the enzyme [10,11]. To lower the cost of the enzymatic process, a cheap immobilized enzyme capable of effectively catalyzing the transformation of refined corn oil to biodiesel was prepared by simple adsorption of the crude lipase from Penicillium expansum (PEL) on resin D4020 [12]. Herein, we extend our interest to the production of biodiesel from waste oil with high acid value by the immobilized PEL. It has been reported that water from enzymatic esterification of methanol with free fatty acids present in waste oil with high acid value could foster the hydrolysis of fatty acid methyl esters (ME), thus lowering the yield of ME [13]. For this reason, efforts have been made to control the water concentration in the reaction mixture by adding adsorbents. 2. Materials and methods 2.1. Materials Crude lipase from P. expansum (PEL, 6700 U/g of hydrolytic activity), whose expression was described in a previous report [14], was kindly donated by Shenzhen Leveking Bioengineering Co. Ltd., China. Resin D4020 was from the Chemical Co. of Nankai University, China. PEL was immobilized on resin D4020 according to previous method [12]. The immobilized lipase showed a transesterification activity of 400 U/g (1 unit corresponds to the amount of enzyme that produces 1 mmol methyl oleate from triolein and methanol in 1 min at 35 8C) and an esterification activity of 112 U/g (1 unit corresponds to the amount of enzyme producing 1 mmol methyl oleate from oleic acid and methanol in 1 min at 35 8C). Waste oil with an acid value of 54.3 mg KOH/g and a saponification number of 191 mg KOH/g was collected from the waste oil pool of a local restaurant and treated with activated clay to give a decoloration rate of 82.4% and a yield of 91.0%.
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Refined corn oil was from local market. Methyl palmitate, methyl stearate, methyl oleate, methyl linoleate, methyl linolenate and methyl heptadecanoate were of chromatographic purity and from Sigma, USA. Adsorbents were purchased from Shanghai Runjie Chemical Co. Ltd., China. All other chemicals were also obtained commercially and of analytical grade. 2.2. General procedure for enzymatic reaction In a typical experiment, the reaction was carried out in a 25 ml flask capped with a septum at 35 8C and 200 rpm. The reaction mixture contained 2 g waste oil with high acid value, 0.4 g t-amyl alcohol, 168 U immobilized PEL based on transesterification activity. Methanol was added by a three-step procedure and each one molar equivalent of methanol was added at the reaction time of 0, 4, and 10 h, respectively. For some specified reactions, adsorbent was also added to the reaction mixture to remove the by-product water. In order to assess the operational stability of immobilized PEL, the immobilized enzyme and the adsorbent were filtered after each batch reaction, followed by removing solvent from the enzyme and the adsorbent via airing at room temperature. The adsorbent was separated from the immobilized enzyme by sieving owing to its much bigger size and then was dehydrated at 180 8C for 2 h. After that, the adsorbent and the enzyme were added to the fresh substrate for the next batch. The relative activity of the enzyme was defined as the ratio of ME yield of each batch to the corresponding value of the first batch. Samples of 100 ml were withdrawn at predetermined time intervals and centrifuged (12,000 rpm, 10 min) and the upper layer was mixed with methyl heptadecanoate (as an internal standard) for gas chromatographic analysis.
Fig. 1. Effect of enzyme dosage on the reaction. The reactions were carried out at 35 8C and 200 rpm for 24 h. The reaction mixture contained 2 g waste oil, 0.8 g tamyl alcohol and different amounts of immobilized PEL. At reaction time of 0, 4, and 10 h, one molar equivalent of methanol was added. Symbol:36 U/g (^), 48 U/g (^), 60 U/g (~), 72 U/g (&), 84 U/g (*), 96 U/g (*), 108 U/g (&).
2.3. GC analysis The ME yield of the biotransformation was assayed with a GC2010 gas chromatography (Shimadzu Co., Kyoto) equipped with a HP-5 capillary column (0.53 mm 15 m, HP, USA). The column temperature was hold at 180 8C for 1 min, raised to 186 8C at 0.8 8C/min and kept at 186 8C for 1 min, then upgraded to 280 8C at the rate of 20 8C/min. Nitrogen was used as the carrier gas at 2 ml/min. Split ratio was 100:1 (v/v). The temperatures of the injector and the detector were 250 and 280 8C, respectively.
3. Results and discussion As reported recently, the self-made immobilized PEL displayed a high transesterification activity, although free PEL is unable to catalyze the transesterification of corn oil with methanol due to its inactivation by methanol [12]. Owing to the high cost of corn oil, immobilized PEL-catalyzed biodiesel production from waste oil with high acid value was explored for the first time. To convert waste oil to biodiesel efficiently, a catalyst with both high transesterification activity and high esterification activity is needed and immobilized PEL was expected to meet this requirement. A solvent system was adopted in this work because it has been demonstrated that immobilized PEL did not show any transesterification activity in a solvent-free system [12]. As predicted, the immobilized enzyme could catalyze the formation of methyl esters from waste oil, in spite of the lower ME yield (70.2%) than that with refined corn oil (85.0%). In order to get an indepth understanding of this observation and an improved result, the effects of some important variables on the transformation were examined.
biodiesel in our previous study [12] and so was used as the reaction medium in this work. Similar to a previous report [15], the concentration of t-amyl alcohol had a clear influence on ME yield. As can be seen in Fig. 2, when t-amyl alcohol concentration was below 0.2 g/g of oil, an increase of t-amyl alcohol concentration resulted in a dramatic increase in ME yield. This is because t-amyl alcohol could significantly improve the solubility of both methanol and by-product glycerol in the reaction mixture and so remarkably reduce or eliminate their inactivation effects on the enzyme. On the other hand, the viscosity of the reaction mixture could be substantially lowered by adding t-amyl alcohol, leading to a less mass transfer limitation. When the concentration of t-amyl alcohol was higher than 0.2 g/g of oil, however, ME yield decreased with increasing t-amyl alcohol concentration. The dilution of the substrates by excessive t-amyl alcohol in the system might contribute to this. 3.3. Effect of the reaction temperature Generally speaking, the reaction temperature has a significant effect on the activity and stability of a biocatalyst and the
3.1. Effect of enzyme dosage As shown in Fig. 1, within the range from 36 to 84 U/g of oil, higher enzyme dosage resulted in both the higher initial reaction rate and higher ME yield. Further rise in enzyme dosage brought a slight effect on the ME yield. The optimum enzyme dosage is higher than that with refined corn oil as feedstock (84 U/g of oil vs 60 U/g of oil) [12]. This could be accounted for by the low esterification activity of the enzyme as compared with its transesterification activity. 3.2. Effect of t-amyl alcohol concentration t-Amyl alcohol has been shown to be the optimal reaction medium for immobilized PEL-mediated conversion of corn oil to
Fig. 2. Effect of t-amyl alcohol concentration on the reaction. The reactions were carried out at 35 8C and 200 rpm for 24 h, and the reaction mixture contained 2 g waste oil with high acid value, 168 U immobilized PEL and different concentrations of t-amyl alcohol. At reaction time of 0, 4, and 10 h, one molar equivalent of methanol was added.
N.-W. Li et al. / Process Biochemistry 44 (2009) 685–688
Fig. 3. Effect of temperature on the reaction. The reactions were performed at 200 rpm and different temperatures for 24 h. The reaction mixture contained 2 g waste oil with high acid value, 0.4 g t-amyl alcohol and 168 U immobilized PEL. At reaction time of 0, 4, and 10 h, one molar equivalent of methanol was added.
thermodynamic equilibrium of a reaction as well. Hence, the effect of the temperature on the reaction was examined within the range from 25 to 55 8C (Fig. 3). As shown in Fig. 3, higher ME yield was achieved with the increment of the temperature when the temperature was below 35 8C. However, a slight drop in ME yield was observed with the rise in the temperature from 35 to 50 8C. Further increase in the temperature led to a drastic drop in ME yield, which is in agreement with our previous study [12]. It might stem from the inactivation of the lipase at a high temperature. Obviously, the optimal temperature of the reaction was 35 8C. Under the optimized conditions, a ME yield of 80.1%, which was slightly lower than that (85.0%) with refined corn oil as raw material, could be obtained after 24 h. As there exists a high content of free fatty acid (FFA) in waste oil with high acid value compared with corn oil and water from the esterification reaction of FFA and methanol prohibits a high methyl ester yield [13], efforts to improve the methyl ester yield by removing water from the reaction mixture were made. 3.4. Effect of adsorbents on the reaction As the FFA concentration in waste oil is quite high, a considerable amount of water would be produced during the esterification of fatty acids with methanol. Klibanov and coworkers reported that excess water not only resulted in the aggregation of the enzyme in hydrophobic media, thus reducing its catalytic activity, but also had a negative effect on enzyme’s stability [16]. Furthermore, the presence of water would promote the hydrolysis of the formed esters. Adsorbents could effectively control the water concentration during the reaction process, and thus improve the ME yield [17]. Therefore, several adsorbents, including 3, 4, 5 A´˚ molecular sieve and blue silica gel were tested and it was found that higher ME yield could be achieved with all the tested adsorbents as compared with the control (Fig. 4). Among the adsorbents examined, blue silica gel was regarded as the best one, affording a ME yield of 90.5%.
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Fig. 4. Effect of various adsorbents on the reaction. The reactions were carried out at 35 8C and 200 rpm for 24 h, and the reaction mixture contained 2 g waste oil with high acid value, 0.4 g t-amyl alcohol, 168 U immobilized PEL and 0.8 g different adsorbents. At reaction time of 0, 4, and 10 h, one molar equivalent of methanol was added.
increase in blue silica gel quantity, however, resulted in a lower ME yield. This could be attributed to the lower methanol concentration in the reaction system due to the adsorption of methanol onto blue silica gel [17]. Therefore, the optimal quantity of blue silica gel was thought to be 0.48 g/g. 3.6. Operational stability of PEL Fig. 6 depicts the time course of the conversion. In order to reduce the negative effect of methanol on the enzyme, a three-step methanolysis protocol was used. One molar equivalent of methanol was added at the beginning of the reaction (0 h) and the methanol conversion reached above 90% after reaction for 1 h (ME yield > 30%, theoretical value 33.3%). Further elongation of reaction time resulted in a slow increase in the methanol conversion and the ME yield. To shorten the reaction time and improve the catalytic efficiency of the lipase, another one molar equivalent of methanol was added at 1 h. Similarly, the other one molar equivalent of methanol was added at 3 h when methanol conversion reached about 90% after reaction for another 2 h (ME
3.5. Effect of adsorbent quantity on the reaction The effect of adsorbent quantity on the reaction was subsequently investigated. As can be seen in Fig. 5, the ME yield went up with increasing blue silica gel quantity when it was below 0.48 g/g of oil, due to the removal of water in the mixture by blue silica gel, pushing the reaction toward the synthesis of esters. Further
Fig. 5. Effect of the adsorbent quantity on the reaction. The reactions were carried out at 35 8C and 200 rpm for 24 h, and the reaction mixture contained 2 g waste oil with high acid value, 0.4 g t-amyl alcohol, 168 U immobilized PEL and different amounts of blue silica gel. At reaction time of 0, 4, and 10 h, one molar equivalent of methanol was added.
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could protect the enzyme molecules covered by them from deactivation [19]. 4. Conclusion Immobilized PEL, an inexpensive self-made lipase preparation, could catalyze the methanolysis of waste oil with high acid value for biodiesel production and the process could be greatly improved by using blue silica gel as adsorbent to achieve a ME yield of 92.8% after optimization. Moreover, waste oil with high acid value was more promising feedstock for biodiesel production than refined corn oil, because of its low cost and high ME yield. In addition, the enzyme preparation was more stable in waste oil than in corn oil. This research suggests the applicability of immobilized PEL to biodiesel production. Doubtlessly, more work has to be done to justify its industrial application in the future. Fig. 6. Time course of the reaction. The reaction was carried out at 35 8C and 200 rpm for 7 h, and the reaction mixture contained 2 g waste oil with high acid value, 0.4 g t-amyl alcohol, 0.96 g blue silica gel and 168 U immobilized PEL. At reaction time of 0, 1, and 3 h, one molar equivalent of methanol was added.
yield > 60%, theoretical value 66.7%). Hence, the reaction time was reduced to 7 h and the highest ME yield of 92.8% was achieved, which was much higher than the corresponding ME yield with refined corn oil (85.0%). One of the major advantages of immobilized enzyme is that it can be repeatedly used. To further examine the potential of immobilized PEL for biodiesel production from waste oil with high acid value, its operational stability was investigated. As shown in Fig. 7, immobilized PEL exhibited a fairly good operational stability and 68.4% of its original activity was retained after being repeatedly used for 10 batches, in contrast to 62.8% with corn oil [12]. The reason might be that FFA could improve the solubility of methanol in waste oil, thus lowering its concentration in the microenvironment of the lipase [18]. It is also worth-noting that the catalytic activity of immobilized PEL dropped sharply during the first batch. Nevertheless, no apparent loss in its catalytic activity was observed from the second to the tenth batch. This is quite similar to our previous observation [12,19] and consistent with other researchers’ reports [9,20]. There are several possible reasons for this. First, enzyme molecules not adsorbed firmly on the support was desorbed during the first batch as confirmed by our experiments. Second, the denatured enzyme molecules present in the outer layer of the enzyme preparation
Fig. 7. Operational stability of immobilized PEL. The reaction was performed at 35 8C and 200 rpm by adding one molar equivalent of methanol into the mixture containing 2 g waste oil, 0.4 g t-amyl alcohol, 0.96 g blue silica gel and 168 U immobilized PEL. Each one molar equivalent of methanol was added at 1 and 3 h, respectively. The three steps served as one batch with total reaction time being 7 h.
Acknowledgment This work was financially supported by the Science and Technology Project of Guangzhou city (Grant No. 2005Z3-D0471). References [1] Fukuda H, Kondo A, Noda H. Biodiesel fuel production by transesterification of oils. J Biosci Bioeng 2001;92:405–16. [2] van Kasteren JMN, Nisworo AP. A process model to estimate the cost of industrial scale biodiesel production from waste cooking oil by supercritical transesterification. Resour Conserv Recy 2007;50:442–58. [3] Hill J, Nelson E, Tilman D, Polasky S, Tiffany D. Environmental, economic, and energetic costs and benefits of biodiesel and ethanol biofuels. Proc Natl Acad Sci USA 2006;103:11206–10. [4] Zhang Y, Dub MA, McLean DD, Kates M. Biodiesel production from waste cooking oil: 2. Economic assessment and sensitivity analysis. Bioresour Technol 2003;90:229–40. [5] Xue FY, Miao JX, Zhang X, Luo H, Tan TW. Studies on lipid production by Rhodotorula glutinis fermentation using monosodium glutamate wastewater as culture medium. Bioresour Technol 2008;99:5923–7. [6] Nelson LA, Foglia TA, Marmer WN. Lipase-catalyzed production of biodiesel. J Am Oil Chem Soc 1996;73:1191–5. [7] Wu H, Zong MH, Lou WY. Transesterification of waste oil to biodiesel in solvent free system catalyzed by immobilized lipase. Chin J Catal 2004;25:903–8. [8] Ranganathan SV, Narasimhan SL, Muthukumar K. An overview of enzymatic production of biodiesel. Bioresour Technol 2008;99:3975–81. [9] 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 Enzyme 2001;16:53–8. [10] 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;25:194–200. [11] Shimada Y, Watanabe Y, Samukawa T, Sugihara A, Noda H, Fukuda H, et al. Conversion of vegetable oil to biodiesel using immobilized Candida antarctica lipase. J Am Oil Chem Soc 1999;76:789–93. [12] Li NW, Wu H, Zong MH, Lou WY. Immobilization of lipase from Penicillium expansum and its application to transesterification of corn oil. Chin J Catal 2007;28:333–8. [13] Kaieda M, Samukawa T, Kondo A, Fukuda H. 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 2001;91:12–5. [14] Yuan C, Lin L, Shi QQ, Wu SG. Overexpression of Penicillium expansum lipase gene in Pichia pastoris. Chin J Biotechnol 2003;19:231–5. [15] Li L, Du W, Liu D, Wang L, Li Z. Lipase-catalyzed transesterification of rapeseed oils for biodiesel production with a novel organic solvent as the reaction medium. J Mol Catal B Enzyme 2006;43:58–62. [16] Zaks A, Klibanov AM. The effect of water on enzyme action in organic media. J Biol Chem 1988;263:8017–21. [17] Wang L, Du W, Liu DH, Li LL, Dai NM. Lipase-catalyzed biodiesel production from soybean oil deodorizer distillate with absorbent present in tert-butanol system. J Mol Catal B Enzyme 2006;43:29–32. [18] Huang SY, Chang HL, Goto M. Preparation of surfactant-coated lipase for the esterification of geraniol and acetic acid in organic solvents. Enzyme Microb Technol 1998;22:552–7. [19] Wang Y, Wu H, Zong MH. Improvement of biodiesel production by lipozyme TL IM-catalyzed methanolysis using response surface methodology and acyl migration enhancer. Bioresour Technol 2008;99:7232–7. [20] Soumanou MM, Bornscheuer UT. Improvement in lipase-catalyzed synthesis of fatty acid methyl esters from sunflower oil. Enzyme Microb Technol 2003;33:97–103.
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Highly efficient transformation of waste oil to biodiesel by immobilized lipase from Penicillium expansum Nan-Wei Li a,b, Min-Hua Zong a,*, Hong Wu a a b
Lab of Applied Biocatalysis, South China University of Technology, Guangzhou 510640, China College of Light Industry and Food, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 June 2008 Received in revised form 2 December 2008 Accepted 18 February 2009
An inexpensive self-made immobilized lipase from Penicillium expansum was shown to be an efficient biocatalyst for biodiesel production from waste oil with high acid value in organic solvent. It was revealed that water from the esterification of free fatty acids and methanol prohibited a high methyl ester yield. Adsorbents could effectively control the concentration of water in the reaction system, resulting in an improved methyl ester yield. Silica gel was proved to be the optimal adsorbent, affording a ME yield of 92.8% after 7 h. Moreover, the enzyme preparation displayed a higher stability in waste oil than in corn oil, with 68.4% of the original enzymatic activity retained after being reused for 10 batches. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Biodiesel Waste oil High acid value Immobilized lipase Penicillium expansum Adsorbent
1. Introduction Recently, the price of fossil oil has been soaring up and this important resource will eventually be exhausted if its large-scale use continues [1]. Biodiesel, defined as monoalkyl esters of long chain fatty acids, is one of the alternative fuels derived from renewable lipid feedstock, such as vegetable oils or animal fats [2]. It has attracted much attention due to its renewable nature, improved exhaust emissions, and biodegradability [3]. However, one of the major obstacles to its wide application is its high cost as compared to fossil diesel [4]. It has been reported that the cost of raw materials amounts to about 75% of the total biodiesel production cost [5]. Therefore, waste oil was used as the feedstock in the present work in order to reduce the cost of biodiesel production. Enzymatic approaches serve as a promising technology for biodiesel production due to the mild reaction conditions, easy recovery of product, being environmentally friendly and low demanding on raw materials compared with chemical methods [6–8]. However, the high cost of commercially available enzymes limits the enzymatic production of biodiesel on an industrial scale [8,9]. On the other hand, poor solubility of methanol in oil and adsorption of glycerol onto the lipase lead to the accumulation of
* Corresponding author. Fax: +86 20 22236669. E-mail address: [email protected] (M.-H. Zong). 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.02.012
methanol around the enzyme, resulting in the inactivation of the enzyme [10,11]. To lower the cost of the enzymatic process, a cheap immobilized enzyme capable of effectively catalyzing the transformation of refined corn oil to biodiesel was prepared by simple adsorption of the crude lipase from Penicillium expansum (PEL) on resin D4020 [12]. Herein, we extend our interest to the production of biodiesel from waste oil with high acid value by the immobilized PEL. It has been reported that water from enzymatic esterification of methanol with free fatty acids present in waste oil with high acid value could foster the hydrolysis of fatty acid methyl esters (ME), thus lowering the yield of ME [13]. For this reason, efforts have been made to control the water concentration in the reaction mixture by adding adsorbents. 2. Materials and methods 2.1. Materials Crude lipase from P. expansum (PEL, 6700 U/g of hydrolytic activity), whose expression was described in a previous report [14], was kindly donated by Shenzhen Leveking Bioengineering Co. Ltd., China. Resin D4020 was from the Chemical Co. of Nankai University, China. PEL was immobilized on resin D4020 according to previous method [12]. The immobilized lipase showed a transesterification activity of 400 U/g (1 unit corresponds to the amount of enzyme that produces 1 mmol methyl oleate from triolein and methanol in 1 min at 35 8C) and an esterification activity of 112 U/g (1 unit corresponds to the amount of enzyme producing 1 mmol methyl oleate from oleic acid and methanol in 1 min at 35 8C). Waste oil with an acid value of 54.3 mg KOH/g and a saponification number of 191 mg KOH/g was collected from the waste oil pool of a local restaurant and treated with activated clay to give a decoloration rate of 82.4% and a yield of 91.0%.
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Refined corn oil was from local market. Methyl palmitate, methyl stearate, methyl oleate, methyl linoleate, methyl linolenate and methyl heptadecanoate were of chromatographic purity and from Sigma, USA. Adsorbents were purchased from Shanghai Runjie Chemical Co. Ltd., China. All other chemicals were also obtained commercially and of analytical grade. 2.2. General procedure for enzymatic reaction In a typical experiment, the reaction was carried out in a 25 ml flask capped with a septum at 35 8C and 200 rpm. The reaction mixture contained 2 g waste oil with high acid value, 0.4 g t-amyl alcohol, 168 U immobilized PEL based on transesterification activity. Methanol was added by a three-step procedure and each one molar equivalent of methanol was added at the reaction time of 0, 4, and 10 h, respectively. For some specified reactions, adsorbent was also added to the reaction mixture to remove the by-product water. In order to assess the operational stability of immobilized PEL, the immobilized enzyme and the adsorbent were filtered after each batch reaction, followed by removing solvent from the enzyme and the adsorbent via airing at room temperature. The adsorbent was separated from the immobilized enzyme by sieving owing to its much bigger size and then was dehydrated at 180 8C for 2 h. After that, the adsorbent and the enzyme were added to the fresh substrate for the next batch. The relative activity of the enzyme was defined as the ratio of ME yield of each batch to the corresponding value of the first batch. Samples of 100 ml were withdrawn at predetermined time intervals and centrifuged (12,000 rpm, 10 min) and the upper layer was mixed with methyl heptadecanoate (as an internal standard) for gas chromatographic analysis.
Fig. 1. Effect of enzyme dosage on the reaction. The reactions were carried out at 35 8C and 200 rpm for 24 h. The reaction mixture contained 2 g waste oil, 0.8 g tamyl alcohol and different amounts of immobilized PEL. At reaction time of 0, 4, and 10 h, one molar equivalent of methanol was added. Symbol:36 U/g (^), 48 U/g (^), 60 U/g (~), 72 U/g (&), 84 U/g (*), 96 U/g (*), 108 U/g (&).
2.3. GC analysis The ME yield of the biotransformation was assayed with a GC2010 gas chromatography (Shimadzu Co., Kyoto) equipped with a HP-5 capillary column (0.53 mm 15 m, HP, USA). The column temperature was hold at 180 8C for 1 min, raised to 186 8C at 0.8 8C/min and kept at 186 8C for 1 min, then upgraded to 280 8C at the rate of 20 8C/min. Nitrogen was used as the carrier gas at 2 ml/min. Split ratio was 100:1 (v/v). The temperatures of the injector and the detector were 250 and 280 8C, respectively.
3. Results and discussion As reported recently, the self-made immobilized PEL displayed a high transesterification activity, although free PEL is unable to catalyze the transesterification of corn oil with methanol due to its inactivation by methanol [12]. Owing to the high cost of corn oil, immobilized PEL-catalyzed biodiesel production from waste oil with high acid value was explored for the first time. To convert waste oil to biodiesel efficiently, a catalyst with both high transesterification activity and high esterification activity is needed and immobilized PEL was expected to meet this requirement. A solvent system was adopted in this work because it has been demonstrated that immobilized PEL did not show any transesterification activity in a solvent-free system [12]. As predicted, the immobilized enzyme could catalyze the formation of methyl esters from waste oil, in spite of the lower ME yield (70.2%) than that with refined corn oil (85.0%). In order to get an indepth understanding of this observation and an improved result, the effects of some important variables on the transformation were examined.
biodiesel in our previous study [12] and so was used as the reaction medium in this work. Similar to a previous report [15], the concentration of t-amyl alcohol had a clear influence on ME yield. As can be seen in Fig. 2, when t-amyl alcohol concentration was below 0.2 g/g of oil, an increase of t-amyl alcohol concentration resulted in a dramatic increase in ME yield. This is because t-amyl alcohol could significantly improve the solubility of both methanol and by-product glycerol in the reaction mixture and so remarkably reduce or eliminate their inactivation effects on the enzyme. On the other hand, the viscosity of the reaction mixture could be substantially lowered by adding t-amyl alcohol, leading to a less mass transfer limitation. When the concentration of t-amyl alcohol was higher than 0.2 g/g of oil, however, ME yield decreased with increasing t-amyl alcohol concentration. The dilution of the substrates by excessive t-amyl alcohol in the system might contribute to this. 3.3. Effect of the reaction temperature Generally speaking, the reaction temperature has a significant effect on the activity and stability of a biocatalyst and the
3.1. Effect of enzyme dosage As shown in Fig. 1, within the range from 36 to 84 U/g of oil, higher enzyme dosage resulted in both the higher initial reaction rate and higher ME yield. Further rise in enzyme dosage brought a slight effect on the ME yield. The optimum enzyme dosage is higher than that with refined corn oil as feedstock (84 U/g of oil vs 60 U/g of oil) [12]. This could be accounted for by the low esterification activity of the enzyme as compared with its transesterification activity. 3.2. Effect of t-amyl alcohol concentration t-Amyl alcohol has been shown to be the optimal reaction medium for immobilized PEL-mediated conversion of corn oil to
Fig. 2. Effect of t-amyl alcohol concentration on the reaction. The reactions were carried out at 35 8C and 200 rpm for 24 h, and the reaction mixture contained 2 g waste oil with high acid value, 168 U immobilized PEL and different concentrations of t-amyl alcohol. At reaction time of 0, 4, and 10 h, one molar equivalent of methanol was added.
N.-W. Li et al. / Process Biochemistry 44 (2009) 685–688
Fig. 3. Effect of temperature on the reaction. The reactions were performed at 200 rpm and different temperatures for 24 h. The reaction mixture contained 2 g waste oil with high acid value, 0.4 g t-amyl alcohol and 168 U immobilized PEL. At reaction time of 0, 4, and 10 h, one molar equivalent of methanol was added.
thermodynamic equilibrium of a reaction as well. Hence, the effect of the temperature on the reaction was examined within the range from 25 to 55 8C (Fig. 3). As shown in Fig. 3, higher ME yield was achieved with the increment of the temperature when the temperature was below 35 8C. However, a slight drop in ME yield was observed with the rise in the temperature from 35 to 50 8C. Further increase in the temperature led to a drastic drop in ME yield, which is in agreement with our previous study [12]. It might stem from the inactivation of the lipase at a high temperature. Obviously, the optimal temperature of the reaction was 35 8C. Under the optimized conditions, a ME yield of 80.1%, which was slightly lower than that (85.0%) with refined corn oil as raw material, could be obtained after 24 h. As there exists a high content of free fatty acid (FFA) in waste oil with high acid value compared with corn oil and water from the esterification reaction of FFA and methanol prohibits a high methyl ester yield [13], efforts to improve the methyl ester yield by removing water from the reaction mixture were made. 3.4. Effect of adsorbents on the reaction As the FFA concentration in waste oil is quite high, a considerable amount of water would be produced during the esterification of fatty acids with methanol. Klibanov and coworkers reported that excess water not only resulted in the aggregation of the enzyme in hydrophobic media, thus reducing its catalytic activity, but also had a negative effect on enzyme’s stability [16]. Furthermore, the presence of water would promote the hydrolysis of the formed esters. Adsorbents could effectively control the water concentration during the reaction process, and thus improve the ME yield [17]. Therefore, several adsorbents, including 3, 4, 5 A´˚ molecular sieve and blue silica gel were tested and it was found that higher ME yield could be achieved with all the tested adsorbents as compared with the control (Fig. 4). Among the adsorbents examined, blue silica gel was regarded as the best one, affording a ME yield of 90.5%.
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Fig. 4. Effect of various adsorbents on the reaction. The reactions were carried out at 35 8C and 200 rpm for 24 h, and the reaction mixture contained 2 g waste oil with high acid value, 0.4 g t-amyl alcohol, 168 U immobilized PEL and 0.8 g different adsorbents. At reaction time of 0, 4, and 10 h, one molar equivalent of methanol was added.
increase in blue silica gel quantity, however, resulted in a lower ME yield. This could be attributed to the lower methanol concentration in the reaction system due to the adsorption of methanol onto blue silica gel [17]. Therefore, the optimal quantity of blue silica gel was thought to be 0.48 g/g. 3.6. Operational stability of PEL Fig. 6 depicts the time course of the conversion. In order to reduce the negative effect of methanol on the enzyme, a three-step methanolysis protocol was used. One molar equivalent of methanol was added at the beginning of the reaction (0 h) and the methanol conversion reached above 90% after reaction for 1 h (ME yield > 30%, theoretical value 33.3%). Further elongation of reaction time resulted in a slow increase in the methanol conversion and the ME yield. To shorten the reaction time and improve the catalytic efficiency of the lipase, another one molar equivalent of methanol was added at 1 h. Similarly, the other one molar equivalent of methanol was added at 3 h when methanol conversion reached about 90% after reaction for another 2 h (ME
3.5. Effect of adsorbent quantity on the reaction The effect of adsorbent quantity on the reaction was subsequently investigated. As can be seen in Fig. 5, the ME yield went up with increasing blue silica gel quantity when it was below 0.48 g/g of oil, due to the removal of water in the mixture by blue silica gel, pushing the reaction toward the synthesis of esters. Further
Fig. 5. Effect of the adsorbent quantity on the reaction. The reactions were carried out at 35 8C and 200 rpm for 24 h, and the reaction mixture contained 2 g waste oil with high acid value, 0.4 g t-amyl alcohol, 168 U immobilized PEL and different amounts of blue silica gel. At reaction time of 0, 4, and 10 h, one molar equivalent of methanol was added.
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could protect the enzyme molecules covered by them from deactivation [19]. 4. Conclusion Immobilized PEL, an inexpensive self-made lipase preparation, could catalyze the methanolysis of waste oil with high acid value for biodiesel production and the process could be greatly improved by using blue silica gel as adsorbent to achieve a ME yield of 92.8% after optimization. Moreover, waste oil with high acid value was more promising feedstock for biodiesel production than refined corn oil, because of its low cost and high ME yield. In addition, the enzyme preparation was more stable in waste oil than in corn oil. This research suggests the applicability of immobilized PEL to biodiesel production. Doubtlessly, more work has to be done to justify its industrial application in the future. Fig. 6. Time course of the reaction. The reaction was carried out at 35 8C and 200 rpm for 7 h, and the reaction mixture contained 2 g waste oil with high acid value, 0.4 g t-amyl alcohol, 0.96 g blue silica gel and 168 U immobilized PEL. At reaction time of 0, 1, and 3 h, one molar equivalent of methanol was added.
yield > 60%, theoretical value 66.7%). Hence, the reaction time was reduced to 7 h and the highest ME yield of 92.8% was achieved, which was much higher than the corresponding ME yield with refined corn oil (85.0%). One of the major advantages of immobilized enzyme is that it can be repeatedly used. To further examine the potential of immobilized PEL for biodiesel production from waste oil with high acid value, its operational stability was investigated. As shown in Fig. 7, immobilized PEL exhibited a fairly good operational stability and 68.4% of its original activity was retained after being repeatedly used for 10 batches, in contrast to 62.8% with corn oil [12]. The reason might be that FFA could improve the solubility of methanol in waste oil, thus lowering its concentration in the microenvironment of the lipase [18]. It is also worth-noting that the catalytic activity of immobilized PEL dropped sharply during the first batch. Nevertheless, no apparent loss in its catalytic activity was observed from the second to the tenth batch. This is quite similar to our previous observation [12,19] and consistent with other researchers’ reports [9,20]. There are several possible reasons for this. First, enzyme molecules not adsorbed firmly on the support was desorbed during the first batch as confirmed by our experiments. Second, the denatured enzyme molecules present in the outer layer of the enzyme preparation
Fig. 7. Operational stability of immobilized PEL. The reaction was performed at 35 8C and 200 rpm by adding one molar equivalent of methanol into the mixture containing 2 g waste oil, 0.4 g t-amyl alcohol, 0.96 g blue silica gel and 168 U immobilized PEL. Each one molar equivalent of methanol was added at 1 and 3 h, respectively. The three steps served as one batch with total reaction time being 7 h.
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