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Biodiesel Preparation by Lipase-Catalyzed Transesterification of Jatropha Oil Shweta Shah, Shweta Sharma, and M. N. Gupta* Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India Received April 4, 2003. Revised Manuscript Received October 22, 2003
Alkyl esters of long chain fatty acid are called biodiesel. These esters can be obtained from vegetable oils by transesterification with methanol/ethanol. The transesterification can be carried out chemically or enzymatically. In the present work three different lipases (Chromobacterium viscosum, Candida rugosa, and Porcine pancreas) were screened for a transesterification reaction of Jatropha oil in a solvent-free system to produce biodiesel; only lipase from Chromobacterium viscosum was found to give appreciable yield. Immobilization of lipase (Chromobacterium viscosum) on Celite-545 enhanced the biodiesel yield to 71% from 62% yield obtained by using free tuned enzyme preparation with a process time of 8 h at 40 °C. Further addition of water to the free (1%, w v-1) and immobilized (0.5%, w v-1) enzyme preparations enhanced the yields to 73 and 92%, respectively. Immobilized Chromobacterium viscosum lipase can be used for ethanolysis of oil. It was seen that immobilization of lipases and optimization of transesterification conditions resulted in adequate yield of biodiesel in the case of the enzyme-based process.
Introduction The limited (and fast diminishing) resources of fossil fuels, increasing prices of crude oil, and environmental concerns have been the diverse reasons for exploring the use of vegetable oils as alternative fuels. However, their direct use has not been satisfactory because of their viscous nature and low ignition quality. However, methyl/ethyl esters of fatty acids present in such oils have proved promising enough to be called biodiesel.1 Derived from renewable sources, they can be used without any modification in engine design. Also, they produce much lower levels of most of the pollutants and “potential or probable carcinogens”.1 Biodiesel has been produced from a variety of vegetable sources such as soybean,2 sunflower,3,4 cottonseed,5 rapeseed,6 and palm oil.7 Jatropha (Euphorbiaceae) is a genus comprising 70 species growing in tropical and subtropical countries. Nine species are reported to occur in India. The seed kernel contains 40-60% (w w-1) oil.8 Saturated fatty acids constitute 20% of this, whereas those remaining are unsaturated ones. Oleic acid is the most abundant (44.8%) followed by linoleic acid (34%), palmitic acid * Author to whom correspondence should be addressed. Tel.: 9111-2659 1503; 91-11-2659 6568. Fax: 91-11-2658 1073. E-mail:
[email protected]. (1) Ma, F.; Hanna, M. A. Bioresour. Technol. 1999, 70, 1. (2) Kaieda, M.; Samukawa, T.; Kondo, A.; Fukuda, H. 2001, 91, 12. (3) Mittelbach, M. J. Am. Oil Chem. Soc. 1990, 67, 168. (4) Antolin, G.; Tinaut, F. V.; Briceno, Y.; Castano, V.; Perez, C.; Ramirez, A. I. Bioresour. Technol. 2002, 83, 111. (5) O ¨ znur, K.; Tuten, M.; Aksoy, H. A. Bioresour. Technol. 2002, 83, 125. (6) Linko, Y. Y.; La¨msa¨, M.; Wu, X.; Vosukainen, W.; Sappa¨la¨, J.; Linko, P. J. Biotechnol. 1998, 66, 41. (7) Crabbe, E.; Nolasco-Hipolito, C.; Kobayashi, G.; Sonomoto, K.; Ishizaki, A. Process Biochem. 2001, 37, 65. (8) Makkar, H. P. S.; Becker, K.; Sporen, F.; Wink, M. J. Agric. Food Chem. 1997, 45, 3152.
(12.8%), and stearic acid (7.3%). While composition of the oil is similar to other oils, which are used for edible purposes, the presence of some antinutritional factors such as toxic phorbol esters renders this oil unsuitable for use in cooking.9 Thus, it is a good choice as the starting oil for production of biodiesel. In fact, the seed oil of Jatropha was used as a diesel fuel substitute during World War II. Later, its blends with diesel fuel were tested.10 It is interesting to note that Jatropha seeds themselves are reported to contain lipase activity which could also catalyze transesterification reactions.11 The possibility of using enzymes in organic solvents has opened up several exciting avenues for biotransformations.12-14 One frequently used strategy has been the use of lipase for transesterification reactions. The transesterification reaction, which is employed for biodiesel preparation, is shown in Scheme 1. Two approaches for transesterification of vegetable oils for production of biodiesel are suggested.15 The first is a chemical one in which alcoholysis of oil by methyl or ethyl alcohol in the presence of a strong acid or base produces biodiesel and glycerol.16 The base-catalyzed transesterification is much faster and less corrosive than the acid-catalyzed reaction. Thus alkali hydroxides are the most commonly used catalysts. However, if the (9) Gubitz, G. M.; Mittelbach, M.; Trabi, M. Bioresour. Technol. 1998, 67, 73. (10) Pramanik, K. Renewable Energy 2003, 28, 240. (11) Staubmann, R.; Ncube, I.; Gubitz, G. M.; Steiner, W.; Read, J. S. J. Biotechnol. 1999, 75, 117. (12) Clapes, P.; Torres, J. L.; Adlercreutz, P. Bioorg. Med. Chem. 1995, 3, 245. (13) Carrea, G.; Riva, S. Angew. Chem., Int. Ed. 2000, 39, 2226. (14) Gupta, M. N. In Methods in Nonaqueous Enzymology; Gupta, M. N., Ed.; Birkhauser Verlag: Basel, 2000; p 1. (15) Haas, M. J.; Piazza, G. J.; Foglia, T. A. Lipid Biotechnol. 2002, 587. (16) Fukuda, H.; Kondo, A.; Noda, H. J. Biosci. Bioeng. 2001, 92, 405.
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Scheme 1. Transesterification Reaction of Triglyceride
feedstock has a high free fatty acid (FFA) content (as is common with rendered fats and spent restaurant oils), excess of alkali causes loss of the free fatty acids as their insoluble soaps. This decreases the final yield of ester and consumes alkali. As an alternative, in these cases, one can conduct an acid-catalyzed reaction that requires higher reaction temperatures (100 °C) and longer reaction times than alkali-catalyzed transesterification. Foidl et al.17 have evaluated J. curcas as a source for the production of biofuel in Nicaragua using base- and acid-catalyzed transesterification. The second approach is the enzymatic one, in which lipase-catalyzed transesterification is carried out in nonaqueous environments. Chemical transesterification is efficient in terms of reaction time; however, the chemical approach to synthesize biodiesel from triglyceride has drawbacks, such as difficulty in the recovery of glycerol and the energy-intensive nature of the process. In contrast, biocatalysts allow synthesis of specific alkyl esters, easy recovery of glycerol, and transesterification of glycerides with high free fatty acid content.18 Much work has been done on the lipasecatalyzed transesterification of triglyceride.2,5,18-22 One common drawback with the use of enzyme-based processes is the high cost of the enzyme. Immobilization of enzymes has generally been used to obtain reusable enzyme derivatives.23 This enables recycling of the biocatalyst and hence lowers the cost. In the case of biocatalysts in nonaqueous media, immobilization is also reported to result in better activity. Thus, many transesterification processes employing lipases have used an immobilized form of the enzyme.21-25 In the present work, three different lipases in both free and immobilized (on Celite) forms were screened for transesterification activity and the most promising lipase from Chromobacterium viscosum was used for conversion of Jatropha oil into biodiesel. (17) Foidl, N.; Foidl, G.; Sanchez, M.; Mittelbach, M.; Hackel, S. Bioresour. Technol. 1996, 58, 77. (18) Nelson, L. A.; Fogolia, T. A.; Marmer, W. N. J. Am. Oil Chem. Soc. 1996, 73, 1191. (19) Wu, W. H.; Fogolia, T. A.; Marmer, W. N.; Phillips, J. G. J. Am. Oil Chem. Soc. 1999, 76, 517. (20) Abigor, R. D.; Uadia, P. O.; Fogolia, T. A.; Haas, M. J.; Jones, K. C.; Okpefa, F.; Obibuzor, J. U.; Bafor, M. E. Biochem. Soc. Trans. 2000, 28, 979. (21) Belafi-Bako, K.; Kovacs, F.; Gubicza, L.; Hancsok, J. Biocat. Biotransf. 2002, 20, 437. (22) Shieh, C. J.; Liao, H. F.; Lee, C. C. Bioresour. Technol. 2003, 88, 103. (23) Hsu, A. F.; Jones, K.; Fogolia, T. A.; Marmer, W. N. Biotechnol. Appl. Biochem. 2002, 36, 181. (24) Iso, M.; Chen, B. X,; Eguchi, M.; Kudo, T.; Shreestha, S. J. Mol. Catal. B Enzymatic 2001, 16, 53. (25) Shimada, Y.; Watanabe, Y.; Samukawa, T.; Sugihara, A.; Noda, H.; Fukuda, H.; Tominaga, Y. J. Am. Oil Chem. Soc. 1999, 76, 789.
Figure 1. Effect of the source of lipase on the production of biodiesel at 40 °C. Each of the above reactions was carried out in duplicate and the yields between duplicates were found to agree within 3%.
Materials Jatropha oil was obtained from Dr. Jayaveera, Jawaharlal Nehru Technological University Oil Technological Research Institute, Anatapur, India. Oil was obtained from Jatropha seed by mechanical pressing and was used as such without any pretreatment or analysis. Celite-545 was obtained from Central Drug House, Mumbai, India. Candida rugosa lipase was purchased from Sigma Chemical Co. (St. Louis, USA). Porcine pancreatic lipase was procured from Sisco Research Laboratories, Mumbai, India. Lipase from a microbial source (Chromobacterium viscosum) was purchased from Asahi Chemical Industry Co., Tokyo, Japan. Ethanol and KOH used were of analytical grade (E. Merck, Mumbai, India). All other chemicals and solvents used were of analytical grade. All solvents were used after having been dried overnight with 3 Å molecular sieves (E. Merck, Mumbai, India). Methods Biodiesel Production. Enzyme Preparation. (1) Tuned Enzyme Preparations. Candida rugosa lipase (50 mg) was dissolved in 0.5 mL of 20 mM sodium phosphate buffer, pH 7.3. Chromobacterium viscosum lipase (50 mg) was dissolved in 0.5 mL of 20 mM sodium phosphate buffer, pH 7.8. Porcine pancreatic lipase (100 mg) was dissolved in 0.5 mL 20 mM sodium phosphate buffer, pH 8.0. All enzyme preparations were immediately frozen at -20 °C and lyophilized for 48 h. These are referred to as “tuned” enzyme. (2) Immobilized Enzyme Preparations. Candida rugosa lipase (50 mg) was dissolved in 0.5 mL of 20 mM sodium phosphate buffer pH 7.3 and mixed with 125 mg of Celite. Chromobacterium viscosum powder (50 mg) was dissolved in 0.5 mL of 20 mM sodium phosphate buffer pH 7.8 and mixed with 125 mg of Celite. Porcine pancreatic lipase (100 mg) was dissolved in 0.5 mL of 20 mM sodium phosphate buffer pH 8.0 and mixed with 125 mg of Celite. All enzyme preparations were immediately frozen at -20 °C and lyophilized for 48 h. Transesterification. (1) Chemical Transesterification. The chemical transesterification was carried out in a
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Figure 2. TLC analysis of the ethyl esters. TLC analysis of the reaction mixture during the ethanolysis reaction using tuned Chromobacterium viscosum. Lanes 1-3: reaction mixtures after 24, 8, and 4 h, respectively. JO: Jatropha oil.
round-bottom flask (fitted with a reflux condenser) carrying Jatropha oil (1 g), excess ethanol, and a catalytic amount of KOH. The contents were refluxed for 1 h at 70 °C followed by addition of 10 mL of distilled water and 3-4 drops of concentrated sulfuric acid. The ethyl esters of the oil were extracted with chloroform. The chloroform was then removed by evaporation.26 The product (ethyl esters) was dried over anhydrous Na2SO4. Formation of ethyl esters from Jatropha oil was analyzed by carrying out thin-layer chromatography (TLC) and gas chromatography (GC). (2) Enzyme-Catalyzed Transesterification. Jatropha seed oil (0.5 g) and ethanol were taken in the ratio of 1:4 (mol mol-1) in a screw-capped vial. To this mixture, 50 mg of enzyme preparation (tuned or immobilized) was added and incubated at 40 °C with constant shaking at 200 rpm. The progress of the reaction was monitored by removing aliquots (20 µL) at various time intervals. The aliquots were appropriately diluted (with hexane), and to the diluted aliquots lauric acid was added as an internal standard before analysis by gaschromatography and thin-layer chromatography. (3) Effect of Varied Amount of Buffer on Ethanolysis Reaction. Jatropha seed oil (0.5 g) and ethanol were (26) Kandpal, J. P.; Madan, M. Renew. Ener. 1995, 6, 159.
taken in the ratio of 1:4 (mol mol-1) in a screw-capped vial. The tuned enzymes were prepared by adding varying amount of buffer (20 mM sodium phosphate buffer, pH 7.8) viz., 0.2, 0.5, 1.0, 2.0, and 5.0 mL, to Chromobacterium viscosum lipase (50 mg) as discussed in tuned enzyme preparation. To the reaction mixture, these enzyme preparations were added and incubated at 40 °C with constant shaking at 200 rpm for 8 h. The aliquots were appropriately diluted (with hexane) and to the diluted aliquots lauric acid was added as an internal standard before analysis by gas-chromatography and thin-layer chromatography. (4) Effect of Varied Amount of Enzyme on Ethanolysis Reaction. Jatropha seed oil (0.5 g) and ethanol were taken in the ratio of 1:4 (mol mol-1) in a screw capped vial. To this mixture, varied amounts of separately lyophilized enzymes, viz. 10, 50, 75 and 100 mg, were added. The reaction mixture was incubated at 40 °C with constant shaking at 200 rpm for 8 h. The aliquots were appropriately diluted (with hexane) and to the diluted aliquots lauric acid was added as an internal standard before analysis by gas-chromatography and thin-layer chromatography. Each of the above reactions was carried out in duplicate, and the yields between duplicates were found to agree within 3%.
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Analysis of Esters. Gas-Chromatography Analysis.27 The formation of ethyl esters of Jatropha oil was analyzed on a Nucon-5700 gas chromatograph with a flame-ionization detector. The capillary column (70% phenyl polysilphenylenesiloxane) used had a length of 30 m with an internal diameter of 0.25 mm. Nitrogen was used as the carrier gas at a constant flow rate of 4 kg cm-2. The column oven temperature was programmed from 150 to 250 °C (at the rate of 10 °C min-1) with injector and detector temperatures at 240 and 250 °C, respectively. Thin-Layer Chromatography. The formation of ethyl esters of Jatropha oil in the reaction mixture was also analyzed by thin-layer chromatography with silica gel 60 F254 (E. Merck, Mumbai, India). The solvent system consisted of hexane/ethyl acetate/acetic acid in the ratio of 90:10:1 (v v-1).28 The spots were detected in the iodine chamber. Results and Discussion Transesterification with pH-Tuned Lipases. Choice of the Source. The lipases from different sources (which were commercially obtained) were screened for transesterification. All the lipases were pH tuned before being used. Figure 1 shows that out of three different lipases from Chromobacterium viscosum, Candida rugosa, and porcine pancreas, only the lipase from Chromobacterium viscosum was capable of transesterification. With this enzyme, a yield of 62% for the ethyl ester could be obtained after 8 h. The reaction could be qualitatively followed by TLC (Figure 2) and quantitatively by GC (Figure 3). The formation of biodiesel did not increase beyond 8 h, which is likely to be due to inactivation of the lipase by substrate ethyl alcohol. The inactivation of lipases during transesterification reactions has also been observed by others.21 On the basis of these results, Chromobacterium lipase was selected for further study. Effect of Amount of Buffer Salt on Ethyl Ester Formation. It has also been reported that the amount of buffer salts (apart from buffer pH) present during lyophilization (while tuning the enzyme) also affects the reaction rates. Figure 4 shows that this optimization was useful and maximum yield was obtained when the amount of buffer salt was 0.2 mmol/g of enzyme. Effect of Varying Amount of Enzyme. Figure 5 shows the effect of varying amount of enzyme under these optimized conditions. The best results were obtained with 50 mg or 75 mg of enzyme. The higher amount of enzyme (i.e., 100 mg) in fact decreased the product yield, this was presumably due to increase in the viscosity (the solution was found to be quite viscous in this case) which reduced the reaction rate to the extent that an additional amount of enzyme did not help. As there was not much difference between the yield obtained with 50 mg and 75 mg, 50 mg of enzyme was used further in order to save on the cost of the enzyme. Transesterification with Immobilized Enzyme. In biotransformation involving use of lipases in nonaqueous media, the enzyme has frequently been ad(27) Ban, K.; Kaieda, M.; Matsumoto, T.; Kondo, A.; Fukuda, H. Biochem. Eng. J. 2001, 8, 39. (28) Samukawa, T.; Kaieda, M.; Matsumoto, T.; Ban, K.; Kondo, A.; Shimada, Y.; Noda, H.; Fukuda, H. J. Biosci. Bioeng. 2000, 90, 180.
Figure 3. Fatty acid composition of the Jatropha oil ester using gas chromatography. Peak 1: n-hexane, Peak 2: methyl laurate (internal standard), Peaks 3-6 correspond to ethyl esters of palmitic, stearic, oleic, and linoleic acid, respectively.
Figure 4. Effect of the amount of the buffer salt on biodiesel synthesis. Chromobacterium viscosum lipase (50 mg) was lyophilized in 20 mM sodium phosphate buffer (0.2, 0.5, 1.0, 2.0, 5.0 mL), pH 7.8. The separately lyophilized enzyme powders were added to reactions media containing Jatropha oil and ethanol in the molar ratio of 1:4. Each of the above reactions was carried out in duplicate, and the yields between duplicates were found to agree within 3%.
sorbed on Celite.29 Table 1 shows that the percent yield increased to 71% when immobilized enzyme preparation from Chromobacterium viscosum was used for transesterification. Adding a larger amount of immobilized enzyme was not practical since the matrix and enzyme together made the solution extremely viscous. The immobilized preparations with other lipases (Candida rugosa and Porcine pancreas) did show some detectable ethyl ester formation but the yield was extremely poor. Table 1 summarizes this result. The chemical process carried out for comparative purpose yielded 93% ethyl (29) Mustranta, A.; Forssell, P.; Poutanen, K. Enzyme Microb. Technol. 1993, 15, 133.
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Figure 5. Effect of amount of enzyme addition on biodiesel synthesis. Varied amounts of Chromobacterium viscosum lipase (10, 50, 75, 100 mg) were lyophilized in 20 mM sodium phosphate buffer (0.5 mL), pH 7.8. The separately lyophilized enzyme powders were added to reaction media containing Jatropha oil and ethanol in the molar ratio of 1:4. Each of the above reactions was carried out in duplicate and the yields between duplicates were found to agree within 3%.
Figure 6. Effect of incubation time on biodiesel production using immobilized Chromobacterium viscosum lipase. Each of the above reactions was carried out in duplicate and the yields between duplicates were found to agree within 3%.
Table 1. Optimization of the Biodiesel Preparation by an Enzymatic Route with a Process Time of 8 ha total ethyl ester yield (% w w-1) chemically enzymatically free tuned Chromobacterium free tuned Candida rugosa free tuned Porcine pancreas immobilized Chromobacterium immobilized Candida rugosa immobilized Porcine pancreas
93 62 0 0 71 1.0 1.1
a Each of the reactions was carried out in duplicate, and the yields in duplicates were found to agree within 3%.
esters. In general, the reported yield with chemical transesterification is between 90 and 95%.7,30 Foidl et al. have reported an yield of 88.4% by chemical transesterification of Jatropha oil.17 Figure 6 shows the rate obtained with immobilized lipase from Chromobacterium viscosum under the optimized conditions. Thus, a yield of 89% could be obtained in 10 h using the immobilized enzyme preparation. Biodiesel production using lipases has been tried both in nonaqueous solvent18 and in a solvent-free system.5 Methanolysis of tallow oil, using Mucor mehei lipase in hexane, had led to 77.8% ester yield.18 Lately, a solvent-free transesterification reaction is favored by many workers, since it is more economical.2,21 O ¨ znur et al.5 reported alcoholysis of cotton seed oil in a solvent-free medium, using immobilized Candida antarctica lipase with 92% of the total ester yield. A more extensive list of such efforts has been summarized by us elsewhere recently.31 The yield obtained by us by following a solvent-free approach is comparable to the yield obtained by other workers with different approaches.5,31 Effect of Percent Water in Reaction Media on Biodiesel Production. Amount of water present in the (30) Freedman, E.; Pryde, E. H.; Mounts, T. L. J. Am. Oil Chem. Soc. 1984, 61, 1638. (31) Shah, S.; Sharma, S.; Gupta, M. N. Indian J. Biochem. Biophys., in press.
Figure 7. Effect of percent water (added to the reaction medium) on biodiesel production using tuned and immobilized Chromobacterium viscosum lipases. Each of the above reactions was carried out in duplicate and the yields between duplicates were found to agree within 3%.
media is another critical parameter, which is known to influence biotransformations in nonaqueous media.32 The general picture available is that less than a monolayer of water is required for an enzyme to show biological activity. As the water level increases, it increases the enzyme flexibility and the expressed activity.33 After an optimum level of water, hydrolytic reactions become significant and transesterification yield is expected to go down. Figure 7 shows the effect of different amounts of water present in the solventfree system on biodiesel yield. Addition of 1% (w v-1) water in the case of free enzyme and 0.5% (w v-1) water in the case of immobilized Chromobacterium viscosum lipase gave the maximum yields of 73 and 92%, respectively. Iso et al.24 have also reported that water content is an important parameter for lipase-catalyzed transesterification reaction. Finally, these results show the viability of using lipases in a solvent-free system for obtaining biodiesel (32) Chowdary, G. V.; Prapulla, S. G. Process Biochem. 2002, 38, 393. (33) Triantafyllou, A. O.; Wehtje, E.; Adlercreutz, P.; Mattiasson, B. Biotech. Bioeng. 1995, 45, 406.
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from Jatropha oil. It is also worth noting that the overall yields by chemical transesterification (93%) and enzymatic transesterification (92%) were nearly the same. Acknowledgment. The financial support provided by the Council of Scientific and Industrial Research (CSIR) to Shweta Sharma and Shweta Shah in the form of Senior Research Fellowships is duly acknowledged.
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The funds provided by Council of Scientific and Industrial Research (Extramural Division and Technology Mission on Oilseeds, Pulses and Maize), Department of Science and Technology (DST) and Department of Biotechnology (DBT), Government of India organizations, are gratefully acknowledged. EF030075Z
