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Lipase catalyzed methanolysis of vegetable oils in aqueous medium by Cryptococcus spp. S-2
Process Biochemistry 37 (2001) 405– 410 www.elsevier.com/locate/procbio
Lipase catalyzed methanolysis of vegetable oils in aqueous medium by Cryptococcus spp. S-2 N.R. Kamini, H. Iefuji * National Research Institute of Brewing, 3 -7 -1 Kagamiyama, Higashi-Hiroshima 739 -0046, Japan Received 17 April 2001; received in revised form 30 April 2001; accepted 23 May 2001
Abstract A number of factors affecting the methanolysis of vegetable oils in aqueous medium by Cryptococcus spp. S-2 lipase were investigated. The crude lipase from the yeast efficiently catalyzed the methanolysis of vegetable oils (oil/methanol molar ratio of 1:1) in the presence of 40 wt.% water. The methyl ester content was high with rice bran oil at 30 °C for 96 h and further optimization studies were carried out with varying amount of enzyme, water or methanol. The enzyme was not inactivated by shaking in a mixture containing 4 Meq of methanol against the oil and 100 wt.% water by weight of the substrate and the methyl ester contents increased with increasing molar equivalents of methanol and water contents from 60 to 100 wt.%. The optimal methanolysis conditions were an oil/methanol molar ratio of 1:4, a water content of 80 wt.% by weight of the substrate containing 2000 U of crude lipase with shaking at 160 rpm for 120 h at 30 °C. Thus, the reaction was conducted in a single step to avoid the stepwise addition of methanol and the methyl ester contents reached 80.2 wt.% at 120 h. These same conditions were applied for alcoholysis of rice bran oil and primary alcohols to their respective alkyl ester derivatives which are excellent substitutes for diesel fuel. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Lipase; Methanolysis; Vegetable oils; Aqueous medium; Cryptococcus spp.; Rice bran oil
1. Introduction Interest in lipase catalyzed biosynthesis is rapidly increasing because of the possibility of obtaining a wide variety of high quality products under mild reaction conditions utilizing the substrate selectivity of such biocatalysts [1,2]. Lipases have great potential in food related lipid modifications, in the production of esters, wax and fatty acids [3 – 6]. Lipases (triacylglycerol acylhydrolases, E.C.3.1.1.3) are esterases that catalyze hydrolysis and synthesis of glycerol esters. Ester synthesis is favoured under restricted water availability [7], although a minimum quantity of water is necessary for enzyme catalysis to take place [8]. Alcoholysis of vegetable oils and animal fats is an important reaction that produces fatty acid alkyl esters that are valuable intermediates in oleochemistry, and * Corresponding author. Tel.: + 81-824-200-818; fax: + 81-824200-806. E-mail address: [email protected] (H. Iefuji).
methyl and ethyl esters are excellent substitutes for diesel fuel [9]. Although, conventional chemical technology using alkaline catalysts has been applied to alkyl ester production, there are several drawbacks to this approach including difficulties in the recovery of glycerol, the need for removal of catalyst and the energy intensive nature of the process [8]. The utilization of lipase in alcoholysis is considered as an effective means of circumventing these problems. Several reports describe enzymic alcoholysis of vegetable oils. When ethanol, isopropanol, butanol and long-chain alcohols were used as substrates, the oils were efficiently converted to their fatty acid esters and the efficiency of conversion was low with methanol [1,9 –12]. Lipase catalyzed alcoholysis in the absence of solvent is important in industrial applications. Studies on methyl ester synthesis in aqueous medium have been reported with lipases from Geotrichum candidum [13] and Candida deformans [14]. However, the yields of the reaction were 62 and 58%, respectively, with the substrate oleic acid and triolein. Shimada et al. [15] devel-
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N.R. Kamini, H. Iefuji / Process Biochemistry 37 (2001) 405–410
oped a three-step reaction for the methanolysis of a mixture of soybean and rapeseed oil because the Candida antarctica lipase was inactivated in reaction mixture containing more than 1.5 Meq of methanol and showed a methyl ester yield of over 95%. Later, Watanabe et al. [16] developed a two-step reaction for the methanolysis of vegetable oils. Therefore, it is necessary to find a lipase that efficiently catalyses methanolysis reaction in aqueous medium and in a single step without an additional organic solvent. In an earlier study, the crude lipase from the yeast, Cryptococcus spp. S-2 (strain CS2) was shown to possess considerable stability in solvents and it could be used for the hydrolysis of vegetable oils [17]. This paper describes the methanolysis of vegetable oils in aqueous medium and the factors affecting the methanolysis of rice bran oil by CS2 lipase for the production of methyl esters, which are excellent substitutes for diesel fuel.
incubated at 30 °C for 96 h with shaking at 160 rpm in a bioshaker. Samples were taken at 24 h intervals and analyzed for the methanolysis products by capillary gas chromatography (cGC) as described below.
2. Materials and methods
2.6. cGC analysis
2.1. Materials
Aliquots of 300 ml samples were taken from the reaction mixture at specified times in triplicates and centrifuged to obtain the upper layer. For analysis, 100 ml of the upper layer and 60 ml of tricaprylin were mixed in a 15 ml bottle, to which a specified amount of anhydrous sodium sulfate as a dehydrating agent and 3.0 ml of hexane were added. Tricaprylin served as the internal standard for cGC. One microlitre of the sample was injected to a Shimadzu (Kyoto) GC-17A gas chromatograph connected to a DB-5 capillary column (0.25 mm× 15 m; J and W Scientific, Forsom, CA, USA) for the determination of ME and free fatty acid (FFA) contents in the reaction mixture. The column temperature was maintained at 150 °C for 0.5 min, increased to 210 °C at a rate of 5 °C/min and to 300 °C at a rate of 10 °C/min. The temperatures of the injector and detector were set at 245 and 250 °C, respectively.
Olive oil, rapeseed oil, rice bran oil and soybean oil were purchased from Katayama Chemical Industries Co. Ltd. (Osaka, Japan). The molar amount of the oil was calculated from its fatty acid composition. Tricaprylin, fatty acids and methyl esters (ME) were purchased from Sigma Chemical Co, USA. All other chemicals used were of reagent grade.
2.2. Preparation of enzyme solution The yeast, CS2 was grown in the production medium [17] for 120 h at 25 °C. The culture was centrifuged at 8000 rpm for 20 min and the supernatant was concentrated by ultrafiltration using a disc membrane (YM-10, Amicon) of 10,000 Da cut off and used as the enzyme solution.
2.5. Optimization studies Consecutive optimization studies were carried out by varying the amount of enzyme (from 500 to 2500 U), water (from 13 to 200 wt.% by weight of the substrate) and methanol concentration (molar ratios of oil to methanol= 1:1–1:4). The effect of temperature on methanolysis was also investigated with the optimal conditions. The effect of addition of various organic solvents at a concentration of 10% w/v was studied for optimal methyl ester production. Using optimal parameters, alcoholysis was carried out with short chain alcohols to produce their respective alkyl ester derivatives.
2.3. Lipase acti6ity 3. Results and discussion Lipase activity was estimated using a spectrophotometric assay with p-nitrophenyl laurate (p-NPL) as a substrate [18]. One unit of lipase activity was defined as the amount of enzyme that liberated 1 mmol/min of p-nitrophenol under the standard assay conditions.
2.4. Methanolysis The methanolysis reaction was carried out with a mixture of oil and methanol (9.65/0.35 g, molar ratio of 1:1) as the starting material. About 4 ml of the enzyme solution containing 500 U of CS2 lipase was added to the reaction mixture in a 100 ml stoppered flask and
3.1. Methanolysis of 6egetable oil The methanolysis reactions were carried out from the commercial preparations of the immobilized lipases from C. antarctica, Rhizopus delemar, Rhizomucor miehei and Aspergillus niger [15] and lipase powders from Geotrichum candidum and Pseudomonas cepacia [19]. The crude enzyme from yeast, Cryptococcus spp. S-2 was used for the methanolysis of vegetable oils. The enzyme efficiently catalyzed the methanolysis reaction and the ester conversion was high with rice bran oil and it was 24.9% in 96 h at 30 °C (Table 1). The percent
N.R. Kamini, H. Iefuji / Process Biochemistry 37 (2001) 405–410 Table 1 Methanolysis of vegetable oil by Cryptococcus spp. S-2 lipase Oils
Olive oil Rapeseed oil Rice bran oil Soybean oil
Ester conversion (%)a 24 h
48 h
72 h
96 h
7.5 9.4 9.8 9.6
9.7 14.3 16.6 12.9
12.5 17.6 20.2 16.9
16.7 21.1 24.9 21.1
a
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creased by 8.9 and 16.5 wt.% when the water content was increased to 30 and 50 wt.%, respectively, at 48 h, and there was no significant difference in ME content at the end of 96 h (Table 2). The FFA content was increased from 24.0 to 42.1 wt.%, upon increasing the water content from 13 to 50 wt.%, respectively, at 96 h. Similar results were reported for the methanolysis of a mixture of soybean and rapeseed oil [15].
Ester conversion is expressed as the ratio of methyl esters converted to the oil used as a substrate.
3.4. Cumulati6e effect of water content and methanol concentration on methanolysis of rice bran oil
conversion increased with increase in time with the other oils. Since the percent conversion was high with rice bran oil, it was selected for further study.
At least 3 Meq of methanol are required for the complete conversion of the oil to its corresponding ME. However, the lipase of Rhizopus oryzae [8] was inactivated in reaction mixture containing more than 1 Meq of methanol and the reaction was performed by the stepwise addition of methanol. In this study, the methanolysis reaction was carried out in a single step with different molar equivalents of methanol (molar ratios of oil:methanol= 1:1–1:4) using water contents from 20 to 200 wt.% by weight of the substrate to determine the efficiency of methanolysis. As shown in Fig. 2A, the ME contents increased with increasing molar equivalents of methanol (molar ratios of oil:methanol= 1:1–1:4) and increasing water contents (from 60 to 100 wt.% by weight of the substrate) in the reaction mixture. However, the ME contents were significantly low in reaction mixture containing more than one molar equivalent of methanol and water contents of 20 and 40 wt.% by weight of the substrate. An insufficient amount of water in the reaction mixture probably results in the inactivation of the lipase, which may be due to denaturation of the enzyme by methanol. The ME content was comparatively low, when the reaction mixture contained more than 100 wt.% water by weight of the substrate. The ME content was 62.6 and 66.4 wt.% with a water content of 80 wt.% in the reaction mixture consisting of a molar ratio of oil:methanol=1:3 and 1:4, respectively, at 96 h and with FFA content of 18.1 and 12.6 wt.%. There was no significant increase in ME content, when the water content was increased from 80 to 100 wt.% in the reaction mixture. An opposite effect was observed in the methanolysis reaction, using lipases of Pseudomonas fluorescens [9] and Mucor miehei [19], leading to lower ester conversions when water was added to the reaction mixture. The content of FFA decreased with increasing molar equivalents of methanol and increased with increasing water contents from 20 to 100 wt.% (Fig. 2B). Hence, subsequent experiments were carried out with a combination of molar ratio of oil:methanol=1:3 and a water content of 80 wt.% by weight of the substrate.
3.2. Effect of amount of lipase added on methanolysis of rice bran oil The effect of amount of lipase added on methanolysis of rice bran oil (molar ratio of oil:methanol= 1:1) is shown in Fig. 1. The percent conversion increased proportionally with increase in lipase concentration from 500 to 2000 U and remained constant at higher amounts of lipase. The amount of enzyme is probably the rate determining factor of the reaction up to 2000 U per 4 ml of the enzyme solution. The percent conversion reached 33.3% at 96 h with 2000 U of lipase and subsequent experiments were carried out with 2000 U of lipase.
3.3. Effect of water content on methanolysis of rice bran oil The ME content was 31.5 wt.% at 48 h using a water content of 13 wt.% (1.3 ml of the enzyme solution =13 wt.% by weight of the substrate), showed that 94.6% of the methanol was consumed in the reaction mixture. However, ester formation was de-
Fig. 1. Effect of amount of lipase added on methanolysis of rice bran oil.
N.R. Kamini, H. Iefuji / Process Biochemistry 37 (2001) 405–410
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Table 2 Effect of water content on methanolysis of rice bran oil Water content (wt.%)
13 20 30 40 50
Methyl ester content (wt.%)
Fatty acid content (wt.%)
24 h
48 h
72 h
96 h
24 h
48 h
72 h
96 h
16.4 22.6 23.0 23.1 22.5
31.5 31.4 28.7 26.6 26.3
33.3 33.5 32.4 30.1 28.5
34.0 34.0 33.3 33.3 32.4
4.7 12.5 13.5 18.7 22.5
10.3 21.1 24.2 31.5 35.4
19.9 30.5 32.4 38.0 39.1
24.0 33.3 35.4 40.0 42.1
The reaction was conducted in a mixture of 9.65 g oil and 0.35 g methanol (molar ratio of 1:1) and various amounts of water as enzyme solution (1.3–5.0 ml =13–50 wt.% by weight of the substrate) containing 2000 U of lipase.
3.5. Effect of temperature on methanolysis of rice bran oil
3.7. Effect of organic sol6ents on methanolysis of rice bran oil
To investigate the effect of temperature on methanolysis of rice bran oil, the reaction was conducted over the temperature range of 25– 40 °C (Fig. 3). The ester contents at 24 h decreased with increasing temperature, with the optimal reaction temperature of 25– 30 °C. However, when the reaction was extended to 96 h, the ester content was high (62.3 wt.%) at 30 °C.
The effect of various organic solvents on methanolysis of rice bran oil is shown in Table 3. The ME content was increased from 4.8 to 7.0 wt.% in presence of DMSO, petroleum ether and n-hexane when compared with the ester content of 80.2 wt.% with water. The methanolysis was inhibited in presence of diethylether and the ME content was 70.4 wt.% at 120 h. Nelson et al. [19] investigated alcoholysis with various lipases in
3.6. Effect of methanol content on methanolysis of rice bran oil One of the most important variables affecting the yield of ester is the molar ratio of alcohol to vegetable oil employed [20]. The stoichiometry of this reaction requires 3 moles of alcohol per mole of vegetable oil to yield 3 moles of fatty acid ester and 1 mole of glycerol. However, in the ethanolysis of peanut oil, the ester conversion was high with a molar ratio of oil:ethanol= 1:6 [21]. Hence, the methanolysis of rice bran oil was carried out with increasing molar equivalents of methanol. As shown in Fig. 4, the ME content was increased by 11.8 wt.%, when the molar ratio of oil:methanol was increased from 1:3 to 1:4 at 120 h and there was no significant difference in ME content at a molar ratio of 1:5. The ME content was high (79.7 wt.%) with a molar ratio of oil:methanol= 1:4 at 120 h and similar results were reported for the methanolysis of sunflower oil using NaOH as catalyst [19]. The ME content was low (65.2 wt.%) in the reaction mixture consisting of a molar ratio of oil:methanol= 1:6, which could be due to denaturation of the enzyme by methanol at this concentration. Freedman et al. [22] reported an optimal molar ratio of methanol:soybean oil of 6:1 for the methanolysis of soybean oil. On the basis of the above results, the optimum conditions were determined as follows: a reaction mixture consisting of a molar ratio of oil to methanol of 1:4, a water content of 80 wt.% by weight of the substrate containing 2000 U of lipase and incubation temperature of 30 °C for 120 h.
Fig. 2. Cumulative effect of water content and methanol concentration on methanolysis of rice bran oil. (A), ME content (wt.%) and (B), FFA content (wt.%). The reaction was performed at 30 °C for 96 h in a mixture consisting of molar ratios of oil:methanol = 1:1–1:4 and water contents from 20 to 200 wt.% by weight of the substrate containing 2000 U of lipase.
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Table 3 Effect of organic solvents (10%, w/v) on methanolysis of rice bran oil Organic solvent
ME content (wt.%)
None DMSO Diethylether n-Hexane Petroleum ether
80.2 85.0 70.4 86.1 87.2
Table 4 Effect of primary alcohols on alcoholysis of rice bran oil Fig. 3. Effect of temperature on methanolysis of rice bran oil.
hexane and reported a ME yield of 75.4% in methanolysis of soybean oil with lipase from M. miehei. However, the lipase from CS2 showed an ester yield of 86.1 wt.% in presence of hexane. Though the ester content is high in presence of organic solvent, from an economical point of view, a reaction system without an organic solvent is desirable for the industrial production of diesel fuel and to eliminate the risk of explosion.
3.8. Effect of primary alcohols on alcoholysis of rice bran oil The optimal conditions established for the methanolysis were tested for the alcoholysis of rice bran oil with various alcohols. The alkyl ester contents were less than 30 wt.% with ethanol, propanol and butanol and the ester content was 65.0 and 80.7 wt.% with isobutanol and methanol, respectively, at 120 h (Table 4). Hence, the lipase from Cryptococcus spp. S-2 could efficiently be used for the industrial production of biodiesel from rice bran oil and methanol. However, the conditions used for alcoholysis with ethanol, propanol and butanol might be sensitive to the amount of water and molar ratio of alcohol added in the reaction mixture.
Fig. 4. Effect of methanol content on methanolysis of rice bran oil. The reaction was performed at 30 °C for 120 h in a mixture consisting of molar ratios of oil: methanol = 1:2–1:6 and water content of 80 wt.% by weight of the substrate containing 2000 U of lipase.
Alcohol
Alkyl ester (wt.%)
Methanol Ethanol Propanol Butanol Isobutanol
80.7 27.5 3.5 18.8 65.0
Work is in progress to maximize the production of alkyl esters with rice bran oil and specific alcohols.
Acknowledgements The financial assistance extended by the Science and Technology Agency (STA), JISTEC, Japan to N.R. Kamini is gratefully acknowledged.
References [1] Linko YY, Lamsa M, Huhtala A, Linko P. Lipase-catalyzed transesterification of rapeseed oil and 2-ethyl-1-hexanol. J Am Oil Chem Soc 1994;71:1411 – 4. [2] Steinke G, Kirchhoff R, Mukherjee KD. Lipase-catalyzed alcoholysis of crambe oil and camelina oil for the preparation of long-chain esters. J Am Oil Chem Soc 2000;77:361 – 6. [3] Yang TH, Jang Y, Han JJ, Rhee JS. Enzymatic synthesis of low-calorie structured lipids in a solvent-free system. J Am Oil Chem Soc 2001;78:291 – 6. [4] Bloomer S, Adlercreutz P, Mattiasson B. Facile synthesis of fatty acid esters in high yields. Enzyme Microb Technol 1992;14:546 – 52. [5] Chen JP, Wang JB. Wax ester synthesis by lipase-catalyzed esterification with fungal cells immobilized on cellulose biomass support particles. Enzyme Microb Technol 1997;20:615 –22. [6] Yamamoto K, Fujiwara N. The hydrolysis of castor oil using a lipase from Pseudomonas spp. f-B-24: positional and substrate specificity of the enzyme and optimum reaction conditions. Biosci Biotech Biochem 1995;59:1262 – 6. [7] Macrae AR, Hammond RC. Present and future applications of lipases. Biotechnol Genet Eng Rev 1985;3:193 – 219. [8] Kaieda M, Samukawa T, Matsumoto T, Ban K, Kondo A, Shimada Y, Noda H, Nomoto F, Ohtsuka K, Izumoto E, Fukuda H. Biodiesel fuel production from plant oil catalyzed by Rhizopus oryzae lipase in a water-containing system without an organic solvent. J Biosci Bioeng 1999;88:627 – 31.
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[9] Mittelbach M. Lipase catalyzed alcoholysis of sunflower oil. J Am Oil Chem Soc 1990;67:168 – 70. [10] Show JF, Wang DL. Lipase-catalyzed ethanolysis and isopropanolysis of triglycerides with long-chain fatty acids. Enzyme Microb Technol 1991;13:544 –6. [11] Selmi B, Thomas D. Immobilized lipase-catalyzed ethanolysis of sunflower oil in a solvent-free medium. J Am Oil Chem Soc 1998;75:691 – 5. [12] Abigor RD, Uadia PO, Foglia TA, Haas MJ, Jones KC, Okpefa E, Obibuzor JU, Bafor ME. Lipase-catalyzed production of biodiesel fuel from some Nigerian lauric oils. Biochem Soc Trans 2000;28:979 – 81. [13] Okumura S, Iwai M, Tsujisaka Y. Synthesis of various kinds of esters by four microbial lipases. Biochem Biophys Acta 1979;575:156 – 65. [14] Boutur O, Dubreucq E, Galzy P. Methyl esters production, in aqueous medium, by an enzymatic extract from Candida deformans (Zach) Langeron and Guerra. Biotechnol Lett 1994;16:1179 –82. [15] Shimada Y, Watanabe Y, Samukawa T, Sugihara A, Noda H, Fukuda H, Tominaga Y. Conversion of vegetable oil to biodiesel using immobilized Candida antarctica lipase. J Am Oil Chem Soc 1999;76:789 – 93.
[16] Watanabe Y, Shimada Y, Sugihara A, Noda H, Fukuda H, Tominaga Y. Continuous production of biodiesel fuel from vegetable oil using immobilized Candida antarctica lipase. J Am Oil Chem Soc 2000;77:355 – 60. [17] Kamini NR, Fujii T, Kurosu T, Iefuji H. Production, purification and characterization of an extracellular lipase from the yeast, Cryptococcus spp. S-2. Process Biochem 2000;36:317 – 24. [18] Isobe K, Akiba T, Yamaguchi S. Crystallization and characterization of lipase from Penicillium cyclopium. Agric Biol Chem 1988;52:41 – 7. [19] Nelson LA, Foglia TA, Marmer WN. Lipase-catalyzed production of biodiesel. J Am Oil Chem Soc 1996;73:1191 – 5. [20] Freedman B, Pryde EH, Mounts TL. Variables affecting the yields of fatty esters from transesterified vegetable oils. J Am Oil Chem Soc 1984;61:1638 – 43. [21] Feuge RO, Gros AT. Modification of vegetable oil VII: alkali catalyzed interesterification of peanut oil with ethanol. J Am Oil Chem Soc 1949;26:97 – 102. [22] Freedman B, Butterfield RO, Pryde EH. Transesterification kinetics of soybean oil. J Am Oil Chem Soc 1986;63:1375 – 80.
Lipase catalyzed methanolysis of vegetable oils in aqueous medium by Cryptococcus spp. S-2 N.R. Kamini, H. Iefuji * National Research Institute of Brewing, 3 -7 -1 Kagamiyama, Higashi-Hiroshima 739 -0046, Japan Received 17 April 2001; received in revised form 30 April 2001; accepted 23 May 2001
Abstract A number of factors affecting the methanolysis of vegetable oils in aqueous medium by Cryptococcus spp. S-2 lipase were investigated. The crude lipase from the yeast efficiently catalyzed the methanolysis of vegetable oils (oil/methanol molar ratio of 1:1) in the presence of 40 wt.% water. The methyl ester content was high with rice bran oil at 30 °C for 96 h and further optimization studies were carried out with varying amount of enzyme, water or methanol. The enzyme was not inactivated by shaking in a mixture containing 4 Meq of methanol against the oil and 100 wt.% water by weight of the substrate and the methyl ester contents increased with increasing molar equivalents of methanol and water contents from 60 to 100 wt.%. The optimal methanolysis conditions were an oil/methanol molar ratio of 1:4, a water content of 80 wt.% by weight of the substrate containing 2000 U of crude lipase with shaking at 160 rpm for 120 h at 30 °C. Thus, the reaction was conducted in a single step to avoid the stepwise addition of methanol and the methyl ester contents reached 80.2 wt.% at 120 h. These same conditions were applied for alcoholysis of rice bran oil and primary alcohols to their respective alkyl ester derivatives which are excellent substitutes for diesel fuel. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Lipase; Methanolysis; Vegetable oils; Aqueous medium; Cryptococcus spp.; Rice bran oil
1. Introduction Interest in lipase catalyzed biosynthesis is rapidly increasing because of the possibility of obtaining a wide variety of high quality products under mild reaction conditions utilizing the substrate selectivity of such biocatalysts [1,2]. Lipases have great potential in food related lipid modifications, in the production of esters, wax and fatty acids [3 – 6]. Lipases (triacylglycerol acylhydrolases, E.C.3.1.1.3) are esterases that catalyze hydrolysis and synthesis of glycerol esters. Ester synthesis is favoured under restricted water availability [7], although a minimum quantity of water is necessary for enzyme catalysis to take place [8]. Alcoholysis of vegetable oils and animal fats is an important reaction that produces fatty acid alkyl esters that are valuable intermediates in oleochemistry, and * Corresponding author. Tel.: + 81-824-200-818; fax: + 81-824200-806. E-mail address: [email protected] (H. Iefuji).
methyl and ethyl esters are excellent substitutes for diesel fuel [9]. Although, conventional chemical technology using alkaline catalysts has been applied to alkyl ester production, there are several drawbacks to this approach including difficulties in the recovery of glycerol, the need for removal of catalyst and the energy intensive nature of the process [8]. The utilization of lipase in alcoholysis is considered as an effective means of circumventing these problems. Several reports describe enzymic alcoholysis of vegetable oils. When ethanol, isopropanol, butanol and long-chain alcohols were used as substrates, the oils were efficiently converted to their fatty acid esters and the efficiency of conversion was low with methanol [1,9 –12]. Lipase catalyzed alcoholysis in the absence of solvent is important in industrial applications. Studies on methyl ester synthesis in aqueous medium have been reported with lipases from Geotrichum candidum [13] and Candida deformans [14]. However, the yields of the reaction were 62 and 58%, respectively, with the substrate oleic acid and triolein. Shimada et al. [15] devel-
0032-9592/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 1 ) 0 0 2 2 0 - 5
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N.R. Kamini, H. Iefuji / Process Biochemistry 37 (2001) 405–410
oped a three-step reaction for the methanolysis of a mixture of soybean and rapeseed oil because the Candida antarctica lipase was inactivated in reaction mixture containing more than 1.5 Meq of methanol and showed a methyl ester yield of over 95%. Later, Watanabe et al. [16] developed a two-step reaction for the methanolysis of vegetable oils. Therefore, it is necessary to find a lipase that efficiently catalyses methanolysis reaction in aqueous medium and in a single step without an additional organic solvent. In an earlier study, the crude lipase from the yeast, Cryptococcus spp. S-2 (strain CS2) was shown to possess considerable stability in solvents and it could be used for the hydrolysis of vegetable oils [17]. This paper describes the methanolysis of vegetable oils in aqueous medium and the factors affecting the methanolysis of rice bran oil by CS2 lipase for the production of methyl esters, which are excellent substitutes for diesel fuel.
incubated at 30 °C for 96 h with shaking at 160 rpm in a bioshaker. Samples were taken at 24 h intervals and analyzed for the methanolysis products by capillary gas chromatography (cGC) as described below.
2. Materials and methods
2.6. cGC analysis
2.1. Materials
Aliquots of 300 ml samples were taken from the reaction mixture at specified times in triplicates and centrifuged to obtain the upper layer. For analysis, 100 ml of the upper layer and 60 ml of tricaprylin were mixed in a 15 ml bottle, to which a specified amount of anhydrous sodium sulfate as a dehydrating agent and 3.0 ml of hexane were added. Tricaprylin served as the internal standard for cGC. One microlitre of the sample was injected to a Shimadzu (Kyoto) GC-17A gas chromatograph connected to a DB-5 capillary column (0.25 mm× 15 m; J and W Scientific, Forsom, CA, USA) for the determination of ME and free fatty acid (FFA) contents in the reaction mixture. The column temperature was maintained at 150 °C for 0.5 min, increased to 210 °C at a rate of 5 °C/min and to 300 °C at a rate of 10 °C/min. The temperatures of the injector and detector were set at 245 and 250 °C, respectively.
Olive oil, rapeseed oil, rice bran oil and soybean oil were purchased from Katayama Chemical Industries Co. Ltd. (Osaka, Japan). The molar amount of the oil was calculated from its fatty acid composition. Tricaprylin, fatty acids and methyl esters (ME) were purchased from Sigma Chemical Co, USA. All other chemicals used were of reagent grade.
2.2. Preparation of enzyme solution The yeast, CS2 was grown in the production medium [17] for 120 h at 25 °C. The culture was centrifuged at 8000 rpm for 20 min and the supernatant was concentrated by ultrafiltration using a disc membrane (YM-10, Amicon) of 10,000 Da cut off and used as the enzyme solution.
2.5. Optimization studies Consecutive optimization studies were carried out by varying the amount of enzyme (from 500 to 2500 U), water (from 13 to 200 wt.% by weight of the substrate) and methanol concentration (molar ratios of oil to methanol= 1:1–1:4). The effect of temperature on methanolysis was also investigated with the optimal conditions. The effect of addition of various organic solvents at a concentration of 10% w/v was studied for optimal methyl ester production. Using optimal parameters, alcoholysis was carried out with short chain alcohols to produce their respective alkyl ester derivatives.
2.3. Lipase acti6ity 3. Results and discussion Lipase activity was estimated using a spectrophotometric assay with p-nitrophenyl laurate (p-NPL) as a substrate [18]. One unit of lipase activity was defined as the amount of enzyme that liberated 1 mmol/min of p-nitrophenol under the standard assay conditions.
2.4. Methanolysis The methanolysis reaction was carried out with a mixture of oil and methanol (9.65/0.35 g, molar ratio of 1:1) as the starting material. About 4 ml of the enzyme solution containing 500 U of CS2 lipase was added to the reaction mixture in a 100 ml stoppered flask and
3.1. Methanolysis of 6egetable oil The methanolysis reactions were carried out from the commercial preparations of the immobilized lipases from C. antarctica, Rhizopus delemar, Rhizomucor miehei and Aspergillus niger [15] and lipase powders from Geotrichum candidum and Pseudomonas cepacia [19]. The crude enzyme from yeast, Cryptococcus spp. S-2 was used for the methanolysis of vegetable oils. The enzyme efficiently catalyzed the methanolysis reaction and the ester conversion was high with rice bran oil and it was 24.9% in 96 h at 30 °C (Table 1). The percent
N.R. Kamini, H. Iefuji / Process Biochemistry 37 (2001) 405–410 Table 1 Methanolysis of vegetable oil by Cryptococcus spp. S-2 lipase Oils
Olive oil Rapeseed oil Rice bran oil Soybean oil
Ester conversion (%)a 24 h
48 h
72 h
96 h
7.5 9.4 9.8 9.6
9.7 14.3 16.6 12.9
12.5 17.6 20.2 16.9
16.7 21.1 24.9 21.1
a
407
creased by 8.9 and 16.5 wt.% when the water content was increased to 30 and 50 wt.%, respectively, at 48 h, and there was no significant difference in ME content at the end of 96 h (Table 2). The FFA content was increased from 24.0 to 42.1 wt.%, upon increasing the water content from 13 to 50 wt.%, respectively, at 96 h. Similar results were reported for the methanolysis of a mixture of soybean and rapeseed oil [15].
Ester conversion is expressed as the ratio of methyl esters converted to the oil used as a substrate.
3.4. Cumulati6e effect of water content and methanol concentration on methanolysis of rice bran oil
conversion increased with increase in time with the other oils. Since the percent conversion was high with rice bran oil, it was selected for further study.
At least 3 Meq of methanol are required for the complete conversion of the oil to its corresponding ME. However, the lipase of Rhizopus oryzae [8] was inactivated in reaction mixture containing more than 1 Meq of methanol and the reaction was performed by the stepwise addition of methanol. In this study, the methanolysis reaction was carried out in a single step with different molar equivalents of methanol (molar ratios of oil:methanol= 1:1–1:4) using water contents from 20 to 200 wt.% by weight of the substrate to determine the efficiency of methanolysis. As shown in Fig. 2A, the ME contents increased with increasing molar equivalents of methanol (molar ratios of oil:methanol= 1:1–1:4) and increasing water contents (from 60 to 100 wt.% by weight of the substrate) in the reaction mixture. However, the ME contents were significantly low in reaction mixture containing more than one molar equivalent of methanol and water contents of 20 and 40 wt.% by weight of the substrate. An insufficient amount of water in the reaction mixture probably results in the inactivation of the lipase, which may be due to denaturation of the enzyme by methanol. The ME content was comparatively low, when the reaction mixture contained more than 100 wt.% water by weight of the substrate. The ME content was 62.6 and 66.4 wt.% with a water content of 80 wt.% in the reaction mixture consisting of a molar ratio of oil:methanol=1:3 and 1:4, respectively, at 96 h and with FFA content of 18.1 and 12.6 wt.%. There was no significant increase in ME content, when the water content was increased from 80 to 100 wt.% in the reaction mixture. An opposite effect was observed in the methanolysis reaction, using lipases of Pseudomonas fluorescens [9] and Mucor miehei [19], leading to lower ester conversions when water was added to the reaction mixture. The content of FFA decreased with increasing molar equivalents of methanol and increased with increasing water contents from 20 to 100 wt.% (Fig. 2B). Hence, subsequent experiments were carried out with a combination of molar ratio of oil:methanol=1:3 and a water content of 80 wt.% by weight of the substrate.
3.2. Effect of amount of lipase added on methanolysis of rice bran oil The effect of amount of lipase added on methanolysis of rice bran oil (molar ratio of oil:methanol= 1:1) is shown in Fig. 1. The percent conversion increased proportionally with increase in lipase concentration from 500 to 2000 U and remained constant at higher amounts of lipase. The amount of enzyme is probably the rate determining factor of the reaction up to 2000 U per 4 ml of the enzyme solution. The percent conversion reached 33.3% at 96 h with 2000 U of lipase and subsequent experiments were carried out with 2000 U of lipase.
3.3. Effect of water content on methanolysis of rice bran oil The ME content was 31.5 wt.% at 48 h using a water content of 13 wt.% (1.3 ml of the enzyme solution =13 wt.% by weight of the substrate), showed that 94.6% of the methanol was consumed in the reaction mixture. However, ester formation was de-
Fig. 1. Effect of amount of lipase added on methanolysis of rice bran oil.
N.R. Kamini, H. Iefuji / Process Biochemistry 37 (2001) 405–410
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Table 2 Effect of water content on methanolysis of rice bran oil Water content (wt.%)
13 20 30 40 50
Methyl ester content (wt.%)
Fatty acid content (wt.%)
24 h
48 h
72 h
96 h
24 h
48 h
72 h
96 h
16.4 22.6 23.0 23.1 22.5
31.5 31.4 28.7 26.6 26.3
33.3 33.5 32.4 30.1 28.5
34.0 34.0 33.3 33.3 32.4
4.7 12.5 13.5 18.7 22.5
10.3 21.1 24.2 31.5 35.4
19.9 30.5 32.4 38.0 39.1
24.0 33.3 35.4 40.0 42.1
The reaction was conducted in a mixture of 9.65 g oil and 0.35 g methanol (molar ratio of 1:1) and various amounts of water as enzyme solution (1.3–5.0 ml =13–50 wt.% by weight of the substrate) containing 2000 U of lipase.
3.5. Effect of temperature on methanolysis of rice bran oil
3.7. Effect of organic sol6ents on methanolysis of rice bran oil
To investigate the effect of temperature on methanolysis of rice bran oil, the reaction was conducted over the temperature range of 25– 40 °C (Fig. 3). The ester contents at 24 h decreased with increasing temperature, with the optimal reaction temperature of 25– 30 °C. However, when the reaction was extended to 96 h, the ester content was high (62.3 wt.%) at 30 °C.
The effect of various organic solvents on methanolysis of rice bran oil is shown in Table 3. The ME content was increased from 4.8 to 7.0 wt.% in presence of DMSO, petroleum ether and n-hexane when compared with the ester content of 80.2 wt.% with water. The methanolysis was inhibited in presence of diethylether and the ME content was 70.4 wt.% at 120 h. Nelson et al. [19] investigated alcoholysis with various lipases in
3.6. Effect of methanol content on methanolysis of rice bran oil One of the most important variables affecting the yield of ester is the molar ratio of alcohol to vegetable oil employed [20]. The stoichiometry of this reaction requires 3 moles of alcohol per mole of vegetable oil to yield 3 moles of fatty acid ester and 1 mole of glycerol. However, in the ethanolysis of peanut oil, the ester conversion was high with a molar ratio of oil:ethanol= 1:6 [21]. Hence, the methanolysis of rice bran oil was carried out with increasing molar equivalents of methanol. As shown in Fig. 4, the ME content was increased by 11.8 wt.%, when the molar ratio of oil:methanol was increased from 1:3 to 1:4 at 120 h and there was no significant difference in ME content at a molar ratio of 1:5. The ME content was high (79.7 wt.%) with a molar ratio of oil:methanol= 1:4 at 120 h and similar results were reported for the methanolysis of sunflower oil using NaOH as catalyst [19]. The ME content was low (65.2 wt.%) in the reaction mixture consisting of a molar ratio of oil:methanol= 1:6, which could be due to denaturation of the enzyme by methanol at this concentration. Freedman et al. [22] reported an optimal molar ratio of methanol:soybean oil of 6:1 for the methanolysis of soybean oil. On the basis of the above results, the optimum conditions were determined as follows: a reaction mixture consisting of a molar ratio of oil to methanol of 1:4, a water content of 80 wt.% by weight of the substrate containing 2000 U of lipase and incubation temperature of 30 °C for 120 h.
Fig. 2. Cumulative effect of water content and methanol concentration on methanolysis of rice bran oil. (A), ME content (wt.%) and (B), FFA content (wt.%). The reaction was performed at 30 °C for 96 h in a mixture consisting of molar ratios of oil:methanol = 1:1–1:4 and water contents from 20 to 200 wt.% by weight of the substrate containing 2000 U of lipase.
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Table 3 Effect of organic solvents (10%, w/v) on methanolysis of rice bran oil Organic solvent
ME content (wt.%)
None DMSO Diethylether n-Hexane Petroleum ether
80.2 85.0 70.4 86.1 87.2
Table 4 Effect of primary alcohols on alcoholysis of rice bran oil Fig. 3. Effect of temperature on methanolysis of rice bran oil.
hexane and reported a ME yield of 75.4% in methanolysis of soybean oil with lipase from M. miehei. However, the lipase from CS2 showed an ester yield of 86.1 wt.% in presence of hexane. Though the ester content is high in presence of organic solvent, from an economical point of view, a reaction system without an organic solvent is desirable for the industrial production of diesel fuel and to eliminate the risk of explosion.
3.8. Effect of primary alcohols on alcoholysis of rice bran oil The optimal conditions established for the methanolysis were tested for the alcoholysis of rice bran oil with various alcohols. The alkyl ester contents were less than 30 wt.% with ethanol, propanol and butanol and the ester content was 65.0 and 80.7 wt.% with isobutanol and methanol, respectively, at 120 h (Table 4). Hence, the lipase from Cryptococcus spp. S-2 could efficiently be used for the industrial production of biodiesel from rice bran oil and methanol. However, the conditions used for alcoholysis with ethanol, propanol and butanol might be sensitive to the amount of water and molar ratio of alcohol added in the reaction mixture.
Fig. 4. Effect of methanol content on methanolysis of rice bran oil. The reaction was performed at 30 °C for 120 h in a mixture consisting of molar ratios of oil: methanol = 1:2–1:6 and water content of 80 wt.% by weight of the substrate containing 2000 U of lipase.
Alcohol
Alkyl ester (wt.%)
Methanol Ethanol Propanol Butanol Isobutanol
80.7 27.5 3.5 18.8 65.0
Work is in progress to maximize the production of alkyl esters with rice bran oil and specific alcohols.
Acknowledgements The financial assistance extended by the Science and Technology Agency (STA), JISTEC, Japan to N.R. Kamini is gratefully acknowledged.
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