Biotechnology Letters (2006) 28: 637640 DOI 10.

1007/s10529-006-0027-2

Springer 2006

Lipase-mediated transformation of vegetable oils into biodiesel using propan-2-ol as acyl acceptor
Mukesh Kumar Modi, J.R.C. Reddy, B.V.S.K. Rao & R.B.N. Prasad*
Division of Lipid Science and Technology, Indian Institute of Chemical Technology, 500 007 Hyderabad, India *Author for correspondence (Fax: +91-40-27193370; E-mail: rbnprasad@iict.res.in)
Received 21 December 2005; Revisions requested 22 December 2005; Revisions received 26 January 2006; Accepted 30 January 2006

Key words: biodiesel, Candida antarctica, propan-2-ol, transesterication, vegetable oil

Abstract Propan-2-ol was used as an acyl acceptor for immobilized lipase-catalyzed preparation of biodiesel. The optimum conditions for transesterication of crude jatropha (Jatropha curcas), karanj (Pongamia pinnata) and sunower (Helianthus annuus) oils were 10% Novozym-435 (immobilized Candida antarctica lipase B) based on oil weight, alcohol to oil molar ratio of 4:1 at 50 C for 8 h. The maximum conversions achieved using propan-2-ol were 92.8, 91.7 and 93.4% from crude jatropha, karanj and sunower oils, respectively. Reusability of the lipase was maintained over 12 repeated cycles with propan-2-ol while it reached to zero by 7th cycle when methanol was used as an acyl acceptor, under standard reaction conditions.

Introduction Different processes are currently available for transesterication of vegetable oils for the production of biodiesel and include chemical or enzyme catalysis or supercritical alcohol treatment (Fukuda et al. 2001, Zhang et al. 2003, Kusdiana & Saka 2004, Warabi et al. 2004). Although biodiesel is successfully produced in large scale using chemical catalysts, there are several associated problems, such as glycerol recovery and removal of inorganic salts (Mittelbach 1990, Basri et al. 1997). Enzymatic conversion of vegetable oils into biodiesel oers an environmentally more attractive option to the conventional processes. Immobilization is the most widely used method for achieving stability in lipases and to make them more attractive for industrial use (Clark 1994, Cowan 1996). However, immobilized lipase preparations can be deactivated by lower linear alcohols, such as methanol and ethanol, conventionally used in biodiesel process (Samukawa et al. 2000, Chen & Wu 2003). The inhibition of excessive short chain alcohols is caused by the

poor miscibility of triacylglycerol with methanol or ethanol which exists as droplets in the oil and being polar in nature gets adsorbed to the acrylic resin used as immobilization support in the lipase, Novozym 435, thereby blocks the entry of triacylglycerols, causing the reaction to stop (Chen & Wu 2003). Several measures have been adopted to overcome this problem. Nelson et al. (1996) used hexane as a diluent to prevent the deactivation of lipase by a lower alcohol such as methanol. Samukawa et al. (2000) suggested maintaining a low concentration of methanol (oil to methanol molar ratio of 3:1) during the reaction. Soumanou & Bornscheuer (2003) suggested a three-step addition of methanol to lessen lipase inactivation and achieve higher conversion levels than in a solvent-free medium. However, using a diluent decreases the reaction rate and maintaining a low concentration of methanol in the reaction mixture is not a practical approach for industrial production of biodiesel. Of late it has been reported that the activity of the lipase deactivated by methanol can be restored to a certain extent by washing the enzyme with secondary

638 and tertiary alcohols such as propan-2-ol, butane-2-ol and tert-butanol (Chen & Wu 2003). However, there was no detailed study reported on enzymatic approach for the preparation of biodiesel using the above alcohols. In the present work, propan-2-ol was used as acyl acceptor and explored for its suitability in improving the operational stability of the immobilized lipase over repeated cycles of transesterication so as to bring the enzymatic approaches to biodiesel production from crude jatropha, karanj and sunower oils under the realm of industrial feasibility. Further, the fatty acid 2propyl esters promise improved cold weather performance (Lee et al. 1995). washed with distilled water to remove the glycerol and excess propan-2-ol. The ester phase was then dried using anhydrous sodium sulfate and nally concentrated under reduced pressure to get crude 2-propyl esters. The fatty acid 2-propyl esters (biodiesel) were quantied by silicic acid column chromatography using petroleum ether/ethyl acetate (99.5:0.5, v/v) as eluent. Analysis of 2-propyl esters The 2-propyl esters of crude jatropha, karanj and sunower oils were analyzed by GC, for their fatty acid composition, using a non-bonded cyanosilicone column (DB-23, 30 m0.25 mm0.2 lm). The oven was increased from 170 to 250 C at 5 C min)1 with a holding time of 5 min. The injector and detector were set at 225 and 275 C, respectively. Results and discussion Crude jatropha, karanj and sunower oils were procured from a local market and used as a source of triacylglycerols. Two hundred mg of Novozym 435 (Candida antarctica lipase B immobilized on macroporous acrylic resin, gifted by M/s Novozym, South Asia Pvt. Ltd., Bangalore, India) with an activity of 10 000 PLU g)1 was added to the mixture of oil (2 g) and propan-2-ol (alcohol to oil molar ratio of 4:1) held in a screw-capped test-tube. The molar amount of the oil was calculated from its saponication value which was determined using a standard method (Ocial Methods of AOCS 2004). The contents were stirred magnetically (150 rpm) at 50 C after tightly closing the tube. The reaction conditions were optimized by carrying out dierent sets of experiments with varying alcohol to oil molar ratio, dosage of enzyme and reaction period. At the end of reaction, the contents were shaken with hexane (25 ml) and the enzyme was separated by ltration. The hexane extract was Biodiesel was prepared in the form of 2-propyl esters of fatty acids from three vegetable oils namely crude jatropha, karanj and sunower oils (Table 1), employing propan-2-ol as an acyl acceptor to enhance the operational stability of lipase. An excess of alcohol was required in order to shift the reaction to the right. The optimum alcohol to oil ratio was 4:1 (mol mol)1; Figure 1). The optimum dosage of enzyme was 10% (Figure 2) and this was used in all future work. Using the above reaction conditions, maximum 2-propyl esters conversion was obtained after 8 h (Figure 3). In the present work, the operational stability of immobilized lipase, Novozym 435, over repeated cycles of transesterication was investigated using crude jatropha oil with methanol and propan-2-ol as acyl acceptors. The relative activity of lipase was dened as the percent conver-

Materials and methods Preparation of fatty acid 2-propyl esters

Table 1. Fatty acid composition of 2-propyl esters prepared from the vegetable oils. Oil 16:0 Sunower Jatropha Karanj
*

Relative % (w/w) fatty acyl composition 16:1 1 18:0 3 6 7 18:1 46 37 52 18:2 45 39 18 18:3* 4 20:0 2 20:1 1 22:0 5 24:0 1

6 17 10

n)3.

639
2-propyl esters conversion (% w/w) 100 80 60 40 20 0 Sunflower Jatropha Karanj
2-propyl esters conversion (% w/ w) 100 80 60 40 20 0 2 4 6 8 Reaction time (h) 10
Sunflower Jatropha Karanj

2 3 4 5 Molar ratio (propan-2-ol:oil)

Fig. 1. Eect of propan-2-ol to oil molar ratio on 2-propyl esters conversion in alcoholysis of vegetable oils catalyzed by Novozym-435 (200 mg lipase added to 2 g oil) at 50 C and 150 rpm for 8 h with single step addition of alcohol. Conversions shown on y-axis are meanS.E. of three individual experiments.

Fig. 3. Time course of the 2-propanolysis of vegetable oils catalyzed by Novozym 435 (200 mg) at 50 C and 150 rpm using propan-2-ol to oil molar ratio of 4:1 (0.55, 0.53 and 0.54 g of propan-2-ol added to 2 g of jatropha, karanj and sunower oils, respectively). Conversions shown on y-axis are meanS.E. of three individual experiments.

Relative activity of lipase (%)

2-propyl esters conversion (% w/w)

100 80 60 40 20 0 1 2 3 4 5 6 7 8 9 10 11 12
Cycle 2-Propanolysis Methanolysis

100 80 60 40 20 0 5 10 20 30 Dosage of enzyme (%w/w of oil) Sunflower Jatropha Karanj

Fig. 2. Eect of enzyme dosage on 2-propyl esters conversion in alcoholysis of vegetable oils using propan-2-ol to oil molar ratio of 4:1 (0.55, 0.53 and 0.54 g of propan-2-ol added to 2 g of jatropha, karanj and sunower oils, respectively) at 50 C and 150 rpm for 8 h with single step addition of alcohol. Conversions shown on y-axis are meanS.E. of three individual experiments.

Fig. 4. Operational stability of lipase over repeated cycles in alcoholysis of crude jatropha oil using alcohol to oil molar ratio of 4:1, catalyzed by Novozym 435 (200 mg) at 50 C and 150 rpm for 8 h. For 2-propanolysis 0.55 g of propan-2ol added to 2 g of oil in single step. For methanolysis 0.29 g methanol added to 2 g oil in four steps (72.5 mg in each step). 100% Relative activity of lipase in absolute terms corresponds to 93.4 and 92.8% alkyl esters conversion in methanolysis and 2-propanolysis, respectively.

sion into alkyl esters at a given time compared with the maximum alkyl esters conversion. There was no signicant loss in the relative activity of lipase over twelve repeated cycles when propan2-ol was used as acyl acceptor (Figure 4). A plausible explanation for this behavior is that contrary to short linear chain alcohols (methanol and ethanol), propan-2-ol displays better miscibility in triacylglycerols and is less polar (polarity index being 3.9, 5.2 and 5.1 for propan-2-ol, ethanol and methanol, respectively; http:// www.wcrl.ars.usda.gov/cec/java/solvents.htm) and

therefore ensures better operational stability of immobilized lipase. In conclusion, propan-2-ol is a promising acyl acceptor for immobilized lipase-catalyzed production of biodiesel as it shows virtually no negative eect on lipase activity. Acknowledgements Mukesh Kumar Modi is grateful to the Director, IICT for permitting to carry out the present

640 piece of work. This work (IICT Communication No. 051207) was supported by a nancial grant from Rain Shadow Areas Development Department, Government of Andhra Pradesh, India.
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