Enzymatic transesterification of Jatropha oil
© Kumari et al. 2009
Received: 17 July 2008
Accepted: 14 January 2009
Published: 14 January 2009
Transesterification of Jatropha oil was carried out in t-butanol solvent using immobilized lipase from Enterobacter aerogenes. The presence of t-butanol significantly reduced the negative effects caused by both methanol and glycerol. The effects of various reaction parameters on transesterification of Jatropha oil were studied.
The maximum yield of biodiesel was 94% (of which 68% conversion was achieved with respect to methyl oleate) with an oil:methanol molar ratio of 1:4, 50 U of immobilized lipase/g of oil, and a t-butanol:oil volume ratio of 0.8:1 at 55°C after 48 h of reaction time. There was negligible loss in lipase activity even after repeated use for seven cycles.
To the best of our knowledge this is the first report on biodiesel synthesis using immobilized E. aerogenes lipase.
Biodiesel is a renewable fuel that can be synthesized from edible, non-edible and waste oils. Due to diminishing petroleum reserves, vegetable oils have attracted attention as a potential renewable source for the production of alternatives to petroleum-based diesel fuel. A number of processes have been developed for biodiesel production involving chemical or enzyme catalysis or supercritical alcohol treatment [1–4]. Enzymatic transesterification of triglycerides is a good alternative to chemical processes due to its eco-friendly, selective nature and low temperature requirements [5–9].
Many starting materials such as soybean oil [10, 11], sunflower oil [12, 13], cotton seed oil , rapeseed oil , palm oil [16, 17] and restaurant kitchen wastes  have been evaluated for preparation of biodiesel by the enzymatic route. In many countries, like India, where edible oils are not in surplus supply, there is a need to search for alternative starting materials, such as from non-edible oils. Oil of Jatropha curcas (Euphorbiaceae), a non-edible oil, has been chosen for the present investigation. The seeds and oil are toxic due to the presence of toxic phorbol esters. The oil content of Jatropha seed ranges from 30 to 50% by weight, whereas in kernel the oil content ranges from 45 to 60%. The fatty acid composition of Jatropha oil consists of oleic acid 43.1%, linoleic acid 34.3%, stearic acid 6.9%, palmitic acid 4.2% and other acids 1.4%. Jatropha curcas is a low-growing tree, generally planted as a hedge for protecting crops from animals. It can be grown on barren land under harsh conditions and can be cultivated as a part of the strategy for reclaiming degraded lands . Keeping all this in view, the Indian Government has announced a 'National Mission on Biodiesel' for Jatropha plantations in wasteland regions that is to be implemented on an area of 400,000 ha over the next five years .
There are many reports on biodiesel production using enzyme catalysis by free or immobilized lipases [7, 8, 10, 15, 18, 21–23]. Immobilized lipase in particular is suitable for continuous biodiesel production because of the ease of its recovery from the reaction mixture. There are two major limitations of lipase-catalyzed biodiesel synthesis. One is higher cost (which can be reduced up to a certain extent by immobilization) and another is its inactivation by methanol and glycerol. It has been reported that as methanol is insoluble in vegetable oils, it inhibits the immobilized lipases and thereby decreases the catalytic activity of the transesterification reaction. Further, the hydrophilic by-product glycerol is also insoluble in the oil, so it is easily adsorbed onto the surface of the immobilized lipase leading to a negative effect on lipase activity and operational stability . Use of several solvents such as n-hexane and petroleum ether in the reaction medium has been reported  but the problem persisted since the inhibition of lipases still occurred due to poor solubility of methanol and glycerol in the hydrophobic solvents . There are some reports on enhanced biodiesel synthesis in presence of t-butanol as a solvent [27–29]. As both methanol and glycerol are soluble in t-butanol, the inhibitory effect of methanol and glycerol on lipase activity is reduced. Moreover, t-butanol is not a substrate for the lipases because it does not act on tertiary alcohols.
Biodiesel synthesis from Jatropha oil has been reported by Chromobacterium viscosum and Pseudomonas cepacia lipases. In both the cases the ethanolysis of Jatropha oil for biodiesel synthesis has been carried out [20–30]. In the present investigation, methanolysis of Jatropha oil was performed using immobilized lipase from Enterobacter aerogenes in the presence of t-butanol as solvent.
Materials and methods
Jatropha oil (HPLC grade) was kindly gifted by Professor P Das. Methanol was purchased from Qualigens. Methyl oleate was procured from Sigma. All other solvents and reagents were of AR grade and were obtained from Merck.
All experiments were carried out using lipase from E. aerogenes. The extracellular lipase production from E. aerogenes was carried out in 250 ml Erlenmeyer flasks each containing 50 ml of a medium composed of peptone (0.5%), yeast extract (0.3%), NaCl (0.25%), MgSO4 (0.05%) and coconut oil (3.0%) at pH 7.0. Medium was sterilized and inoculated with 3.5 ml (4 × 108 cells/ml) of inoculum followed by incubation for 60 h at 34°C with shaking at 200 rpm. At the end of the incubation period, supernatant from the fermentation media was collected by centrifugation at 6987 g for 10 min. Supernatant was treated with acetone (1:4 v/v) for 1 h at 4°C followed by centrifugation at 6987 g for 10 min. The precipitate was dissolved in 50 mM phosphate buffer (pH 5.0) and lyophilized for use as a crude lipase preparation for subsequent immobilization.
The lipase assay was performed spectrophotometrically using p-nitro phenyl palmitate as substrate . p-nitro phenol was liberated from p-nitro phenyl palmitate by lipase mediated hydrolysis. One unit (U) of lipase was defined as the amount of enzyme that liberates one micromole of p-nitro phenol per minute under the assay conditions.
Lipase from E. aerogenes was immobilized on silica activated with ethanolamine followed by cross-linking with glutaraldehyde, as described previously .
Reaction setup for transesterification reaction
Transesterification reaction was carried out at 30°C in screw-capped vials placed inside a reciprocal shaker. The initial reaction mixture consisted of oil:methanol molar ratio of 1:2, t-butanol:oil volume ratio of 0.2, immobilized E. aerogene lipase 20 U and 200 rpm (unless otherwise stated), along with the respective controls (samples without enzyme). All the experiments were performed in triplicate and the results were reported as the mean ± standard deviation.
Sampling and analysis
Samples were taken from the reaction mixture at specified time intervals. The samples were centrifuged at 6987 g for 10 min at 4°C to remove the carrier containing the immobilized enzyme (thus negating the possibilities of additional reaction) followed by 100-fold dilution of the initial sample with n-hexane. The stability tests were performed in t-butanol in each cycle (up to 20 cycles) and the supernatant and residual immobilized enzyme activities were tested for enzyme leaching for more than seven cycles; no leaching of enzyme either in the supernatant or in the residual immobilized enzyme was observed.
Synthesis of fatty acid methyl ester was analyzed by injecting the diluted aliquots of the reaction mixture in a gas chromatograph (Agilent 6820). The column temperature was kept at 150°C for 0.5 min, raised to 250°C at 15°C/min and was maintained at this temperature for 6 min. The temperatures of the injector and detector were set at 245 and 350°C respectively. The % molar conversion of products was identified by comparing the peak areas of standard methyl esters at particular retention times. Quantification of the final products (fatty acid methyl esters) was done from the calibration curves of pure fatty acid methyl esters (methyl oleate, methyl linoleate, methyl stearate and methyl palmitate).
Results and discussion
Effect of substrate molar ratio
Effect of agitation speed
Effect of t-butanol quantity
Effect of reaction temperature
Effect of additional water content
Effect of enzyme concentration
Reusability of lipase
Fuel properties of Jatropha oil methyl esters
Fuel properties of methyl esters of Jatropha oil
Biodiesel standard ASTM 6751-02
Biodiesel standard EN 14214
1.9 to 6.0
3.5 to 5.0
Flash point (°C)
Pour point (°C)
-15 to 10
Calorific value (MJ/kg)
33 to 40
The present study shows that the efficient methanolysis of Jatropha oil is possible by lipase catalysis in presence of t-butanol as solvent. The present work is a comprehensive study on the reaction parameters influencing the enzymatic synthesis of biodiesel. Immobilized E. aerogenes lipase was employed to catalyze the transesterification reaction. The amount of enzyme and temperature were found to have an immense effect on biodiesel synthesis. The conversion increased with increasing temperature up to 55°C, which was near the boiling point of the reaction mixture. About 94% yield of biodiesel was obtained (of which 68% conversion was achieved with respect to methyl oleate) using 50 U of immobilized E. aerogenes lipase with 1:4 oil to methanol molar ratio at 55°C for 48 h. The high operational stability of immobilized lipase also indicates the efficiency of the process. From the present work, it has been demonstrated that methanolysis of Jatropha oil could be effectively carried out in this novel system with a good operational stability of the lipases. However, further research and development on additional fuel property measures, long-term run and wear analysis of biodiesel-fuelled engines is necessary.
The authors gratefully acknowledge CSIR, Govt of India and Department of Biotechnology, India for providing research fellowship to Annapurna Kumari and Paramita Mahapatra respectively.
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