154 Energy & Fuels 2004, 18, 154-159

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 (12.8%), and stearic acid (7.3%). While composition of

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.: 91-
11-2659 1503; 91-11-2659 6568. Fax: 91-11-2658 1073. E-mail:
mn_gupta@hotmail.com.
(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) Öznur, K.; Tuten, M.; Aksoy, H. A. Bioresour. Technol. 2002,
83, 125.
(6) Linko, Y. Y.; Lämsä, M.; Wu, X.; Vosukainen, W.; Sappälä, 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.
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 biotrans-
formations.12-14 One frequently used strategy has been
the use of lipase for transesterification reactions. The
transesterification reaction, which is employed for bio-
diesel 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.

10.1021/ef030075z CCC: $27.50 © 2004 American Chemical Society

Published on Web 01/03/2004
Biodiesel Preparation
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 reac-
tion 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 triglyc-
eride 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 lipase-
catalyzed 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.
Energy & Fuels, Vol. 18, No. 1, 2004 155
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, Jawa-
harlal 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. Etha-
nol 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 phos-
phate 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 im-
mediately frozen at -20 °C and lyophilized for 48 h.
These are referred to as “tuned” enzyme.
(2) Immobilized Enzyme Preparations. Candida ru-
gosa 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 phos-
phate 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
156 Energy & Fuels, Vol. 18, No. 1, 2004
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 Na2-
SO4. 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 gas-
chromatography 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.
Shah et al.
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-chromatogra-
phy 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%.
Biodiesel Preparation Energy & Fuels, Vol. 18, No. 1, 2004 157

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 pro-
grammed 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 ru- Figure 3. Fatty acid composition of the Jatropha oil ester
gosa, and porcine pancreas, only the lipase from Chro- using gas chromatography. Peak 1: n-hexane, Peak 2: methyl
mobacterium viscosum was capable of transesterifica- laurate (internal standard), Peaks 3-6 correspond to ethyl
tion. With this enzyme, a yield of 62% for the ethyl ester esters of palmitic, stearic, oleic, and linoleic acid, respectively.
could be obtained after 8 h. The reaction could be
qualitatively followed by TLC (Figure 2) and quantita-
tively 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 reac-
tions 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 Forma-
tion. It has also been reported that the amount of buffer
salts (apart from buffer pH) present during lyophiliza-
tion (while tuning the enzyme) also affects the reaction
rates. Figure 4 shows that this optimization was useful
Figure 4. Effect of the amount of the buffer salt on biodiesel
and maximum yield was obtained when the amount of synthesis. Chromobacterium viscosum lipase (50 mg) was
buffer salt was 0.2 mmol/g of enzyme. lyophilized in 20 mM sodium phosphate buffer (0.2, 0.5, 1.0,
Effect of Varying Amount of Enzyme. Figure 5 shows 2.0, 5.0 mL), pH 7.8. The separately lyophilized enzyme
the effect of varying amount of enzyme under these powders were added to reactions media containing Jatropha
optimized conditions. The best results were obtained oil and ethanol in the molar ratio of 1:4. Each of the above
with 50 mg or 75 mg of enzyme. The higher amount of reactions was carried out in duplicate, and the yields between
duplicates were found to agree within 3%.
enzyme (i.e., 100 mg) in fact decreased the product yield,
this was presumably due to increase in the viscosity (the
sorbed on Celite.29 Table 1 shows that the percent yield
solution was found to be quite viscous in this case) which
increased to 71% when immobilized enzyme preparation
reduced the reaction rate to the extent that an ad-
from Chromobacterium viscosum was used for transes-
ditional amount of enzyme did not help. As there was
terification. Adding a larger amount of immobilized
not much difference between the yield obtained with 50
enzyme was not practical since the matrix and enzyme
mg and 75 mg, 50 mg of enzyme was used further in
together made the solution extremely viscous. The
order to save on the cost of the enzyme.
immobilized preparations with other lipases (Candida
Transesterification with Immobilized Enzyme.
rugosa and Porcine pancreas) did show some detectable
In biotransformation involving use of lipases in non-
ethyl ester formation but the yield was extremely poor.
aqueous media, the enzyme has frequently been ad-
Table 1 summarizes this result. The chemical process
(27) Ban, K.; Kaieda, M.; Matsumoto, T.; Kondo, A.; Fukuda, H.
carried out for comparative purpose yielded 93% ethyl
Biochem. Eng. J. 2001, 8, 39.
(28) Samukawa, T.; Kaieda, M.; Matsumoto, T.; Ban, K.; Kondo, A.; (29) Mustranta, A.; Forssell, P.; Poutanen, K. Enzyme Microb.
Shimada, Y.; Noda, H.; Fukuda, H. J. Biosci. Bioeng. 2000, 90, 180. Technol. 1993, 15, 133.
158 Energy & Fuels, Vol. 18, No. 1, 2004 Shah et al.

Figure 5. Effect of amount of enzyme addition on biodiesel

synthesis. Varied amounts of Chromobacterium viscosum Figure 6. Effect of incubation time on biodiesel production
lipase (10, 50, 75, 100 mg) were lyophilized in 20 mM sodium using immobilized Chromobacterium viscosum lipase. Each of
phosphate buffer (0.5 mL), pH 7.8. The separately lyophilized the above reactions was carried out in duplicate and the yields
enzyme powders were added to reaction media containing between duplicates were found to agree within 3%.
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%.

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 93
enzymatically
free tuned Chromobacterium 62
free tuned Candida rugosa 0
free tuned Porcine pancreas 0
immobilized Chromobacterium 71
immobilized Candida rugosa 1.0
immobilized Porcine pancreas 1.1
a Each of the reactions was carried out in duplicate, and the

yields in duplicates were found to agree within 3%.

Figure 7. Effect of percent water (added to the reaction

esters. In general, the reported yield with chemical medium) on biodiesel production using tuned and immobilized
transesterification is between 90 and 95%.7,30 Foidl et Chromobacterium viscosum lipases. Each of the above reac-
al. have reported an yield of 88.4% by chemical trans- tions was carried out in duplicate and the yields between
esterification of Jatropha oil.17 Figure 6 shows the rate duplicates were found to agree within 3%.
obtained with immobilized lipase from Chromobacte-
media is another critical parameter, which is known to
rium viscosum under the optimized conditions. Thus, a
influence biotransformations in nonaqueous media.32
yield of 89% could be obtained in 10 h using the
The general picture available is that less than a mono-
immobilized enzyme preparation. Biodiesel production
layer of water is required for an enzyme to show
using lipases has been tried both in nonaqueous sol-
biological activity. As the water level increases, it
vent18 and in a solvent-free system.5 Methanolysis of
increases the enzyme flexibility and the expressed
tallow oil, using Mucor mehei lipase in hexane, had led
activity.33 After an optimum level of water, hydrolytic
to 77.8% ester yield.18 Lately, a solvent-free transes-
reactions become significant and transesterification
terification reaction is favored by many workers, since
yield is expected to go down. Figure 7 shows the effect
it is more economical.2,21 Öznur et al.5 reported alco-
of different amounts of water present in the solvent-
holysis of cotton seed oil in a solvent-free medium, using
free system on biodiesel yield. Addition of 1% (w v-1)
immobilized Candida antarctica lipase with 92% of the
water in the case of free enzyme and 0.5% (w v-1) water
total ester yield. A more extensive list of such efforts
in the case of immobilized Chromobacterium viscosum
has been summarized by us elsewhere recently.31 The
lipase gave the maximum yields of 73 and 92%, respec-
yield obtained by us by following a solvent-free approach
tively. Iso et al.24 have also reported that water content
is comparable to the yield obtained by other workers
is an important parameter for lipase-catalyzed trans-
with different approaches.5,31
esterification reaction.
Effect of Percent Water in Reaction Media on
Finally, these results show the viability of using
Biodiesel Production. Amount of water present in the
lipases in a solvent-free system for obtaining biodiesel
(30) Freedman, E.; Pryde, E. H.; Mounts, T. L. J. Am. Oil Chem. (32) Chowdary, G. V.; Prapulla, S. G. Process Biochem. 2002, 38,
Soc. 1984, 61, 1638. 393.
(31) Shah, S.; Sharma, S.; Gupta, M. N. Indian J. Biochem. Biophys., (33) Triantafyllou, A. O.; Wehtje, E.; Adlercreutz, P.; Mattiasson,
in press. B. Biotech. Bioeng. 1995, 45, 406.
Biodiesel Preparation
from Jatropha oil. It is also worth noting that the overall
yields by chemical transesterification (93%) and enzy-
matic 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.
Energy & Fuels, Vol. 18, No. 1, 2004 159
The funds provided by Council of Scientific and Indus-
trial 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 organiza-
tions, are gratefully acknowledged.
EF030075Z