Biochemical Engineering Journal 39 (2008) 185–189

Enzymatic production of biodiesel from Jatropha oil:

A comparative study of immobilized-whole cell and
commercial lipases as a biocatalyst
Sriappareddy Tamalampudi a , Mahabubur Rahman Talukder e , Shinji Hama d ,
Takao Numata b , Akihiko Kondo b , Hideki Fukuda a,c,∗
a Department of Molecular Science and Material Engineering, Faculty of Engineering, Kobe University,
1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan
b Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University,

1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan

c Organization of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-ku,

Kobe 657-8501, Japan

d Bio-energy Corporation, 9-7-2 Minaminanamatsu, Amgasaki 660-0053, Japan
e Institute of Chemical and Engineering Sciences, 1 Pesek Road, Jurong Island, Singapore 627833, Singapore

Received 27 March 2007; received in revised form 17 July 2007; accepted 15 September 2007

Abstract
The large percentage of biodiesel fuel (BDF) cost associated with feedstock oil and enzyme. In order to reduce the cost of BDF production, the
lipase producing whole cells of Rhizopus oryzae (ROL) immobilized onto biomass support particles (BSPs) was used for the production of BDF
from relatively low cost non-edible oil from the seeds of Jatropha curcas. The activity of ROL was compared with that of commercially available
most effective lipase (Novozym 435). Different alcohols as a hydroxyl donor are tested, and methanolysis of Jatropha oil progresses faster than
other alcoholysis regardless of lipases used. The maximum methyl esters content in the reaction mixture reaches 80 wt.% after 60 h using ROL,
whereas it is 76% after 90 h using Novozym 435. Both the lipases can be used for repeated batches and both lipases exhibit more than 90% of their
initial activities after five cycles. Our results suggest that whole-cell ROL immobilized on BSP is a promising biocatalyst for producing BDF from
oil.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Lipase; Transesterification; Filamentous fungi; Immobilized cells

1. Introduction
Biodiesel (BDF) produced by alcoholysis of vegetables oils
or animal fats is viewed as promising renewable resources of
fuel. The use of BDF is becoming increasingly important due to
diminishing petroleum reserves and environmental regulations.
BDF is expensive in comparison with petroleum-based fuel and
60–75% of the cost is associated with feedstock oil [1]. There-
fore, the exploring ways to reduce the cost of BDF with respect
∗ Corresponding author at: Department of Molecular Science and Material
Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-
ku, Kobe 657-8501, Japan. Tel.: +81 78 803 6192; fax: +81 78 803 6192.
E-mail address: fukuda@kobe-u.ac.jp (H. Fukuda).
to enzyme and substrate oils are of prime interest in the recent
BDF research.
Jatropha curcas, an agro-forestry crop is a genus comprising
70 species growing in topical and sub-tropical countries. Jat-
ropha grows as a natural habitat across sub-Sahara Africa, India,
South East Asia and China. It grows rapidly, takes approximately
2–3 years to reach maturity and generate economic yields. It has
a productive lifespan in excess of 30 years. The fatty acid com-
position of Jatropha oil is similar to other edible oils but the
presence of some anti-nutritional factors such as toxic phorbol
esters renders this oil unsuitable for cooking purposes [2]. Jat-
ropha oil is thus a promising candidate for BDF production in
terms of availability and cost.
BDF is industrially produced via chemical catalysis using
strong bases as a catalyst. The strong base process suffers

1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2007.09.002
186 S. Tamalampudi et al. / Biochemical Engineering Journal 39 (2008) 185–189
from several drawbacks such as difficulty in recovery of glyc-
erol, removal of base catalyst from product and the treatment
of alkaline wastewater. The utilization of lipases for the pro-
duction of BDF has been reported as an effective means of
circumventing the aforementioned problems [3,4]. The first dif-
ficulty of using lipase is that it is more expensive than the
base catalyst like NaOH. Immobilized lipase is distinguished
from free lipase because of its easy recovery from the reac-
tion mixture facilitating its repeated use. Several researchers
[5–7] have reported that the commercially available Novozym
435 (Candida antarctica lipase B immobilized on acrylic
resin) was the most effective catalyst among tested lipases
for the production of BDF. However, laborious and expen-
sive purification processes of this lipase from culture broth
are restricting its application for BDF production in industrial
scale.
Here, we report that whole cells of lipase producing Rhizo-
pus oryzae (ROL) immobilized onto biomass support particles
(BSPs) made of reticulated polyurethane foam can catalyze the
alcoholysis of Jatropha oil more effectively than Novozym 435.
The advantage of using whole cells of R. oryzae immobilized
onto BSP over Novozym 435 is that no labor intensive and cost
associated lipase purification steps prior to the immobilization
is required because the whole cells of R. oryzae can sponta-
neously immobilized onto BSPs during cultivation. Moreover,
Jatropha oil makes the biodiesel fuel production more feasi-
ble for industrial applications than the other edible vegetable
oils.
2. Materials and methods
2.1. Materials
Jatropha oil was obtained as a gift from Dr. Jayaveera,
Jawaharlal Technological University (JNTU), Oil Technolog-
ical Research Institute, Anantapur, India. The saponification
value of Jatropha oil is 210. The water content in Jatropha
oil is 1.5 wt.%. Candida antarctica lipase B immobilized on
macro-porous acrylic resin (Novozym 435) was purchased from
Sigma–Aldrich Japan K.K, Tokyo, Japan. According to the man-
ufacturer, the enzyme belongs to the class of triacylglycerol
hydrolases (EC 3.1.3.3), with a declared activity of ≥10,000 U/g
(propyl laurate units per gram). All other chemicals are of ana-
lytical grade.
2.2. Microorganism and culture medium
R. oryzae IFO 4697 which has 1,3-positional specificity
lipase was used as the whole-cell biocatalyst. The organism was
maintained on 4% potato dextrose agar (Difco, Sparks, MD,
USA) slants. R. oryzae was grown in basal medium containing
1% glucose/olive oil, polypepton 70 g, NaNO3 1.0 g, KH2 PO4
1.0 g and MgSO4 ·7H2 O 0.5 g in 1 l distilled water. Reticulated
polyurethane foam particles (Bridge Stone Co. Ltd., Osaka,
Japan) with a particle voidage of more than 97% and a pore
size of 50 pores per linear inch were used for the immobiliza-
tion of R. oryzae. In all the methanolysis experiments, 0.2 g of
BSPs containing immobilized R. oryzae and Novozym 435 were
used.
2.3. Air-lift bioreactor cultivation
Seed culture of R. oryzae was grown in 500 ml Sakaguchi
flask containing 100 ml basal medium with 1% glucose. After
cultivation for 24 h, the seed culture was transferred to air-lift
bioreactor containing 10 l basal medium with 30 g/l olive oil
and 12,000 BSPs. The bioreactor was maintained at 2.5 vvm at
30 ◦ C. During the growth, R. oryzae cells were naturally immo-
bilized in BSPs during the cultivation in air-lift bioreactor. After
cultivation, the BSP-immobilized cells were separated from the
culture medium by filtration, washed with tap water and dried
at room temperature for around 24 h followed by cross linking
with glutaraldehyde.
2.4. Glutaraldehyde treatment of immobilized cells
To stabilize the lipase activity, separated whole-cell bio-
catalysts were incubated with 0.1 vol.% glutaraldehyde (GA)
solution at 25 ◦ C for 1 h. BSPs were separated from the GA
solution and were shaken in phosphate buffer at 4 ◦ C for few
minutes, washed with tap water for 1 min followed by drying at
room temperature for 1 day.
2.5. Alcoholysis
Alcoholysis was carried out at 30 ◦ C in 50 ml screw-capped
vessel with reciprocal shaking at 150 rpm. A typical reac-
tion mixture consisted of Jatropha oil (5 g), alcohol–oil molar
ratio (3:1) and lipases (0.2 g) for the complete conversion of
triglycerides to methyl esters. Reaction was started by adding
lipase into pre-incubated reaction mixture. The alkyl ester con-
tents were analyzed by capillary gas chromatography (GC) as
described below [8]. The activity of lipases is expressed as
amount of methyl esters (ME) produced per hour per gram
lipase.
2.6. Analysis
Samples (150 ␮l) were taken from the reaction mixture at
specified time and centrifuged at 12,000 rpm for 5 min to obtain
the upper layer. The upper layer (80 ␮l) and tricaprylin (20 ␮l)
were precisely weighed into a 10 ml bottle, to which a small
amount of anhydrous sodium sulfate and hexane (3 ml) were
added. Tricaprylin and sodium sulfate served as the internal
standard and dehydrating agent, respectively. A 1.0 ␮l aliquot
of the treated sample was injected into GC-18A gas chromato-
graph (Shimadzu Corp., Kyoto, Japan) connected to a DB-5
capillary column (0.25 mm × 10 m, J&W Scientific, Folsom,
CA, USA). The column temperature was held at 150 ◦ C for
0.5 min, raised to 300 ◦ C at 10 ◦ C/min, and maintained at this
temperature for 3 min. The temperature for injector and flame
ionization detector (FID) were set at 245 and 250 ◦ C, respec-
tively.
 S. Tamalampudi et al. / Biochemical Engineering Journal 39 (2008) 185–189 187

Fig. 2. Effect of methanol–oil molar ratios on lipases activities. Reaction con-

Fig. 1. Effect of different alcohols on lipase activities. Reaction conditions:
Jatropha oil 5 g; alcohol–oil molar ratio 1:1; lipases 0.2 g; reaction temperature
30 ◦ C; reaction time 60 min.
3. Results and discussion
3.1. Effect of alcohol type on biodiesel production
More commonly used alcohols for biodiesel production are
methanol, ethanol, propanol and butanol. The short chain nor-
mal alcohols were employed in the transesterification reaction in
order to know their effect on whole-cell R. oryzae and Novozym
435 (Fig. 1). Although, both the lipases can able to catalyze the
reaction, ROL is more efficient than Novozym 435 regardless
of alcohol type. Among alcohols tested, methanol is most active
for production of biodiesel from Jatropha oil and the activities
of lipases decrease with the increase in alcohol chain length.
Due to low molecular weight and higher polarity, methanol may
easily diffuse and access the lipase enzyme localized in the cell
membrane of R. oryzae [9] resulting in higher reaction rate.
ditions: Jatropha oil 5 g; lipases 0.2 g; reaction temperature 30 ◦ C; reaction time
60 min.
foam may adsorb more methanol than acrylic resin particles
so that activity of ROL decreased more rapidly than that of
Novozym 435.
3.3. Effect of lipases loaded
In order to investigate the effect of the weight of lipase
enzyme on methanolysis of Jatropha oil, the amount of lipase is
varied while keeping the amount of oil constant. Fig. 3 shows
that the specific activity of ROL remains constant up to lipase
amount equivalent to 6 wt.% of Jatropha oil, while it is only
2 wt.% in case of Novozym 435. This result suggested that
the amount of Novozym 435 added was much greater than the
required and external mass transfer resistance had limited the
rate of transesterification reaction.
3.4. Time course methanolysis of Jatropha oil

3.2. Comparison of ROL and Novozym 435 resistance to Since the concentration of methanol more than 1/3 Mequiv.
methanol has adverse effects on Novozym 435 and ROL, the time course

For the complete conversion of palm oil to methyl esters,

at least 3 Mequiv. of methanol (i.e. methanol–oil ratio 3:1)
need to be added in the reaction mixture. However, maximum
1/2 Mequiv. of methanol is generally miscible with vegetable
oils and the insoluble methanol in reaction mixture inactivates
lipases [10,11]. The activities of ROL and Novozym 435 at dif-
ferent methanol–oil ratios are thus investigated to compare their
tolerance to methanol (Fig. 2). Activities of lipases increase
with the increase in methanol–oil ratio up to 1 corresponding to
1/3 Mequiv. of methanol. Both lipases activities are decreased
after methanol–oil ratio 1 and compared with Novozym 435,
ROL is more susceptible to be poisoned by methanol. The
porous materials used for immobilizing ROL and Novozym
435 are polyurethane foam and acrylic resin particles, respec-
tively. These materials could adsorb polar compounds such
as methanol. When methanol–oil ratio is high, the immiscible
Fig. 3. Effect of lipase loaded on methanolysis Jatropha oil. Reaction conditions:
methanol droplets attached to the materials reducing or blocking Jatropha oil 5 g; alcohol–oil molar ratio 1:1; reaction temperature 30 ◦ C; reaction
the entry of Jatropha oil (apolar) to lipases [10]. Polyurethane time 60 min.
188 S. Tamalampudi et al. / Biochemical Engineering Journal 39 (2008) 185–189

Fig. 4. Time course methanolysis of Jatropha oil at different water content. The molar equivalent of methanol is added by three successive addition of 1/3 Mequiv.
of methanol at 0, 4 and 17 h for ROL (a) and at 0, 20 and 63 h for Novozym 435 (b). Reaction conditions: Jatropha oil 5 g; lipases 0.2 g; reaction temperature 30 ◦ C.

Table 1
Recyclability of the whole-cell biocatalyst (ROL) and Novozyme 435
Cycle number ROL Novozym 435

ME content (%) Residual activity (%) ME content (%) Residual activity (%)

1 80.2 100 75.1 100

2 78.3 97.6 73.2 97.4
3 78.2 97.5 71.6 95.3
4 75.5 94.1 70.5 93.8
5 73.1 91.1 70.5 93.8

of methyl esters production was carried out by three successive
additions of 1/3 Mequiv. of methanol. The progress of methanol-
ysis reaction catalyzed either by ROL or Novozym 435 is shown
in Fig. 4a and b, respectively. In case of ROL, ME content reaches
80% after 60 h at added water 5% (v/v), above which (e.g. at
10% water) methyl ester yield decreased. Since acyl migration
occurs with the intracellular lipase of immobilized cell and water
may improve the cell permeability, the rate of methanolysis cat-
alyzed by ROL increases in presence of added water. However,
excess water reduces methanolysis as it also acts as a competitive
inhibitor for lipase-catalyzed esterification or transesterification
[12]. In case of Novozym 435, the rate of methanolysis decreases
with increase in water content and ME content reaches 75% after
90 h at 0% added water. The results suggest that ROL catalyzes
methanolysis of Jatropha oil more efficiently than Novozym 435.
In addition, ROL is active even at 5–10% water content in Jat-
ropha oil, while Novozym 435 needs nearly anhydrous reaction
medium. Shimada et al. [11] reported that water (>500 ppm)
in soybean oil decreased the rate of methanolysis catalyzed by
Novozym 435. It should be mentioned that crude vegetable oil
usually contains 3–5% water. Therefore, the step for removing
water from crude Jatropha oil can be avoided when ROL is used
for production of biodiesel.
directly used for the next batch. The time of methanolysis using
ROL and Novozym 435 are kept constant at 60 and 90 h, respec-
tively for each reaction cycle. Table 1 shows that no significant
decrease in BDF content is observed after five repeated cycles
and both lipases exhibit more than 90% of their initial activities.
It is observed that ROL is easier to separate from reaction mix-
ture because of its larger size (4 mm × 4 mm) compared with
Novozym 435 (<1 mm in diameter).
4. Conclusions
Whole-cell R. oryzae immobilized onto BSP catalyzes the
methanolysis of Jatropha oil more efficiently than Novozym 435.
The presence of water in Jatropha oil has significant effect on
the rate of methanolysis and ROL exhibits highest activity in
presence of 5% (v/v) added water. In contrast to ROL, Novozym
435 activity is inhibited by the presence of added water and
it needs nearly anhydrous media for efficient catalyzing. The
results obtained here suggest that whole-cell ROL immobilized
onto BSP can be used as low cost biocatalyst for production of
biodiesel from crude Jatropha oil. Furthermore, expensive down
stream processing steps for lipase enzyme and refining methods
for the Jatropha oil can be avoided.

3.5. Repeated use of ROL and Novozym 435 References

In order to test the reusability of ROL and Novozym 435, [1] T. Krawczyk, Biodiesel-alternative fuel makes inroads but hurdles remain,
both the biocatalysts were filtered from the reaction mixture and Informatics 7 (1996) 801–829.
 S. Tamalampudi et al. / Biochemical Engineering Journal 39 (2008) 185–189
[2] S. Shah, S. Sharma, M.N. Gupta, Biodiesel preparation by lipase-catalyzed
transesterification of Jatropha oil, Energy Fuel 18 (2004) 154–159.
[3] H. Fukuda, A. Kondo, H. Noda, Biodiesel fuel production by transesterifi-
cation of oils—a review, J. Biosci. Bioeng. 92 (2001) 405–416.
[4] L.A. Nelson, T.A. Foglia, W.N. Marmer, Lipase-catalyzed production of
biodiesel, J. Am. Oil Chem. Soc. 73 (1996) 1191–1195.
[5] W. Du, Y. Xu, D. Liu, J. Zeng, Comparative study on lipase-catalyzed
transformation of soybean oil for biodiesel production with different acyl
acceptors, J. Mol. Catal. B: Enzym. 30 (2004) 125–129.
[6] Y. Shimada, Y. Watanabe, T. Samukawa, A. Sugihara, H. Noda, H. Fukuda,
Y. Tominaga, Conversion of vegetable oil to biodiesel using immobilized
Candida antarctica lipase, J. Am. Oil Chem. Soc. 76 (1999) 789–793.
[7] Y. Xu, W. Du, D. Liu, J. Zeng, A novel enzymatic route for biodiesel
production from renewable oils in a solvent-free medium, Biotechnol. Lett.
25 (2003) 1239–1241.
[8] M. Kaieda, T. Samukawa, T. Matsumoto, K. Ban, A. Kondo, Y. Shimada,
H. Noda, F. Nomoto, K. Ohtsuka, E. Izumoto, H. Fukuda, Biodiesel fuel
189
production from plant oil catalyzed by Rhizopus oryzae lipase in a water-
containing system without an organic solvent, J. Biosci. Bioeng. 88 (1999)
627–631.
[9] S. Hama, S. Tamalampudi, T. Fukumizu, K. Miura, H. Yamaji, A. Kondo, H.
Fukuda, Lipase localization in Rhizopus oryzae cells immobilized within
biomass support particles for use as whole-cell biocatalysts in biodiesel
fuel production, J. Biosci. Bioeng. 101 (2006) 328–333.
[10] J.W. Chen, W.T. Wu, Regeneration of immobilized Candida ntarctica lipase
for transesterification, J. Biosci. Bioeng. 95 (2003) 466–469.
[11] Y. Shimada, Y. Watanabe, A. Sugihara, Y. Tominaga, Enzymatic alcohol-
ysis for biodiesel fuel production and application of the reaction to oil
processing, J. Mol. Catal. B: Enzym. 76 (2002) 133–142.
[12] R.H. Valivety, G.A. Johnston, C.J. Suckling, P.J. Halling, Solvent
effects on biocatalysis in organic systems: equilibrium position and
rates of lipase-catalysed esterification, Biotechnol. Bioeng. 38 (1991)
1137–1143.