Bioresource Technology 91 (2004) 7784

Optimization of extracellular lipase production

by Geotrichum sp. using factorial design
J.F.M. Burkert, F. Maugeri, M.I. Rodrigues *

Department of Food Engineering, Universidade Estadual de Campinas, UnicampCampinas SP, CP 6121, Cep 13081-970, Brazil
Received 11 September 2001; received in revised form 15 May 2003; accepted 15 May 2003

Abstract
Response surface methodology was employed to study the eects of carbon source (soy oil, olive oil and glucose) and nitrogen
source concentrations (corn steep liquor and NH4 NO3 ) on the lipase production by Geotrichum sp. The experiment included a 24
central composite rotatable design (CCRD) and four others 23 CCRD. According to the responses from the experimental designs,
the eects of each variable were calculated and the interactions between them were determined. The response surface methodology
was applied for the optimization of the nutrient concentrations in the culture medium for the enzyme production, at 30
C. The
optimum medium composition for lipase production by Geotrichum sp. was ammonium nitrate 2.12.5%, corn steep liquor 1315%
and soy oil 0.6% as carbon source, which lead to a lipase activity of about 20 U/ml. Using olive oil as carbon source, the optimum
composition was ammonium nitrate 0.81%, corn steep liquor 1315% and olive oil 0.6%, leading to an activity of 17 U/ml.
 2003 Elsevier Ltd. All rights reserved.

Keywords: Factorial design; Surface response analysis; Optimization; Lipase production; Geotrichum sp.

1. Introduction lipids from animal skins) and medical (blood triglyceride

Lipolytic enzymes, such as lipases (E.C. 3.1.1.3), can
be found in animals, plants and microorganisms (Ka-
mimura et al., 2001; Elibol and Ozer, 2000). Lipases
hydrolyze triglycerides to fatty acids and glycerol, and
under certain conditions catalyze the reverse reaction,
producing glycerides from glycerol and fatty acids
(Saxena et al., 1999). Some lipases are also able to cata-
lyze esterication, transesterication and enantioselec-
tive hydrolysis reactions (Nini et al., 2001; Shintre et al.,
2002; Nagayama et al., 2002; Piao et al., 2003; Kui et al.,
2003; Raku et al., 2003). The interest in microbial lipase
production has increased in the last decades, because of
its large potential in industrial applications as additives
in foods (avor modication), ne chemicals (synthesis
of esters), detergents (hydrolysis of fats), waste water
treatment (decomposition and removal of oil sub-
stances), cosmetics (removal of lipids), pharmaceuticals
(digestion of oil and fats in foods), leather (removal of
*
Corresponding author. Tel.: +55-197-884031; fax: +55-197-
884027.
E-mail address: bel@fea.unicamp.br (M.I. Rodrigues).
assay) (Elibol and Ozer, 2000; Kamini et al., 2000).
The lipase from Geotrichum candidum is currently the
subject of intense investigation and industrial interest
because it shows a strong preference for fatty acids that
contain a cis-9 double bound (Bertolini et al., 1994;
Jacobsen and Poulsen, 1995; Mac^edo et al., 1997). Re-
cent applications of the cis-9-selective enzyme in the
hydrolysis or esterication of CLA (conjugated linoleic
acid), a mixture of octadecadienoic acids diering in the
position of their conjugated double bonds, has attracted
considerable attention because of its anticarcinogenic
and antiatherogenic properties (Warwel and Borgdorf,
2000). Lipase synthesis and secretion required induction
for some strains of microorganisms (Pokorny et al.,
1997).
In order to increase production levels and purication
factors of lipases from several microorganisms cloning
techniques have been employed (Phillips et al., 1995;
Nagao et al., 1996). Production of lipase by Candida
rugosa was optimized employing a response surface
method (Rao et al., 1993). The optimal nutrient con-
centrations were 0.25% urea, 4.5% maltose and 15% oil
for biomass production, or 0.5% urea, 1.5% maltose and
7.5% oil for lipase production. The medium optimization

0960-8524/$ - see front matter  2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-8524(03)00152-4
78 J.F.M. Burkert et al. / Bioresource Technology 91 (2004) 7784
for lipase production by Rhizopus delemar using indus-
trial grade carbon sources (glucose, yellow dextrin and
amidex) and nitrogen sources (sodium caseinate, soya
paste, soybean our and yeast extract) was studied by
Cruz et al. (1993). These authors selected soya paste and
dextrin as the best carbon and nitrogen sources. Opti-
mization of the medium composition was carried out
with a fractional factorial design in two steps. The lipo-
lytic activity after 48 h of fermentation was 12-fold
higher than the activity with the initial medium. These
results showed that it was necessary to modify the carbon
source-nitrogen source ratio, to reduce olive oil concen-
tration and add Tween 80 to the medium to achieve
maximum production.
The eect of carbon and nitrogen sources on extra-
cellular lipase production from Aspergillus niger was
also studied applying two complete experimental designs
and response surface analysis by Hatzinikolaou et al.
(1996). Four carbon sources (glucose, sucrose, corn oil
and olive oil) and three nitrogen sources (ammonium
phosphate, ammonium sulphate and peptone) were
used. The optimum medium was shown to be a combi-
nation of corn oil and peptone, with a maximum lipase
activity of 42.4 U/ml.
Penicillium cyclopium triacylglycerol lipase produc-
tion was maximized in stationary batch culture using
surface response methodology based on a Doehlert ex-
perimental design. The optimal conditions for high lipase
production were 1% substrate (corn steep), pH 5.5 and
inoculum of 104 spores/ml using 1% of commercial olive
oil. A nine-fold increase of both the predicted and mea-
sured values for lipase was veried (Vanot et al., 2001).
Factorial design and response surface analysis are
important tools to determine the optimal process con-
ditions. Factorial design of a limited set of variables is
advantageous compared to the conventional method,
which handles a single parameter per trial, as such an
approach frequently fails to locate optimal conditions
due to its failure to consider the eect of possible in-
teractions between factors (Kalil et al., 2000). In this
work the eect of carbon source (soy oil, olive oil and
glucose) and nitrogen source concentrations (corn steep
liquor and NH4 NO3 ) on the lipase production by Geo-
trichum sp. were studied at 30
C and the optimal con-
ditions for lipase production were determined.
2. Methods
2.1. Microorganism, medium inoculum and enzyme pro-
duction
A Geotrichum sp. isolated by Mac^edo et al. (1997)
was used in this study. The inoculum was produced in a
medium consisted of 5% corn steep liquor, 1% soy oil or
1% olive oil and 0.5% ammonium nitrate. Flasks of 1000
ml containing 200 ml of culture medium were incubated
in a shaker (120 rpm) at 30
C, for 24 h. Lipase pro-
duction was carried out with a 10% (v/v) inoculum of
Geotrichum sp. in 500 ml shaken-asks with 200 ml of
culture medium, and incubated at 30
C in a rotary
shaker (120 rpm) for about 50 h.
2.2. Lipase assay
Lipase assay was performed with olive oil emulsion,
which was prepared by mixing 25 ml of olive oil and 75
ml of 7% arabic gum solution in a homogenizer for 5
min at 500 rpm. The reaction mixture containing 5 ml of
olive oil emulsion, 2 ml of 10 mM phosphate buer (pH
7.0) and 1 ml of the culture supernatant was incubated
at 37
C for 30 min with orbital shaking. The emulsion
was immediately disrupted after incubation by the ad-
dition of 15 ml of acetoneethanol mixture (1:1 v/v), and
the liberated free fatty acids were titrated with 0.05 N
NaOH. One unit of lipase activity was dened as the
amount of enzyme, which liberated 1 lmol of fatty acid
per minute (Mac^edo et al., 1997).
2.3. Factorial design
Five factorial designs were carried out. In the rst
one, a 24 factorial design was performed in order to
study the eects of glucose concentration (G), corn steep
liquor concentration (CSL) and ammonium nitrate
concentration (NH4 NO3 ) with 1% olive oil (OO) or soy
oil (SO). The lipase production was studied as a re-
sponse. Additionally, two 23 factorial designs were em-
ployed, in which the concentrations of CSL, ammonium
nitrate and soy oil were studied, and two others 23 fac-
torial designs studied the eects of CSL, ammonium
nitrate and olive oil. The independent variables and their
levels are presented in Table 1 (rst factorial design),
Table 2 (second factorial designs), Table 3 (third facto-
rial design), Table 4 (fourth factorial design) and Table 5
(fth factorial design).
In the rst experimental design, 22 experimental runs
were carried out, which included 16 factorial points and
6 central points (3 central points with olive oil and 3
central points with soy oil). Three variables, at ve
levels, were studied in the remaining experimental de-
signs, which required 17 runs, included 8 factorial points
and 3 central points. Central points provide additional
degrees of freedom for error estimating, which increases
power when testing the signicance of eects (Carvalho
et al., 1997). A 23 experimental design with star cong-
uration (6 axial points) and 3 central points, totaling 17
experiments, was carried out to obtain a second order
model. The distance of the axial points was
1.68, cal-
culated from Eq. (1) (Khuri and Cornell, 1987).
1=4
a 2n 1
 J.F.M. Burkert et al. / Bioresource Technology 91 (2004) 7784 79

Table 1
Coded levels and real values (in parentheses) for the rst factorial design (22 trials) and lipase activity (U/ml) during the fermentations
Run Variables (%) Enzyme activity (U/ml) (h)
G CSL NH4 NO3 Oil 9.5 24.0 33.5 48.5
1 )1 (0) )1 (2) )1 (0) )1 (SO) 7.96 9.77 8.69 9.53
2 +1 (1) )1 (2) )1 (0) )1 (SO) 2.71 2.30 2.06 2.36
3 )1 (0) +1 (6) )1 (0) )1 (SO) 6.67 7.37 6.47 8.15
4 +1 (1) +1 (6) )1 (0) )1 (SO) 4.29 4.05 3.76 4.94
5 )1 (0) )1 (2) +1 (0.75) )1 (SO) 8.60 9.86 8.38 5.49
6 +1 (1) )1 (2) +1 (0.75) )1 (SO) 4.87 5.12 6.05 4.74
7 )1 (0) +1 (6) +1 (0.75) )1 (SO) 6.96 8.69 8.46 11.18
8 +1 (1) +1 (6) +1 (0.75) )1 (SO) 7.26 6.77 6.54 6.50
9 )1 (0) )1 (2) )1 (0) +1 (OO) 8.51 8.84 7.68 8.66
10 +1 (1) )1 (2) )1 (0) +1 (OO) 3.29 1.75 2.33 2.36
11 )1 (0) +1 (6) )1 (0) +1 (OO) 7.45 8.88 9.19 12.77
12 +1 (1) +1 (6) )1 (0) +1 (OO) 4.53 3.90 4.45 8.20
13 )1 (0) )1 (2) +1 (0.75) +1 (OO) 9.13 8.79 9.44 10.31
14 +1 (1) )1 (2) +1 (0.75) +1 (OO) 5.87 5.01 4.35 4.09
15 )1 (0) +1 (6) +1 (0.75) +1 (OO) 8.45 8.60 8.13 9.44
16 +1 (1) +1 (6) +1 (0.75) +1 (OO) 7.26 6.88 6.22 6.53
17 0 (0.5) 0 (4) 0 (0.375) 0 (SO) 7.09 6.34 5.79 5.82
18 0 (0.5) 0 (4) 0 (0.375) 0 (SO) 6.95 6.29 5.79 5.72
19 0 (0.5) 0 (4) 0 (0.375) 0 (SO) 6.81 6.26 5.70 5.56
20 0 (0.5) 0 (4) 0 (0.375) 0 (OO) 4.77 6.18 6.12 6.55
21 0 (0.5) 0 (4) 0 (0.375) 0 (OO) 6.18 5.62 5.62 6.96
22 0 (0.5) 0 (4) 0 (0.375) 0 (OO) 6.05 5.53 6.01 6.14

Table 2
Coded levels and real values (in parentheses) for the second factorial design (17 trials) and lipase activity (U/ml) during the fermentations
Run Variables (%) Enzyme activity (U/ml) (h)
SO NH4 NO3 CSL 7.5 24 31.5 48.0 54.0
1 )1 (0.7) )1 (0.2) )1 (2.4) 9.12 8.12 7.95 8.48 8.40
2 +1 (1.3) )1 (0.2) )1 (2.4) 8.45 8.35 8.34 8.02 7.34
3 )1 (0.7) +1 (0.8) )1 (2.4) 10.91 10.18 9.96 7.82 10.11
4 +1 (1.3) +1 (0.8) )1 (2.4) 10.71 10.67 9.90 9.91 9.00
5 )1 (0.7) )1 (0.2) +1 (6.6) 11.78 11.90 11.20 10.32 10.91
6 +1 (1.3) )1 (0.2) +1 (6.6) 11.24 11.70 11.47 11.11 9.89
7 )1 (0.7) +1 (0.8) +1 (6.6) 11.94 12.07 11.84 11.41 10.19
8 +1 (1.3) +1 (0.8) +1 (6.6) 11.82 11.32 11.70 11.53 10.04
9 )1.68 (0.5) 0 (0.5) 0 (4.5) 10.34 10.18 9.55 13.24 12.71
10 +1.68 (1.5) 0 (0.5) 0 (4.5) 10.28 10.42 9.92 10.04 8.91
11 0 (1) )1.68 (0) 0 (4.5) 9.53 10.33 9.07 10.01 8.38
12 0 (1) +1.68 (1) 0 (4.5) 11.30 10.73 10.88 11.35 9.66
13 0 (1) 0 (0.5) )1.68 (1) 7.62 5.36 3.56 2.93 1.67
14 0 (1) 0 (0.5) +1.68 (8) 12.87 14.47 14.04 13.47 12.33
15 0 (1) 0 (0.5) 0 (4.5) 11.75 10.90 11.02 10.84 9.92
16 0 (1) 0 (0.5) 0 (4.5) 11.72 10.48 10.26 10.83 9.50
17 0 (1) 0 (0.5) 0 (4.5) 10.31 9.57 9.19 10.32 8.92

a is the distance of the axial points; n is the number of
independent variables.
All data were treated with the aid of STATISTICA
5.0 from Statsoft Inc. (2325 East 13th Street, Tulsa, OK,
74104, USA).
3. Results and discussion
The experimental conditions and the results for lipase
activity as a function of time, in the rst factorial design,
are shown in Table 1. The statistical analysis was per-
formed with data obtained at 9.67 h of fermentation, as
there was no signicant increase in the lipase activity
after this time, for most of the experiments, as shown in
Fig. 1. Also, as the activity is nearly constant, the pro-
ductivity decreases gradually with fermentation time.
This criterion was used in all experimental designs. The
eect estimates for each variable were determined and
reported in Table 6, for 9.67 h of fermentation. An es-
timate of a main eect is obtained by evaluating the
dierence in process performance caused by a change
80 J.F.M. Burkert et al. / Bioresource Technology 91 (2004) 7784

Table 3
Coded levels and real values (in parentheses) for the third factorial design (17 trials) and lipase activity during the fermentations
Run Variables (%) Enzyme activity (U/ml) (h)
SO NH4 NO3 CSL 8.25 24.08 32 48.42 53.5
1 )1 (0.36) )1 (0.9) )1 (7.02) 11.61 12.41 13.79 13.38 14.20
2 +1 (0.84) )1 (0.9) )1 (7.02) 12.41 13.12 14.46 13.70 14.88
3 )1 (0.36) +1 (2.1) )1 (7.02) 14.72 14.91 15.60 15.60 15.98
4 +1 (0.84) +1 (2.1) )1 (7.02) 14.72 15.33 15.77 15.65 15.68
5 )1 (0.36) )1 (0.9) +1 (12.98) 15.65 17.92 19.09 19.86 20.48
6 +1 (0.84) )1 (0.9) +1 (12.98) 15.59 15.28 17.25 16.86 16.45
7 )1 (0.36) +1 (2.1) +1 (12.98) 17.86 17.02 17.78 17.07 16.94
8 +1 (0.84) +1 (2.1) +1 (12.98) 18.56 18.20 18.18 18.16 18.00
9 )1.68 (0.2) 0 (1.5) 0 (10) 14.67 16.46 18.22 17.60 17.66
10 +1.68 (1.0) 0 (1.5) 0 (10) 14.00 14.96 16.42 16.81 16.14
11 0 (0.6) )1.68 (0.5) 0 (10) 13.43 15.45 16.15 17.97 18.70
12 0 (0.6) +1.68 (2.5) 0 (10) 17.46 17.39 17.52 17.62 17.47
13 0 (0.6) 0 (1.5) )1.68 (5.0) 12.46 13.77 15.64 16.13 16.00
14 0 (0.6) 0 (1.5) +1.68 (15) 18.07 17.30 18.12 17.85 17.75
15 0 (0.6) 0 (1.5) 0 (10) 14.09 15.07 15.86 16.90 16.68
16 0 (0.6) 0 (1.5) 0 (10) 14.17 15.64 16.72 17.63 17.41
17 0 (0.6) 0 (1.5) 0 (10) 13.79 17.11 17.68 17.95 17.73

Table 4
Coded levels and real values (in parentheses) for the fourth factorial design (17 trials) and lipase activity during the fermentations
Run Variables (%) Enzyme activity (U/ml) (h)
OO NH4 NO3 CSL 8 24 33.7 48 56.2
1 )1 (0.7) )1 (0.2) )1 (2.4) 9.31 8.57 9.68 11.25 13.71
2 +1 (1.3) )1 (0.2) )1 (2.4) 9.04 8.58 9.91 9.19 9.45
3 )1 (0.7) +1 (0.8) )1 (2.4) 10.67 9.38 10.94 11.26 12.85
4 +1 (1.3) +1 (0.8) )1 (2.4) 10.64 11.21 11.22 4.63 4.56
5 )1 (0.7) )1 (0.2) +1 (6.6) 13.27 12.11 14.44 18.38 18.16
6 +1 (1.3) )1 (0.2) +1 (6.6) 11.91 11.22 12.67 12.62 13.17
7 )1 (0.7) +1 (0.8) +1 (6.6) 12.51 12.65 12.90 13.81 14.12
8 +1 (1.3) +1 (0.8) +1 (6.6) 13.28 13.12 13.92 14.47 15.73
9 )1.68 (0.5) 0 (0.5) 0 (4.5) 11.01 9.61 10.84 12.95 13.79
10 +1.68 (1.5) 0 (0.5) 0 (4.5) 11.25 10.05 11.24 11.70 11.88
11 0 (1) )1.68 (0) 0 (4.5) 10.56 9.88 11.20 12.03 13.66
12 0 (1) +1.68 (1) 0 (4.5) 13.95 13.13 15.10 16.35 17.63
13 0 (1) 0 (0.5) )1.68 (1) 7.68 6.33 5.95 3.09 2.69
14 0 (1) 0 (0.5) +1.68 (8) 12.91 12.98 14.13 14.20 14.51
15 0 (1) 0 (0.5) 0 (4.5) 10.96 10.48 12.10 11.88 13.17
16 0 (1) 0 (0.5) 0 (4.5) 10.63 9.26 10.14 10.74 10.63
17 0 (1) 0 (0.5) 0 (4.5) 10.77 10.15 11.19 11.94 12.71

from the low ()1) to the high (+1) level of the corre-
sponding factor (Haaland, 1989). The process perfor-
mance was measured by the lipase activity response.
Both the t-test and p-value statistical parameters were
used to conrm the signicance of factors studied (Ta-
bles 6 and 7). The t-value that measured how large the
coecient is in relationship to its standard error was
obtained by dividing each coecient by its standard
error. The p-value is the chance of getting a larger t-
value (in absolute value) by chance alone. A small p-
value suggested that the coecient was a large signal in
comparison to the noise because it was too large to have
arisen by chance alone. In this case, p < 0:05, suggested
signicance at the 0.05 level. This also corresponded to a
95% condence level for a test of the hypothesis that the
eects (or coecients) in question were equal to zero.
Small p-values were associated with larger t-value be-
cause they imply that the eects (or coecients) were
much greater than its standard error. It can be seen that
the most relevant variables concerning the lipase activity
are glucose and ammonium nitrate (Table 6). An in-
crease in glucose concentration from 0% to 1% exhibited
a negative inuence, reduction of lipase activity by 3 U/
ml, at 9.67 h. Glucose was consequently discarded as
carbon source. Similar results were observed for Peni-
cillium restrictum (Freire et al., 1997). On the other
hand, ammonium nitrate increased lipase activity. Fur-
thermore, there were no statistically signicant dier-
 J.F.M. Burkert et al. / Bioresource Technology 91 (2004) 7784 81

Table 5
Coded levels and real values (in parentheses) for the fth factorial design (17 trials) and the lipase activity during the fermentations
Run Variables (%) Enzyme activity (U/ml) (h)
OO NH4 NO3 CSL 8 24.3 32 48.6 54
1 )1 (0.36) )1 (0.9) )1 (7.02) 10.73 12.77 12.70 12.96 14.26
2 +1 (0.84) )1 (0.9) )1 (7.02) 12.63 13.85 13.04 12.75 13.90
3 )1 (0.36) +1 (2.1) )1 (7.02) 12.60 13.57 13.85 14.39 14.61
4 +1 (0.84) +1 (2.1) )1 (7.02) 14.41 15.23 15.22 14.82 15.65
5 )1 (0.36) )1 (0.9) +1 (12.98) 14.47 14.44 15.55 16.10 17.30
6 +1 (0.84) )1 (0.9) +1 (12.98) 14.26 14.06 15.09 15.65 15.35
7 )1 (0.36) +1 (2.1) +1 (12.98) 15.68 17.19 17.79 18.40 20.05
8 +1 (0.84) +1 (2.1) +1 (12.98) 15.23 16.24 16.94 16.30 16.67
9 )1.68 (0.2) 0 (1.5) 0 (10) 13.25 14.10 15.17 16.00 16.64
10 +1.68 (1.0) 0 (1.5) 0 (10) 12.93 13.32 14.30 14.67 14.74
11 0 (0.6) )1.68 (0.5) 0 (10) 12.60 13.84 13.95 13.73 15.39
12 0 (0.6) +1.68 (2.5) 0 (10) 14.32 14.47 14.54 14.49 15.64
13 0 (0.6) 0 (1.5) )1.68 (5.0) 13.38 13.28 13.23 15.02 15.41
14 0 (0.6) 0 (1.5) +1.68 (15) 16.33 17.15 17.71 18.36 18.75
15 0 (0.6) 0 (1.5) 0 (10) 13.06 14.50 15.29 15.10 15.89
16 0 (0.6) 0 (1.5) 0 (10) 12.89 13.33 14.15 14.31 14.73
17 0 (0.6) 0 (1.5) 0 (10) 12.58 13.60 14.39 14.91 16.05

Fig. 1. Lipase activity as a function of time for the rst 24 factorial design: (a) runs 111 and (b) runs 1222.

Table 6 3.1. Lipase production with soy oil

Main eects analysis for lipase activity from the rst factorial design at
9.67 h of fermentation
3.1.1. Eect of the carbon source and nitrogen source
Factor Eect Std. Err. t-value p-value concentrations
% Glucose )2.96 0.431 )6.851 0.0010a The experimental conditions and the results for lipase
% CSL 0.24 0.431 0.559 0.6002 production, according to the second factorial design, are
% NH4 NO3 1.62 0.431 3.763 0.0131a
presented in Table 2. The eect estimates for each
Oil type 0.65 0.431 1.497 0.1944
a
variable, as well as the interactions between them, were
Signicant factors (p < 0:05).
determined and reported for 7.5 h of fermentation
(Table 7). After this time, there was not signicant in-
ences between soy oil and olive oil for enzyme produc- crease in the lipase activity. The increase in soy oil
tion. The variation in the concentration of the corn steep concentration from 0.7% to 1.3% did not show signi-
liquor was not statistically signicant at a 95% con- cant inuence in lipase activity. The variables corn steep
dence level. After these results, the glucose was dis- liquor and ammonium nitrate were shown to be signi-
carded and a comparative study between soy oil and cant and the increase in the concentration of these
olive oil for lipase production was performed. variables led to an increase in lipase activity. The
82 J.F.M. Burkert et al. / Bioresource Technology 91 (2004) 7784

Table 7
Main eects and interactions analysis for lipase activity from the second factorial design after 7.5 h of fermentation, and fourth factorial design at 8 h
of fermentation
Second factorial design Fourth factorial design
Factor Eect Std. Err. t-value p-value Factor Eect Std. Err. t-value p-value
% SO (1) )0.38 0.491 )0.66 0.5784 % OO (1) )0.22 0.12 )1.90 0.1978
% NH4 NO3 (2) 1.20 0.491 2.06 0.1758a % NH4 NO3 (2) 0.89 0.12 7.62 0.0168b
% CSL (3) 1.90 0.491 3.26 0.0825c % CSL (3) 2.83 0.12 24.14 0.0017b
(1) (2) 0.22 0.491 0.38 0.7389 (1) (2) 0.59 0.12 5.06 0.0369b
(1) (3) 0.05 0.491 0.09 0.963 (1) (3) )0.08 0.12 )0.62 0.5990
(2) (3) )0.83 0.491 )1.42 0.2909 (2) (3) )0.59 0.12a )5.02 0.0375b
a
Signicant factors (p < 0:2).
b
Signicant factors (p < 0:05).
c
Signicant factors (p < 0:1).

interactions between these three factors were not sta- Table 8

tistically signicant for lipase activity. In the third ex- ANOVA for the third factorial design at 8.25 h of fermentation
perimental design, the concentration ranges for corn Source of Sum of Degrees of Mean F -ratio (model
variation square freedom square signicance)
steep liquor and ammonium nitrate were increased and,
for soy oil, it was decreased. Regression 67.09 4 16.77 167.70a
Residual 1.170 12 0.10
Lack of t 1.085 10 0.109 2.72b
3.1.2. Model tting Pure error 0.080 2 0.040
A model tting was accomplished for the third ex- Total 68.256 16
perimental design as shown in Table 3. The independent Regression coecient: R 0:991; F0:95;4;12 3:26; F0:95;10;2 19:4.
a
and dependent variables were tted to the second-order F -ratio (regression/residual).
b
model equation and examined in terms of the goodness F -ratio (lack of t/pure error).
of t. The ANOVA were used to evaluate the adequacy
of the tted model. The R-squared value provided a perimental data. Based on the F test, the model is pre-
measure of how much of the variability in the observed dictive, since its calculated F value is higher than the
response values could be explained by the experimental critical F value and the regression coecient (0.991) is
factors and their interactions. A good model (values close to unity. The coded model was used to generate
above 0.9 are considered very good) explains most of the response surfaces (Fig. 2) for the analysis of the variable
variation in the response. The closer the R-squared value eects on lipase activity.
is to 1.00, the stronger the model and the better the re-
sponse predictions (Haaland, 1989). Activity 14:17 1:27NH4 NO3 0:48NH4 NO23
On the basis of the analysis of variance (ANOVA), as 1:73CSL 0:42CSL2 2
shown in Table 8 for 8.25 h of fermentation, a second
order model (Eq. (2)) was established, describing the As can be seen in Fig. 2, an increase in corn steep liquor
enzyme activity as a function of ammonium nitrate and and ammonium nitrate concentrations led to an increase
corn steep liquor concentrations. The pure error was in lipase activity. Therefore, to maximize lipase activity
very low, indicating a good reproducibility of the ex- both variables must be kept at the highest tested levels.

Fig. 2. Response surface (a) and contour diagrams (b) for the lipase activity as a function of CSL and NH4 NO3 concentrations, with 0.6% soy oil,
after 8.25 h of fermentation, according to the third experimental design.
 J.F.M. Burkert et al. / Bioresource Technology 91 (2004) 7784 83

In a subsequent work, not reported here, higher con- Table 9

centrations of corn steep liquor and ammonium nitrate ANOVA for the fth factorial design at 8 h of fermentation
were shown to be inhibitory for the enzyme production. Source of Sum of Degrees of Mean F -ratio
Soy oil did not have a signicant eect, however the variation square freedom square
presence of a minimum amount is necessary as enzyme Regression 27.966 4 6.992 33.61a
Residual 2.496 12 0.208
inductor. The maximum lipase activity predicted in Fig.
Lack of t 2.377 10 0.238 4.00b
2 is about 20 U/ml. This value is approximately 11 times Pure error 0.119 2 0.059
higher than the one obtained by Mac^edo et al. (1997), Total 30.462 16
using the same microorganism. Regression coecient: R 0:958; F0:95;4;12 3:26; F0:95;10;2 19:4.
a
F -ratio (regression/residual).
b
3.2. Lipase production with olive oil F -ratio (lack of t/pure error).

3.2.1. Eect of the carbon source and nitrogen source
concentrations
The trials and results for the fourth factorial design
are shown in Table 4. The eect estimates were deter-
mined and are reported in Table 7. The analysis of the
eects shows that the corn steep liquor is the most im-
portant variable, as an increase in the concentration of
this variable from 2.4% to 6.6% led to an increase in
lipase activity. The variable ammonium nitrate shows
the same inuence as corn steep liquor. The increase in
olive oil concentration did not show signicant inuence
in lipase activity. In the fth experimental design, the
concentration range for corn steep liquor was increased
and that for the olive oil was decreased while ammo-
nium nitrate remained the same.
3.2.2. Model tting
The data from the fth experimental design are
shown in Table 5. On the basis of the analysis of vari-
ance (ANOVA), shown in Table 9, a second order coded
model (Eq. (3)) describing the behavior of the fermen-
tation with olive oil at 95% condence, for 8 h of fer-
mentation, was established.
Activity 13:10 0:64NH4 NO3 1:04CSL
0:63CSL2  0:54OO  CSL
Based on the F test, the model is predictive, since the F
value calculated is ten times higher than the critical F
value and the regression coecient (0.958) is close to
unity. Based on these results the model can be utilized to
generate response surfaces for the analysis of the vari-
able eects on lipase activity. The response surfaces in
Fig. 3 were obtained using Eq. (3). It can be seen that an
increase in corn steep liquor and ammonium nitrate
concentrations led to an increase in lipase activity. The
maximum lipase activity predicted in Fig. 3 is approxi-
mately 18 U/ml.
4. Conclusions
The enzyme production using soy oil was higher than
with olive oil. On the basis of the experimental design
and response surface analysis, the optimum conditions
to obtain high lipase activity using soy oil were ammo-
nium nitrate 2.12.5%, corn steep liquor 1315% and
soy oil 0.6%. At these conditions, 20 U/ml enzyme ac-
tivity was obtained. This represents an increase of more
than eleven times of the activity before the optimization,
as obtained by Mac^edo et al. (1997). With olive oil, the
optimum medium composition was ammonium nitrate
0.81%, corn steep liquor 1315% and olive oil 0.6%.
These conditions yielded 17 U/ml, which represents a
tenfold increase over the level obtained by the same
3
authors above.
The optimization of the culture medium composition,
besides the tenfold increase in the enzyme production,
decreased the fermentation time as well, which means

Fig. 3. Response surface (a) and contour diagrams (b) for the lipase activity as a function of CSL and NH4 NO3 concentrations, with 0.6% olive oil,
after 8 h of fermentation, according to the fth experimental design.
84 J.F.M. Burkert et al. / Bioresource Technology 91 (2004) 7784
that the productivity was greatly increased. The utili-
zation of raw materials with lower costs, for example
corn steep liquor and soy oil, led to a reduction in the
culture medium overall cost for lipase production, which
usually ranges 2550% of the total production costs.
Therefore, with the increase in yield and productivity
and simultaneous cost reduction, the industrial lipase
production by Geotrichum sp. can be regarded as pos-
sible and economically attractive.
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