212 Journal of Basic Microbiology 2009, 49, 212 – 215

Short Communication
Improvement of Yarrowia lipolytica lipase production
by fed-batch fermentation

Patrick Fickers1, Jacqueline Destain2 and Philippe Thonart1, 2

1
Centre Wallon de Biologie Industrielle, Service de Technologie Microbienne, Université de Liège,
Bd du Rectorat Bat. 40, B-4000 Liège, Belgium
2
Unite de Bio-Industrie, Faculté Universitaire des Sciences Agronomiques de Gembloux,
Passage des Déportés, 2, B-5030 Gembloux, Belgium

Two different types of fed-batch fermentation were investigated to improve production yields
of the Lip2 extracellular lipase in Y. lipolytica mutant-strain LgX64.81 grown in a 20l bioreactor.
Compare to batch cultures, culture feeding with the complete medium led to a 2-fold increased
lipase production (2016 ± 76 U ml–1) whereas addition of a combination of glucose and olive oil
led to a 3-fold increase. The high level of lipase production obtained on glucose media with
Y. lipolytica LgX64.81 could be related to its phenotype i.e. a lower sensibility to glucose cata-
bolite repression due to a modification in the level of HXK1 expression.

Keywords: Bioreactor / Fed-batch / Lipase / Yarrowia lipolytica

Received: June 06, 2008; accepted: September 08, 2008

DOI 10.1002/jobm.200800186

30 × 103 min–1, respectively [4]. The Lip2 prefered sub-

*
Introduction
Triacylglycerol hydrolases or lipases (EC 3.1.1.3.) are
enzymes able to catalyze ester bond hydrolysis in acyl-
glycerol at the lipid-water interface with the release of
fatty acids and glycerol [1]. Interest in lipases has been
greatly increased during recent years, mainly because
this class of enzymes presents a broad range of medical
and biotechnological applications such as substitutes
for pancreatic lipases, trans-esterification of oils and
fats, resolution of racemic mixtures, development of
characteristic flavors in food processing industries, use
as ingredients in detergent formulations and the treat-
ment of wastes (for review see [2]).
The yeast Yarrowia lipolytica is frequently isolated
from lipid- or protein-containing substrates such as
cheese or olive oil (for review see [3]). Y. lipolytica pro-
duces a 38 kd extracellular Lip2 lipase, encoded by
the LIP2 gene. The optimal activity was found at 37 °C
and pH 7 whereas Km and kcat values, determined on
purified enzyme, were found equal to 807 mM and
Correspondence: Patrick Fickers; present adresse: Centre d’Ingénierie
des Protéines, Institut de Chimie, Université de Liège, Allée de la Chi-
mie, 3 Bat. B6, B-4000 Liège, Belgium
E-mail: pfickers@ulg.ac.be
Phone: + 32 4 366 33 38
Fax: + 32 4 366 33 64
strates are the saturated triglyceride tricaprylin (C8 : 0),
the unsaturated triglyceride triolein (C18 : 1) and the
long-chain fatty acid methyl ester (C12, C14, C16). The
higher activity observed towards triglycerides com-
pared to hydrophilic fatty acid methyl ester indicate
that Lip2 is a true lipase [5].
Mutants with increased capacities of lipase secretion
were obtained from Y. lipolytica CBS6303 strain by
chemical mutagenesis [6]. One particular isolate, named
LgX64.81, was selected on the basis of its phenotypic
characteristics, i.e. high level of lipase production upon
addition of oleic acid compared to the wild-type strain
and the lipase encoding gene expression uncoupled
from the glucose catabolite repression [7, 8]. This mu-
tant was the focus of a number of investigation espe-
cially for the development of a lipase production pro-
cess in bioreactors [6, 9]. Upon growth of LgX64.81
mutant in a 15 liters bioreactor, lipase activity reached
1000 U ml–1, which represents a 35-fold increase com-
pared to the parental strain. Previous works have dem-
onstrated that lipase production could be significantly
increased using fed-batch operation strategies. With
a constant specific growth rate strategy, lipase pro-
duction by Candida rugosa was enhanced 10-folds
(117 U ml–1) compare to a batch operation whereas a

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Journal of Basic Microbiology 2009, 49, 212 – 215 Improvement of Yarrowia lipolytica lipase production 213

8-fold increased lipase production (23 U ml–1) was ob- Results

tained by high cell densitiy fed-batch culture of Candida
cylindracea [10, 11]. Therefore, similar strategies for the Lipase production in batch culture
production of the extracellular Lip2 lipase by the Y. lipo- Lipase production by Y. lipolytica LgX64.81 was con-
lytica mutant strain LgX64.81 were investigated and ducted in an optimised medium containing glucose as
presented in this paper. primary carbon source and olive oil as inducer [6, 9].
Cell growth, glucose concentration and lipase activity
were measured at various time points for 46 h (Fig. 1A).
Materials and methods Cell growth occurred during the first 23 h of culture to
reach a density of 2.1 × 109 cells ml–1; then after the
The Y. Lipolytica strain LgX64.81 was used in all experi- culture entered into the stationary phase. As expected,
ments. Precultures were carried out successively in dissolved oxygen (pO2) rapidly decreased during the
50 ml and 400 ml of YPD medium (glucose 1.5%, yeast first 20 h of growth and remained close to 10% of satu-
extract 1%, casein peptone 1%) in shaking flask at 29 °C ration. After 46 h, the increase in the pO2 value, to-
for 16 h. Lipase production were conducted in a 20 l gether with glucose exhaustion in the culture broth
bioreactor (LSL Biolafitte, France) at 29 °C with a stir- indicated the end of the culture (Fig. 1A). Lipase activity
ring speed of 350 rpm and an air flow of 0.5 or 1 VVM measurements revealed that enzyme production exhib-
during batch and fed-batch culture, respectively. The ited two distinct phases. During the first 23 h of cul-
working volume was fixed at 15 l of an optimized me- ture, a low level of production of 9.8 U ml–1 h–1 could
dium containing glucose 1.5%, whey powder 3%, yeast
extract 3%, corn steep liquor 1% (v/v), olive oil 0.5%,
(NH4)2SO4 0.8% [9]. A 3-fold concentrated optimized
medium or a mixture of glucose 5% and olive oil 1.5 %
were used during the fed-batch culture. Dissolved oxy-
gen was continuously monitored with a polarographic
oxygen electrode (Ingold, Switzerland). A level probe,
placed 10 cm from the top of the vessel, activated the
addition of the polyether antifoam Tego KS911 (Gold-
schmidt, Germany) when necessary. pH was automati-
cally maintained at 6.5 ± 0.1 by addition of 4 N NaOH or
4 N H3PO4. Yeast growth was estimated by counting the
cells on a Bürker microscope chamber. When cells were
grown on media containing olive oil, samples were
extracted with 2.5 volume of a propanol/butanol mix-
ture (1 : 1, v/v) prior to microscopic observation. Lipase
activity was determined by a titrimetric method as
previously described [12]. One unit of lipase activity
correspond to the amount of enzyme that catalyses the
release of 1 µmol of fatty acid per min at 37 °C. Glucose
concentration in the culture supernatant was deter-
mined by HPLC (HP Agilent 1100 apparatus, Agilent
Technologie, France) using a C-610H column (300 mm ×
7.8 mm, 9 µm packing, Agilent Technologie) and refrac-
tometric detection. For that purpose, olive oil was ex-
tracted from the crude supernatant twice with the
same volume of hexane. Thirty microlitres of the aque-
ous phase were injected and compounds were eluted at
30 °C with H3PO4 at 0.1% in milliQ water at a flow rate
of 0.5 ml min–1. Nitrogen concentration in the culture
Figure 1: Time course of cell growth (䉱), lipase activity ( ),
supernatant was determined using the UniCellTM Re- glucose concentration (䊉) and dissolved oxygen (䊐) during batch
agent set (Hach, Loveland, Co.) as previously described culture (A) and fed-batch culture with the complete medium (B) or
[9]. with a mixture of glucose and olive oil (C).

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214 P. Fickers et al.
be observed. During the second phase, between 23 and
39 h of culture, the production of lipase increased to a
value of 57.3 U ml–1 h–1 (Fig. 1A). A maximal lipase ac-
tivity of approximately 1200 ± 89 U ml–1 was obtained
after 39 h and remained constant until the end of the
culture (46 h). Nitrogen concentration in the culture
supernatant, determined after 39 h of fermentation,
was equal to 0.3 g l–1.
Fed-batch culture
During batch fermentation, the maximal lipase activity
in the culture broth was observed after 39 h of cul-
ture. The decrease in the oxygen uptake observed
at that time suggested that the depletion of carbon
and/or nitrogen sources could be the limiting factor
for lipase production. Therefore, two different types of
fed-batch cultures were set up. In the first one,
the complete medium was added whereas only a com-
bination of glucose and olive oil was added in the
second one. In batch culture, a glucose consumption
rate of about 0.5 g l–1 h–1 was observed during the expo-
nential growth phase. Therefore, for the fed-batch
strategies, a constant feeding flow rate equivalent to
0.5 g l–1 of glucose was used between 20 h and 40 h of
culture.
A significant increase of lipase production was ob-
tained for both types of fed-batch cultures. Feeding
with the whole medium led to an enzymatic activ-
ity of 2000 ± 76 U ml–1 after 62 h of fermentation,
which represents a 2-fold increase compared to the
batch culture (Fig. 1B). During the stationary phase
(i.e. after 46 h), the biomass reached a concentration of
1.7 × 109 cell ml–1. Dissolved oxygen in the culture
broth decreased during the growth phase to a value
close to 15% and the oxygen uptake remained constant
during all the feeding period. Then, it increased as the
glucose concentration decreased in the culture broth.
Nitrogen sources in the culture supernatant were found
in excess (8.7 g l–1) after 66 h of fermentation. There-
fore, culture with feeding of glucose and olive oil were
performed in the same condition. They led to an addi-
tional increase in lipase production. The lipase activity
reached 3044 ± 143 U ml–1 after 63 h of culture. No
further increase could be observed then after. The bio-
mass reached a value of 1.7 × 109 cell ml–1 after 23 h of
culture and microscopic observation showed a modifi-
cation in the cell form. After approximately 40 h of
culture, yeast cells tended to adopt an hyphal morphol-
ogy, with an average value of 80% of filamentous cells
at the end of the culture. Nitrogen concentration in the
culture supernatant, determined after 64 h of fermen-
tation, was equal to 0.2 g l–1.
© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Journal of Basic Microbiology 2009, 49, 212 – 215
Discussion
The Y. lipolytica LgX64.81 mutant was previously iso-
lated by chemical mutagenesis from the wild-type
strain CBS6303. For that particular isolate, an industrial
medium was set up with regards to its phenotype [6].
The composition of the culture medium is one of the
key factors for the development of a fruitful production
process. Batch culture in this medium led to a 35-fold
increased lipase production compared to the parental
strain, confirming thus previous reports [6]. Lipase
production mainly occurred during the stationary
phase and seems to be influenced by glucose concentra-
tion in the culture broth. Indeed, the increase in lipase
activity ended as glucose became exhausted in the cul-
ture broth. We also observed that increasing glucose
concentration at the beginning of the culture did no led
to higher lipase production yield (data not shown). By
contrast, addition of glucose during the stationary
phase led to significant increase in lipase production
indicating that either glucose concentration and the
time of its addition are essential for an efficient synthe-
sis of the enzyme. In the Y. lipolytica wild-type strain,
the production of lipase is detected at relatively low
levels, and only after depletion of this substrate in the
culture broth [7]. By contrast, production of lipase by
Y. lipolytica LgX64.81 mutant was found to be less sensi-
tive to glucose catabolite repression due to a modifica-
tion in the level of HXK1 encoding hexokinase, witch
negatively regulate the expression of the extracellular
lipase LIP2 gene [8]. The mutant phenotype could thus
explain the high level of lipase production in the pres-
ence of glucose. During the fed-batch culture with the
complete medium, a lipase production could be ob-
served even after glucose depletion in the medium. This
could be explain by the positive effect on the lipase
production of the casein hydrolysates present in large
amount in the fermentation broth at the end of that
type of fed-batch culture [9]. Olive oil is a source of oleic
acid which is a positive regulator of LIP2 expression.
However, the sole addition of a source of oleic acid did
not led to the production yield obtained here [8].
In all experiments, dissolved oxygen never reached a
concentration that could limit yeast development. This
indicates that fermentation conditions, such as stirring
and aeration are adequate both for cell growth and
production of lipase in these conditions. The morpho-
logical observation suggests that the hyphal form of
LgX64.81 could also be a key factor for the production
of lipase. These observations are in accordance with the
results of Novotny et al. [12] who demonstrated that the
level of lipase production was modulated by the cell
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Journal of Basic Microbiology 2009, 49, 212 – 215
morphology in Y. lipolytica. However, no clear relation-
ship could be established between the morphologic
state and the culture condition. A high glucose concen-
tration during the stationery phase could only explain
these observations.
In conclusion, it has been demonstrated here that fed
batch culture with glucose and olive oil led to a signifi-
cant increase in lipase production yield.
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