Biotechnology and Bioprocess Engineering 2009, 14: 105-111

DOI/10.1007/s12257-008-0040-5

Effects of Oxygen Volumetric Mass

Transfer Coefficient and pH on Lipase
Production by Staphylococcus
warneri EX17
Giandra Volpato1,2, Rafael C. Rodrigues1,2, Jlio Xandro Heck3, and
Marco Antnio Zchia Ayub1,2*
1

Department of Chemical Engineering, Federal University of Rio Grande do Sul State, Rua Professor Luiz Englert, s/n, ZC
90040-040, Porto Alegre, RS, Brazil
2
Food Science and Technology Institute, Federal University of Rio Grande do Sul State, Av. Bento Gonalves, 9500, P.O. Box
15090, ZC 91501-970, Porto Alegre, RS, Brazil
3
Technical School, Federal University of Rio Grande do Sul State, Rua Ramiro Barcelos, 2777, ZC 90035-007, Porto Alegre,
RS, Brazil
Abstract The principal objectives of this study were to evaluate the kinetics of lipase production by Staphylococcus warneri EX17
under different oxygen volumetric mass transfer coefficients (kLa) and pH conditions in submerged bioreactors, using glycerol (a biodiesel by-product) as a carbon source. Cultivations were conducted at different kLa (26, 38, 50, and 83 h1) and pH
values (6.0, 7.0, and 8.0). The optimal kLa and pH were 38 h1 and 7.0, respectively. Under these conditions, the maximal
cell production obtained was 8.0 g/L, and the volumetric and specific lipase production reached high levels of activity, approximately 800 U/L and 150 U/g cell, respectively, after 12 h of cultivation. This result was approximately five times higher
than that obtained in the shake flask cultures. The relationship between cell growth and lipase production was found to be
associated with growth by the Luedeking-Piret model. KSBB
Keywords: lipase, Staphylococcus warneri, bioreactor, oxygen volumetric mass transfer coefficient (kLa)

INTRODUCTION
Lipases (triacylglycerol hydrolase, EC 3.1.1.3) are lipolytic enzymes, which catalyze the hydrolysis of the ester
linkages of long chain acylglycerols at oil/water interfaces
[1]. These enzymes have the ability to catalyze several reactions of industrial interest, including esterification and transesterification, and can be utilized in food technology, detergents, the chemical industry and the biomedical sciences [24]. Additionally, these enzymes are the most extensively
utilized in organic synthesis [5].
Lipases are produced principally from microbial sources. In
particular, bacterial lipases perform a vital role in commercial
applications. Examples of important lipase-producing bacte*Corresponding author
Tel: +55-51-3308-6685 Fax: +55-51-3308-7048
e-mail: mazayub@ufrgs.br

rial genera include Bacillus, Pseudomonas, and Staphylococcus [6]. Microbial lipase production has been reported to
be influenced by pH and cultivation temperature, medium
composition, and oxygen tension, among a variety of other
factors [7]. Oxygen transfer rates have been identified by
some authors as a key factor influencing the production of
lipase by microorganisms, with a reported correlation between the increase of productivity with the increase of oxygen availability to cells [4,8,9]. The oxygen volumetric mass
transfer rate is generally considered to be a critical parameter
in aerobic cultures of microorganisms, due to its parch solubility and the need for a constant supply. The selection, design, and scale-up of biochemical reactors and the accurate
estimation of mass transfer rates for different scales and different operational conditions are of critical importance in
bioprocesses [10]. The oxygen transfer rate can be described
and analyzed by means of the oxygen volumetric mass transfer coefficient, or kLa. As with any other transport phenom-

106

ena occurring between different phases, the oxygen transfer

rate is profoundly affected by the hydrodynamic conditions
in the bioreactor, which affect the mass transfer coefficient
and the interfacial area. Therefore, variations in the configurations of bioreactors and the physicochemical properties of
the culture medium, including density, diffusivity, rheological properties, and surface tension, can attenuate the oxygen
transfer capacity, resulting in oxygen depletion [11].
Another factor that influences lipase production is the nutritional composition of the media, as these enzymes are
generally produced over lipidic carbon sources, including
oils, fatty acids, or glycerol in the presence of an organic
nitrogen source [12]. Glycerol can be utilized as a carbon
source in microbial bioconversions for the production of a
range of chemicals that might be utilized either as endproducts or as precursors for the production of other chemicals. Although research is limited regarding the use of glycerol in microbial bioconversions, it appears that a broad
range of products might be thereby produced [13]. More
important yet is the fact that glycerol is a by-product of biodiesel synthesis; since this biofuel will be produced on a
large scale in the coming years, large amounts of glycerol
will be discarded and can be used in bioprocesses. In a previous work, we demonstrated that glycerol was better than
glucose for growth and lipase production by Staphylococcus
warneri EX17 (previously identified as S. caseolyticus). It
has also been reported that cultivation using the raw glycerol
acquired by the enzymatic synthesis of biodiesel showed that
it was possible that this abundant, cheap, and surplus byproduct of biodiesel synthesis might be used for lipase production along with this strain [14]. This is essential in regard
to the economic feasibility of biotechnological processes and
the future integration of lipase production and biodiesel synthesis.
In the current study, we assessed the kinetics of lipase
production by S. warneri EX17 with different oxygen transfer rates and medium pH conditions, using glycerol as a carbon source. These were selected because the choice of an
appropriate culture pH and oxygen transfer is crucial, owing
to the profound influence of these variables in culture yields
and operating costs.

MATERIALS AND METHODS

Microorganism, Culture Maintenance, and Inoculum
Preparation

A strain of S. warneri isolated by us from an abattoir fatrich wastewater was utilized in this study. This strain was
identified via 16S rRNA gene sequencing and designated
EX17. The cells were maintained at 4C on tributyrin agar
plates containing (in g/L): peptone, 5; yeast extract, 3; tributyrin, 10. The inoculum was prepared in 125 mL Erlenmeyer
flasks filled with 25 mL of LB medium and inoculated with
a loop from a stock culture. The cells were cultivated for 18
h at 150 rpm at 37C. Following this procedure, 2 mL of
preinoculum was inoculated into 100 mL of LB medium and

grown to an optical density (OD) of 1.0. This was the inoculum for the bioreactor cultivations. This procedure was utilized as the standard inoculum preparation for all experiments.
Bioreactor Cultures

Batch cultivations were conducted in a 2 L working volume stirred bioreactor (Biostat B model, B. Braun Biotech
International, Germany) filled with 2 L of medium. The bioreactor was equipped with temperature, agitation, aeration,
and pH controllers, as well as two Rushton turbines with six
flat-blades. The culture medium was optimized in a previous
study [14] and contained, in g/L: peptone, 10; raw yeast extract (autolysed unpurified yeast extract, Prodesa, Brazil), 5;
glycerol, 30; olive oil, 6; soybean oil, 5. The medium was
sterilized by 20 min of autoclaving at 121C. The media
were emulsified in a blender for 30 s at maximum speed.
The optimized temperature was 37C. The influence of the
pH of the culture media was studied as either uncontrolled
(initial pH 8.0) or controlled at 6.0, 7.0, or 8.0. The dissolved
oxygen concentration in the culture was measured using a
polarographic electrode, and was expressed as a percentage
of O2 saturation. The oxygen transfer rate was assessed as
the oxygen volumetric mass transfer coefficient (kLa). The
kLa value was estimated via the dynamic gassing-out method
[15]. The agitation and aeration rates were set to different
values, characterizing four different oxygen transfer conditions: 26 h1 (400 rpm, 4 vvm), 38 h1 (600 rpm, 4 vvm), 50
h1 (600 rpm, 5 vvm), and 83 h1 (600 rpm, 6 vvm).
Samples were extracted during the cultivation in order to
determine the lipolytic and proteolytic activities, biomass,
and glycerol concentrations. All experiments were conducted
in duplicate.
Analytical Methods

The cell-free supernatant of the culture medium, referred

to as the crude enzyme extract, was used to estimate the enzyme activities and to determine glycerol concentrations.
Lipolytic activity was determined using p-nitrophenyl
palmitate (pNPP) as the substrate [16]. A volume of 0.15 mL
of crude enzyme extract was mixed with 1.35 mL of the prepared substrate solution and incubated for 15 min at 37C in
a water bath. Controls containing heat-inactivated enzyme
were also incubated for each assay. The mixture was centrifuged (14,000 g, 10 min, 10C) and the reaction was measured by absorbance at 410 nm. One unit of enzyme was defined as the quantity of enzyme required to liberate 1 mol
of p-nitrophenol per min at 37C. Under these conditions,
the extinction coefficient of p-nitrophenol is 13.23 103 M1.
Proteolytic activity was evaluated via the azocasein method,
in accordance with the method described by Sarath et al.
[17]. One unit of enzymatic activity was defined as the quantity of enzyme necessary to induce a change of one unit of
absorbance under the conditions described in the method.
Biomass was gravimetrically quantified as the cell dry
weight. Samples were centrifuged, washed twice in cold
distilled water, and dried at 80C to a constant weight. Glyc-

Biotechnol. Bioprocess Eng. 107

erol concentration was determined by HPLC with a refractive index (RI) detector (Perkin Elmer Series 200, USA) and
a Phenomenex RHM monosaccharide column (300 7.8
mm), at 80C, using ultrapure water as an eluent, a flow of
0.6 mL/min, and a sample volume of 20 L.

Luedeking and Piret Models

Microbial cultivations can be analyzed by kinetic models,

which provide us with some insight into the proper design
and operation of the bioreactor. The model developed by
Luedeking and Piret [18] has been widely employed to correlate the production rate with cell growth (Eqs. 1 and 2):
dP
dX
=
+ X
dt
dt

(1)
B

or
qp = +

(2)

In which P is the lipolytic activity, X is the cell density, t is

time, is the specific growth rate, qp is the specific rate of
lipase formation, and and are constants. Eq. (1) is a model
describing the formation of a mixed-growth-associated product. If = 0, the product is growth-associated, whereas for =
0, the product is non-growth-associated.

RESULTS AND DISCUSSION

In a previous work [14], we demonstrated the ability of
one strain of S. warneri isolate EX17 to manifest lipolytic
activity in a submerged cultivation. The culture conditions
were optimized in an orbital shaker, and the maximum
lipolytic activity of 140 U/L was obtained at the end of the
exponential phase, in an uncontrolled pH culture (initial pH
= 8.0). In the current study, we present the scaling-up of the
process to a bioreactor, studying the process variables of
oxygen transfer rates, pH, and glycerol as a carbon source in
relation to enzyme production.
Influence of pH

S. warneri EX17 was cultivated in a 2 L batch bioreactor.

First, we studied different conditions of pH, either uncontrolled (initial pH = 8.0) or controlled throughout cultivations at pH values of 6.0, 7.0, or 8.0. Figs. 1A and 1B demonstrate the time courses of biomass and lipolytic activity,
respectively, for the cultures under different pH conditions.
Exponential growth kinetics were observed (Fig. 1A) and the
highest levels of biomass production were obtained with a
controlled neutral pH, corresponding to approximately 10
fold that observed in cultures at a controlled pH of 8.0.
When the cultivation was conducted under uncontrolled pH
conditions (initial pH = 8.0), biomass formation was more
abundant than in the culture at a controlled pH value of 8.0.

Fig. 1. Influence of pH in the cultivation of S. warneri EX17 under oxygen transfer rate of 38 h1. (A) Cell growth and (B)
lipase volumetric activity. Symbols: pH 6.0 (), pH 7.0
(), pH 8.0 (), uncontrolled pH ().

This is likely attributable to a reduction in the pH caused by

cellular growth, and thus the pH remained in a range around
7.0. The lipolytic activity (Fig. 1B) was approximately 800
U/L at 12 h of cultivation with controlled neutral pH, which
was almost 10-fold that seen with a controlled pH at 8.0 (92
U/L), and five fold that noted with uncontrolled pH (150
U/L). These results show that pH is a crucial factor in the
growth of S. warneri and lipase production. Therefore, all
subsequent experiments were conducted with pH controlled
at 7.0. Notwithstanding, very few studies have attempted to
determine the influence of pH on lipase production. Controlled pH was utilized by Fickers et al. [19] for lipase production by Yarrowia lipolytica (pH 6.5) in a bioreactor, and
by Chen et al. [7] for cultivations of Acinetobacter radioresistens at a pH of 7.0. However, the majority of reports in the
relevant literature have asserted that lipase production occurs
under uncontrolled pH conditions [5,9,20-22]. The rapid pH
reduction in the first hours of cultivation may result in enzyme inactivation as most lipases from bacteria do below

108

Fig. 2. Cultivation of S. warneri EX17 under oxygen transfer rate

of 26 h1, with controlled pH = 7.0. Symbols: cell mass
(), glycerol (), lipase specific activity (), lipase volumetric activity ().

Fig. 3. Cultivation of S. warneri EX17 under oxygen transfer rate

of 38 h1, with controlled pH = 7.0. Symbols: cell mass
(), glycerol (), lipase specific activity (), lipase volumetric activity ().

pH 5.0 [14,23-26].
Influence of Oxygen Transfer

The time courses of cell growth, glycerol consumption,

and lipase production by S. warneri EX17 at a kLa of 26 h1
are depicted in Fig. 2. Under these conditions, the biomass
and the lipolytic enzyme production were similar to the previous results obtained in the shake flasks. However, the incubation time required for maximum lipase specific activity
was reduced from 24 h of cultivation to only 6 h. We noted a
two-fold increase in cell concentration as compared to what
was observed in the shake flask experiments, from 3.0 to 6.0
g/L. Similar profiles were noted by Gupta et al. [27], who
demonstrated that Burkholderia multivorans evidenced a
reduction in the peak of lipase production from 36 to 15 h of
cultivation when scaling up from the shake flask to the bioreactor, while biomass increased by a factor of five.
The increase in oxygen transfer coefficient from 26 to 38
h1 (Fig. 3) resulted in an improved lipase volumetric production of approximately four-fold, whereas the specific
activity increased by approximately three-fold, and the biomass increased from 6.0 to 8.0 g/L. This condition (kLa of 26
h1) appears to be oxygen limiting, impairing cell growth and
lipase production. These findings are generally consistent
with the results reported by Alonso et al. [22] in which a
five-fold increase in lipase production was effected by Y.
lipolytica when the culture kLa was increased from 8.5 to
12.2 h1.
The kinetic profiles of cell growth, glycerol consumption,
and lipase production by S. warneri EX17 at kLa of 50 and
83 h1 are depicted in Figs. 4 and 5, respectively. Increases in
kLa to 50 and 83 h1 resulted in a reduction in biomass concentrations (6.5 and 7.4 g/L, respectively), when compared
with what was seen at a kLa of 38 h1, probably attributable
to the toxic effects of oxygen. It has been demonstrated that
an excess of dissolved oxygen may limit cell growth and,

Fig. 4. Cultivation of S. warneri EX17 under oxygen transfer rate

of 50 h1, with controlled pH = 7.0. Symbols: cell mass
(), glycerol (), lipase specific activity (), lipase volumetric activity ().

consequently, lipase production [28]. Similar behavior was

reported in a study of Candida rugosa growing in a bioreactor at different aeration rate conditions, when the highest cell
concentration (6.0 g/L) and lipase production (1,800 U/L)
were obtained for aeration rates of 3.0 L air/min, with a
sharp reduction in both when the aeration rate was up to 4.0
L air/min [29]. Dominguez et al. [9] reported on lipase production by Thermus thermophilus HB27 in a bioreactor under different air flow conditions (0, 0.5, 1.0, 1.5 and 2.5 L
air/min), and obtained the highest levels of intracellular lipase production (120 U/L) at 1.5 L air/min, and the highest
levels of extracellular lipase production (60 U/L) at 1.0 L
air/min. However, for Rhizopus arrhizus, Elibol and Ozer [5]
demonstrated that the increase in dissolved oxygen increased
both the biomass concentration and lipase production. Fucios et al. [20] observed a similar behavior of lipase produc-

Biotechnol. Bioprocess Eng. 109

Table 1. Influence of kLa on cultivation parameters of growing

and lipase production by S. warneri EX17
1

Parameter
a

max (h )
b

qx (g/Lh)

Oxygen transfer coefficient (h )

26

38

50

83

0.59

0.75

0.71

0.68

0.36

0.69

0.66

0.67

c
s

0.79

1.33

1.10

0.91

22.96
51.6

27.16
150.1

36.48
121.6

26.91
85.8

q (g/Lh)
qp (U/g cellh)
Maximum specific lipolytic
activity (U/g cell)
a

Maximum growth rate; bbiomass productivity; csubstrate consumed; dlipase

productivity.

Fig. 5. Cultivation of S. warneri EX17 under oxygen transfer rate

of 83 h1, with controlled pH = 7.0. Symbols: cell mass
(), glycerol (), lipase specific activity (), lipase volumetric activity ().

Fig. 6. Description of S. warneri EX17 lipase production by

Luedeking-Piret model. Values of kLa: , 26 h1; , 38
h1; , 50 h1; , 83 h1.

tion by T. thermophilus and Thermus aquaticus. These authors confirmed that increases in the aeration rate of cultures
of T. thermophilus, the production of biomass and, particularly, intracellular lipolytic activity increased in a similar
manner. They observed a maximum extracellular lipolytic
activity of 18 U/L for T. thermophilus and 33 U/L for T.
aquaticus, and the highest levels of intracellular lipolytic
activity at 80 and 70 U/L, respectively. It may be speculated
that these differences are a consequence of morphophysiological variability between the microorganisms, reflecting
different oxygen requirements for the maintenance of their
physiological functions or their influence on the rheological
properties of culture media [22].
The results obtained in this study demonstrated that lipase

production by S. warneri was limited by oxygen supply. For

cultures with kLa values of 26 and 38 h1, the dissolved oxygen decreased quickly, reaching zero at 3 and 5 h of cultivation, respectively. In the culture with a kLa of 50 h1, the
dissolved oxygen concentration decreased quickly in the first
4 h and stabilized at approximately 3.5 mg O2/L until the end
of the run, whereas for the kLa of 83 h1, the dissolved oxygen remained at a level of above 5.0 mg O2/L, thereby suggesting a possible toxic effect due to an excess of oxygen. In
order to check for the proteolysis of lipases, the batches were
evaluated for the protease contents of the medium, and the
highest levels of proteolytic activities at the different kLa
values were as follows: 2.79 U/mL for 26 h1, 32.45 U/mL
for 38 h1, 3.58 U/mL for 50 h1, and 2.33 U/mL for 83 h1.
These were very close results, and the highest value for proteases was obtained under the conditions that generated the
most profound lipolytic activities. Therefore, we can rule out
any impact of lipase proteolysis.
We also noted that lipase production was partially growthassociated. This result indicates that the cell generates lipases
in order to obtain the energy required to form new cells from
the available carbon sources, and at the same time it does so
as a normal metabolic activity irrespective of growth, which
can be confirmed by glycerol consumption profiles (Figs.
2~5). We correlated the enzyme production rate with cell
growth using the Luedeking-Piret model described in Eq. (1).
The plot of qp versus is provided in Fig. 6. As can be observed, the data regarding lipase production could be described by the mixed model, and the model obtained was:
qp (U/g cellh) = 33.02 (h1) + 2.20

(3)

From Eq. (3), it can be observed that the lipase production

rate for S. warneri EX17 evidences a linear relationship with
the specific cell growth rate (R2 = 0.95). The results verify
that lipase biosynthesis is partially growth-associated, which
is consistent with findings for other microorganisms [5,7,29].
Table 1 summarizes the major process parameters when
different kLa values were employed. The max value was determined to be highest at a kLa of 38 h1 (0.75 h1), again
demonstrating that this kLa value was favorable for growth
as compared to the other kLa values. At this kLa value, the
biomass productivity, consumed substrate and lipase produc-

110

tivity were at their highest levels. The maximum volumetric

lipolytic activity and specific lipolytic activity were noted at
a kLa of 38 h1 at 12 h of cultivation.

CONCLUSION
In the current study, we report the biotechnological potential of lipase production in a bioreactor by S. warneri EX17,
a strain recently isolated from abattoir wastewater treatment
plants and never before studied. We demonstrated the importance of kLa in order to maximize cell growth and lipase production for this bacterium, and also that oxygen supply was a
key factor controlling the bioconversion of glycerol into
biomass and enzyme. The use of glycerol as the sole carbon
source for lipase production is essential for the new generation of biotechnology products such as biodiesel, as this substrate is going to be produced in very large amounts as a byproduct; therefore, its integration into the process will exert a
profound effect on cost reduction. Under the optimal kLa
conditions, lipase production was five times higher than previously observed in shaking flask cultivations, and glycerol
proved to be better than other commonly utilized carbon
sources, such as glucose, for instance. Further studies will
involve the scale-up of the process to obtain this biocatalyst,
thereby reducing the costs of production and its industrial
applications, such as in biodiesel synthesis.
The authors wish to thank CAPES
and CNPQ for their scholarships and financial support for
this research.

Acknowledgments

Received March 4, 2008; accepted October 25, 2008

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