09-04-2014, 11:34 AM
Lipase Production by Solid-State Fermentation in Fixed-Bed Bioreactors
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ABSTRACT
In the present work, packed bed bioreactors were employed with the aim of increasing productivity and scaling up
of lipase production using Penicillium simplicissimum in solid-state fermentation. The influence of temperature and
air flow rate on enzyme production was evaluated employing statistical experimental design, and an empirical
model was adjusted to the experimental data. It was shown that higher lipase activities could be achieved at lower
temperatures and higher air flow rates. The maximum lipase activity (26.4 U/g) was obtained at the temperature of
27°C and air flow rate of 0.8 L/min.
INTRODUCTION
Lipases are hydrolytic enzymes that act in
aqueous-organic interfaces,
catalysing thecleavage of ester bonds in triglycerides and
producing glycerol and free fatty acids. However,
in environments with low water availability,
lipases are able to catalyse esterification,
interesterification and transesterification reactions,
being thus very versatile biocatalysts (Sharma et
al., 2001; Pandey et al., 1999). Since the mid
1980's, there is a growing interest for lipases,
especially for those of microbial origin. Due to the
different reactions lipases are able to catalyse and
due to their regio and enantio selectivity, lipases
find an increasing range of applications, such as in
the detergent, food, pharmaceutical, fine
chemicals, leather and pulp and paper industries
(Freire and Castilho, 2000).
MATERIALS AND METHODS
Microorganism
Penicillium simplicissimum strain used in this
work was isolated from the solid residues
generated during the industrial processing of
babassu seeds (Freire, 1996). The strain was
maintained in glycerol (30% v/v) at -20 oC. Spores
were obtained through strain propagation for 7
days at 30 oC in a culture medium containing (%
w/v): soluble starch 2.0, MgSO4.7H2O 0.025,
KH2PO4 0.05, CaCO3 0.5, yeast extract 0.1, olive
oil 1.0 and agar 1.0 (Gutarra, 2003).
Solid-State Fermentation
Fermentations were carried out in bench-scale
fixed-bed bioreactors. The glass reactors had a
cylindrical shape (4 cm diameter, 14 cm height)
and a water jacket to maintain the fermentation
temperature under control. Sterile and moist air
was injected in the reactor bottom and air flow rate
was measured with a rotameter.
Lipase Activity Determination
Lipase activity of the extracts was determined as
follows: 1 mL of extract was added to 19 mL of an
emulsion prepared with olive oil (5% w/v) and
arabic gum (10% w/v) in sodium phosphate buffer
(100 mM, pH 7.0). The reaction was carried out
under agitation (200 rpm) at 35oC for 30 min. The
reaction was stopped by the addition of 20 mL of
acetone-ethanol mixture (1:1) and the fatty acids
produced were extracted under agitation (200 rpm)
for 10 min and titrated until end-point (pH 11.0)
with NaOH solution (0.04 N). The blank assays
were performed by adding the extract just after the
addition of the acetone-ethanol solution to the
flask. One unit of lipase activity (U) was defined
as the amount of enzyme.
CONCLUSION
The best conditions for lipase production by P.
simplicissimum in bench-scale fixed-bed
bioreactors were determined (27°C and air flow
rate of 0.8 L/min). Under these conditions, the
cultivation of the fungus in the medium containing
babassu cake and sugar cane molasses led to a
lipase activity of 26.4 U/g. The empirical model
obtained from the statistical analysis of the
experimental data was able to describe the effect
of the independent variables (temperature and air
flow rate) on lipase production. Furthermore, the
model was able to predict experimental lipase
activity levels for a relatively large interval of
operation conditions.