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of acid and ethanol during the cultivation process. On the
other hand, in case of bioreactor culture, the DO decreased
gradually during the growth phase and increased gradually
thereafter as cells entered the stationary phase. As observed
also in the previous experiment, the oxygen was not limited
during the cultivation process.
Cultivation of S. boulardii in completely semi-defined medium
in shake flask and stirred tank bioreactor
Based on the data of the previous experiments, three different
parallel cultivations were conducted using semi-defined
medium supplemented with yeast extract in concentration of
5 g l -1 . Cultivations were conducted in both shake flask and
bioreactors under controlled and uncontrolled pH conditions
(Fig. 3). In case of shake flask cultures, cells grew exponentially
reaching the maximal cell mass of about 3.1 g l -1 after
about 12 h and decreased gradually thereafter. During the
growth phase the pH decreased reaching its minimal value
of 2.9 after 17 h. On the other hand, cells grew in different
manner in case of bioreactor cultures. The rate of glucose
consumption in case of bioreactor culture was higher than
shake flask culture. At the end of cultivation time, 25 % and
Fig. 3 Kinetics of cell growth, glucose consumption and different growth
parameters during the cultivation of S. boulardii in complete defined medium
supplemented by yeast extract (5 g l -1 ) under different cultivation conditions:
(A) shake flask culture, (B) bioreactor culture (uncontrolled pH), (C) bioreactor
culture (controlled pH at 5.5)
12 % of the initial glucose were un-utilized by cells in case
of shake flask and bioreactor cultures, respectively. Whereas,
glucose was completely consumed in bioreactor culture of
controlled pH at 5.5 (Fig. 3C). After only 10 h cultivation,
glucose was the limiting nutrient in this culture and cells
entered the stationary phase as carbon source limitation.
Oxygen was in excess in this culture and the lowest value of
DO was 25 % saturation as cell entered the stationary phase.
In general, the final cell mass in this experiment was significantly
higher than the previous experiment on using completely
defined medium. Yeast extract supplementation to the
cultivation medium was necessary as an important source for
amino acids, peptides and soluble vitamins. However, yeast
extract was found to be necessary for cell growth and the
production of enzymes, primary and secondary metabolites
by different types of microorganisms (Kadowaki et al., 1988;
Costa et al., 2002; López et al., 2003; Lin and Chen, 2007).
Fed-batch cultivation of S. boulardii in semi-defined medium
with different feeding strategies
Based on the data of batch cultivation of S. boulardii in
semi-defined medium under pH-stat in bioreactor, cultivations
were carried out under the same cultivation conditions
with two different methods of feeding strategies. Since cell
viability and its fermentative capacity are important for further
application of therapeutic yeast, fed-batch cultivation
was designed to start glucose feeding before carbon source
starvation in culture. The negative effect of carbon starvation
in culture can induce energy deprivation and loss of
fermentative capacity of yeast (Thomsson et al., 2003).
Fed Batch cultivation with mono-substrate with an increased
feeding rate
Cultivation was carried out as typical batch culture in
bioreactor for the first 9 h. During that time, cells grew
exponentially and reached cell mass of about 4.1 g l -1 . On
the other hand, glucose concentration gradually decreased
during this phase. The glucose consumption rate was of
about 2 g l -1 h -1 and the glucose concentration reached
about 4 g l -1 at the end of batch phase. Therefore, glucose
was fed with the same consumption rate to prevent carbon
limitation. However, the feeding rate of glucose was
increased by 2 g l -1 h -1 every 5 h to compensate the increased
volumetric consumption rate of glucose as a result
of further cell growth. As shown in Figure 4, the cell mass
increased significantly during the glucose feeding phase
reaching about 18 g l -1 after 20 h. The cell growth was
terminated after that time and glucose was accumulated in
culture in high concentration as cell entered the stationary
phase. During the active growth phase of fed-batch culture,
from 9 to 20 h, the value of DO was below 20 %
saturation and increased gradually thereafter and reached
about 80 % after 30 h. The gradual increase in DO value
in culture with very low rate directly indicates the termination
of cell growth and the high viability of cells pro-
426 ı Originalarbeiten Deutsche Lebensmittel-Rundschau ı 104. Jahrgang, Heft 9, 2008