In tern a tio n a l

Sch o la rs
Jo u rn a ls
International Journal of Chemical and Biochemical Engineering ISSN 7682-1366 Vol. 2 (2), pp. 038-046, February,
2016. Available online at www.internationalscholarsjournals.org © International Scholars Journals
Author(s) retain the copyright of this article.

Full Length Research Paper

Intermittent pressurized oxygenation in the fermentation

of Zygosaccharomyces rouxii
Ming-Hui Yang, Wei-Jun Chien*, Chee-Shan Chen
Department of Applied Chemistry Chaoyang University of Technology, Taichung, Taiwan.
Accepted 18 February, 2016

Intermittent pressurized oxygenation was used in the fermentation of Zygosaccharomyces rouxii for biomass
production. Fermentation was carried out in a 1.2 L cylindrical bioreactor and temperature was controlled at
30C. During fermentation, the yeast cells went through pressurization-depressurization cycles. Experimental
data revealed the likelihood that pressurization actively aided oxygen transfer into the cells, and
depressurization facilitated exporting metabolites. Various intermittent and continuous operations under
different pressure were studied and compared. Results showed that mild operation pressure (5 psi) was
beneficial. When further increased the pressure (8 to 10 psi), negative effects began to appear. The modified
Gompertz model was used for fitting the cell growth data to find the growth parameters. In 24 h culturing time,
compared to continuous aeration at atmospheric pressure, the 5 psi-2min-30sec (pressurize 2min, depressurize
-1
30 sec) operation increased the maximum specific growth rate from 0.58 to, 0.67 h , while the final to initial cell
concentration ratio increased from 102 to 316. Higher glucose uptake rate (initial 15%, residual 0.6%) was also
observed in the intermittent modes of pressurized oxygenation.

Key words: Dissolved oxygen, pressurized oxygenation, Zygosaccharomyces rouxii, growth kinetics, stirred tank
bioreactor.

INTRODUCTION

Yeasts are fungi of many industrial applications.
Zygosaccharomyces rouxii is an osmotolerant strain
important in the food industry. It is most widely used (or
naturally occurred) in the soy sauce production process,
in which the yeast provides the characteristic soy sauce
flavor (Wah et al., 2013; Wu et al., 2010; Sluis et al., 2001;
Hamada et al., 1989; Sugiyama, 1984). Major volatile
flavor compounds derived from inoculated Z. rouxii have
been identified as alcohols, furanones, esters, aldehyde,
acid, pyrone and phenols (Lee et al., 2013; Wah et al.,
2013). Together, these compounds contribute to the
desirable flavor profile of soy sauce. Cao et al. (2010)
used genome shuffling technology to accelerate and
enhance the flavor formation in soy sauce production,
____________________________________
*Corresponding author. wjchien@cyut.edu.tw;
oxa9615@yahoo.com.tw
which added a useful tool to improve the soy sauce
making process.
Z. rouxii has applications other than soy sauce
production. It can be used for the production of
non-alcoholic (or low alcoholic) beer, in which the function
of Z. rouxii was to consume the ethanol produced by
Saccharomyces cerevisiae while retaining acceptable
flavor (Sohrabvandi et al., 2009; Dziondziak, 1989). In
another application, Z.rouxii was used for the production
of 1,3-propanediol from glucose by co-culturing with
Klebsiella pneumonia (Ma et al., 2012). In yet another
application, the yeast was used for the production of
glutaminase (Lyer and Singhal, 2010).
Molecular oxygen is an important yeast growth factor (Wu
et al. 2010; Weusthuis et al., 1994; Kuriyama and
Kobayashi, 1993; Visser et al., 1990; Dawson, 1960).
Unfortunately, the solubility of molecular oxygen (DO) in
aqueous fermentation broth is low. Researchers dealt with
Yang et al. 038
this problem in several ways as described below.
Increase aeration rate is a straight forward way to increase
DO. Dawson (1960) reported that in his continuous culture
experiments with Z. rouxii, increasing aeration rate increased
both polyol production and cell growth. In another way,
reducing air bubble size will increase gas-liquid contact area
and increase oxygen transfer rate (OTR). Designing
spargers to produce fine bubbles in agitation system to
increase OTR have been investigated (Zimmerman et al.,
2009; Parakulsuksatid, 2000; Alves and Vasconcelos, 1996).
Using air lift fermenter is another important design which is
well known for improving DO, while keeping shear force
caused by mixing at minimum (Zimmerman et al., 2009;
Chen, 1990; Chisti et al., 1990). Adding emulsified
oxygen-vectors to increase DO by using immiscible liquid
with high oxygen solubility, has also been proposed (Rols et
al., 1990; Folescu, 2011).
In general, OTR is proportional to the difference between the
equilibrium (or saturate) oxygen concentration (C*) and the
instantaneous oxygen concentration (C). The proportional
constant is generally termed as the “volumetric mass transfer
coefficient” (kLa) (Montes et al., 1999; Garcia-Ochoa and
Gomez, 2009; Karimi et al., 2013). While (C* - C) is the
driving force for OTR, kLa is governed by physical
parameters like gas-liquid contact area (bubble size),
viscosity, density, temperature, and chemical composition of
the liquid.
Given the same kLa (fixed operational conditions: design of
the fermenter, temperature, mixing, aeration, chemical
composition etc.), OTR will only be influenced by the driving
force (C* - C). Henry’s law states that C* is proportional to
oxygen’s partial pressure in the gas phase. Considering the
condition that the yeast cells are consuming oxygen fast,
which will keep DO (or C) low, an increase in pressure will
increase C*. Consequently, the OTR’s driving force (C* - C)
will be increased. The main goal of this research is to
investigate the strategy of adjusting the air pressure and
observe the effect(s) on the fermentation of Z. rouxii for
biomass production.
Since the cell wall is a semipermeable membrane, once the
extracellular pressure increased, the intracellular pressure
will increase accordingly. Such pressure equilibration will
develop fast. Once the cross membrane pressure gradient
diminishes, instead of a higher extracellular C*, both sides’
C* will correspond to the same equilibrium value. Then, the
above stated enlarged (C* - C) will not be applicable.
Strategically, if the intracellular pressure was to be released
and then increase the extracellular pressure, presumably the
favorable enlarged (C* - C) will be renewed. It is also this
study’s purpose to see how such intermittent (or periodical)
operation will affect the cell growth.
MATERIALS AND METHODS
Micoorganism
The yeast train CCRC 21873 was obtained from the Cultural
Collection and Research Center, Taiwan. The yeast was
activated in shaker flasks three times (48 h each) before use.
YMP (Difco) fermentation broth containing 0.3% yeast
extract, 0.3% malt extract, 0.5% peptone and 15% glucose
was used throughout the experiments.
Apparatus
The fermenter was constructed using an acrylic tube (100
mm OD, 170 mm long, 3 mm wall thickness), and acrylic
plates of 10 mm thick were screwed to the top and bottom
like flanges in pipe works. To assure suffice turbulence, six
baffles of 1.5 mm thick and 11 mm wide acrylic plates were
evenly distributed inside the fermenter. Total volume of the
fermenter was 1.2 L and the working volume was 700 mL.
Pressure gauge, temperature probe, and venting tube were
mounted on the top plate of the fermenter. Sinusoidal valves
were used to control aeration and venting (pressurization and
depressurization) through a timer. The fermenter and feed
(liquid broth and air) lines were sterilized using bleach (3%
NaOCl) and washed with sterilized water. The air filter was
sterilized separately in autoclave then connected to the
system aseptically. Before experimentations, sterilized
fermentation broth was pumped into the fermenter through a
silicon tube to the bottom of the vessel, and aerated for 3
days to assure sterility. Air flow rate was controlled by a
pressure regulator.
DO meter was used for continuous monitoring oxygen
concentration in the fermentation broth. A membrane type
DO meter (DO-5510, Lutron Electronic Enterprise Co. Ltd.,
Taipei, Taiwan) was horizontally inserted at the mid-point of
the side wall. Data was collected through USB interface at a
frequency of 1 s-1.
Analysis
Glucose and ethanol were analyzed by HPLC method
(PU-2807, refractive index detector, RI-2301, Jasco, Japan).
BIO-RAD HPX-87H Aminex ion exclusion column was used
(column temperature was controlled at 40C). Since ethanol
was detected only at low levels (<1%) in early growth phase,
it was not included in this study. The mobile phase for HPLC
was 0.025 M sulfuric acid at a flow rate of 0.6 mL/min. Cell
count was done under microscope on a hemocytometer.
Methylene blue was used as a dye to count only viable cells.
Mixing Time
For viable dynamic results, it is important that the mixing time
of the fermenter be measured. The mixing time was studied
by injecting methylene blue solution at various injection ports.
The mixing process was recorded as video file and played
back on a computer at 30 frames per second (time interval of
0.033 s). Relative dye concentration variation at the point
geometrically opposite to the injection port was recorded as
gray levels (%). Mixing time was determined by measuring
the time, for dye concentration at the observation point to
reach 2% of a final constant value, on a gray level (relative
dye concentration) vs. time graph.
Mathematical Model
The three-parameter modified Gompertz model (Zwietering
et al., 1990) was used to describe the cell growth, as shown

below.
 N    max e  
(-t)+1
Ln
 N0  = A exp
 - exp
 A  
where
N = cell concentration ( number of cells / mL )
N0 = initial concentration
A = Ln ( Nmax / N0 )
max = maximum specific growth rate ( h-1 )
e = exp (1)
 = lag time (h) of cell growth
RESULTS
Oxygen Dissolving Dynamics
The oxygen dissolving dynamics and mixing were first
studied to validate subsequent experiments. Fig. 1
compared the time courses (at 30C) of DO in water
under three conditions: (A) bubbling to accumulate
pressure to 10 psi and hold pressure with vent valve
closed, (B) bubbling at atmospheric pressure with vent
valve opened and (C) pumping air to 10 psi in head space
and hold (oxygen transfer only through liquid surface).
The final DO levels all reached saturation according to
Henry’s law (only data for 0 to 300 sec were shown).
Curve (A) showed a much faster oxygen transfer rate and
a higher saturation concentration, and this may facilitate
the fermentation as will be discussed shortly.
Mixing Time
For a three-phase (liquid broth, air bubbles and yeast
cells) fermentation system as adopted in this study,
providing enough turbulence (mixing) to assure
homogeneity is important for viable results. The
fermenter’s mixing time was measured as stated in the
Materials and Method section. Fig. 2 showed the time
profile of the relative dye concentration (as percentage
gray level) observed at point opposite to the injection port.
In Fig. 2, the dye concentration at fixed position fluctuated
shortly after injection. The time length for the dye
concentration to reach constant (2%) was taken as the
mixing time. The results for various stirrer rpm were
summarized in Fig. 3. The mixing time decreased with
increasing turbulence (rpm of the stirrer), and the
magnitude of decrease leveled off after 100 rpm. This
trend has been reported in the literature (Campolo and
Soldati, 2004). To assure homogeneity of the fermentation
broth, the stirrer speed was set at 400 rpm and six baffles
were adopted throughout this study.
Time Courses
Time courses of cell growth for different modes of
aeration, dissolved oxygen and glucose consumption
were shown in Fig.4 to 8. Mainly, three modes of aeration
were compared: continuous aeration at atmospheric
pressure, intermittently pressurized aeration, and
continuous aeration under pressure. Data were taken at 2
039 Int. J. Chem. Biochem. Eng.
hour intervals for 24 hours. Cell growth data were subject
to nonlinear regression as described above to find the
growth parameters. Important data were summarized in
Table 1 and will be discussed in the next section. Fig. 4 to
8 showed that after a short lag time of 2 to 4 h, the yeast
entered growth phase and reached steady phase within
10 to 15 h.
DISCUSSIONS
Pressurized OTR
The purpose of exhibiting Fig. 1 is to quantitatively show
the pressure effect on OTR (comparing curve (A) and
curve (B), and the effect of gas-liquid interphase area
(comparing curve (A) and curve (C)). As stated above,
under the same operation condition (k La fixed mainly by
bubble size), OTR is governed by oxygen concentration
gradient (C* - C).
According to Henry’s law, the equilibrium concentration of
DO is proportional to oxygen partial pressure in the gas
phase. Therefore, increasing aeration pressure will
proportionally increase C*, which in turn, will increase the
driving force of oxygen transfer. Due to this, OTR was
progressively accelerated during the process of
pressurization. As observed in Fig. 1, the initial oxygen
transfer rate for 10 psi (curve (A)) was much faster than
that for atmospheric pressure aeration. However, high
OTR and DO are not necessarily beneficial to cell growth,
as will be discussed shortly.
Curve (A) in Fig. 1 showed that for pressurized bubbling
operation (10 psi), the oxygen level reached 90%
saturation within 30 sec; and curve (C) showed that during
pressure holding period, oxygen was still being
transferred through liquid surface at a significant level.
Both OTR and mixing time data for water showed that
they are reasonably fast enough for effective intermittent
pressurized aeration operations for durations of 2 to 4
min.
However, Henry’s law applies only to dilute solution. In the
presence of high concentration of solute (glucose and
metabolites), both OTR and saturate DO will be lower. For
15% glucose solution, the initial OTR was slower than that
for water (5 psi, 30C), and saturate DO is lowered by
about 2 ppm (10.7 ppm for water and 8.6 ppm for 15%
glucose solution). Since this research is focused on
comparing pressurized to atmospheric aeration,
quantitative study of solvent effect is not presented in this
report.
Apart from the above discussed solute’s (glucose) effects.
The influence of pressure and solute on the volumetric
oxygen transfer coefficient (k La) is another concern. As
commented by Karimi et al. (2013), k La is affected by the
physical state of the solution, as well as the design and
operational condition of the bioreactor. Karimi et al. (2013)
attained systematic quantitative relations between (k La)
and various operation conditions (impeller designs, air
Yang et al. 040

(A)

DO (ppm) (B)

(C)

time (s)

Figure 1. Oxygen dissolving dynamics at 30C, (A) bubbling to 10 psi and hold
pressure with vent valve closed; (B) bubbling at atmospheric pressure with vent
valve opened; (C) oxygen transfer only through water surface at 10 psi.

100

80
gray level (%)

60

40

20
0 1 2 3 4
time (s)

Figure 2. Mixing time experiment at 400 rpm stirring speed, gray level (%)
represents the relative dye concentration.

flow rates, impeller speed). In their report, high k La of ca. min air flow rate, with two Standard Rushton turbines with
-1
0.052 s was achieved at 1000 rpm impeller speed, 5 L / 4 vertical Blades (Karimi et al., 2013).
 041 Int. J. Chem. Biochem. Eng.

mixing time (s)

rpm

Figure 3. Relationship between mixing time and agitation speed.

(○) glucose ( % w/v)

( ) Ln ( N / NO )
(  ) DO ( ppm )

time ( h )

Figure 4. Time courses, aeration : 0.44 vvm with vent valve open
(atmospheric pressure), solid line is regression result.

In this study, during the pressurization stage (with vent preset value. Initial DO was low (stripped with nitrogen
valve closed) the pressure increased and cumulated to before test), and (C*-C) was high, leading to fast OTR.
Yang et al. 042
The instantaneous kLa could be obtained from a DO vs
time curve by measuring the slope (dC / dt) at a point (C,
t) and dividing by (C* - C). For example, for set pressure
of 5 psi, C* was 8.6 ppm for 15% glucose solution (10.7
ppm for pure water). At an aeration rate of 1.0 vvm, DO
reached 0.7 ppm at 15 sec, the slope was 0.029 ppm/s,
-1
divided by (C*-C), kLa was 0.037 s (DO-time curve not
shown). At 45 sec, DO reached 1.1 ppm, and the
-1
instantaneous kLa was 0.0009 s . Such drastic decrease
in kLa could be attributed to the decreasing cross bubble
pressure gradient when the vessel pressure was
approaching the set point. During the early stage of
pressurization, intracellular pressure was low. Higher
extracellular pressure might have enhanced the
cross-membrane OTR in a similar manner.
Growth Curves and Modeling
Fig. 4 to 8 showed that in all operation modes tested in
this study, the modified Gompertz model worked well for
describing cell growth, for the yeast under investigation.
As commented by Zwietering et al. (1990), the merit of
this model is that only three parameters were needed to
describe cell growth, and they directly correspond to the
three important kinetic variables in cell growth: lag time,
maximum specific growth rate and the final to initial cell
concentration ratio. In their report (Zwietering et al.,
1990), the model was applied on bacterial systems, the
model worked equally well in describing yeast growth in
this study.
Since the inoculums for all sets of experiments were first
activated in shaker flasks, the lag times for entering
growth phase were not significantly different, and they
were not included in Table 1 for discussion. In Fig. 4,
sugar and DO consumption rates were highest during the
growth phase. At 0.44 vvm aeration rate, DO level
dropped to 2 ppm and began to increase when entering
the steady phase, and the sugar consumption rate leveled
off (residual sugar was 5.7%) during this period of time.
This indicated that sugar and DO was not fully utilized,
probably due to product inhibition and/or low OTR into the
cells.
Intermittent Pressurized Aeration
Applying intermittent aeration in Z. rouxii fermentation for
soy sauce production has been reported in the literature.
Wu et al. (2010) applied 10 min. aeration in a 3-day period
to assure good growth of the yeast. Beatrice Food Co.
(1972) reported that intermittent aeration facilitates
maturing the soy sauce. This study investigates
intermittently pressurized aeration for biomass production
of the yeast.
In the intermittently pressurized aeration modes (Fig. 5 to
7), the patterns of DO and sugar consumption rates were
similar to that in Fig. 4. However, Fig. 5 to 7 showed that
the final to initial cell concentration (Nmax / No) ratios were
all much higher than that in Fig. 4 (see also Table 1).
Meanwhile, residual sugar concentrations were all
significantly lower than that in Fig. 4 (see also Table 1)
indicating better sugar utilizing than Fig. 4. Although at
this point, it would be too hasty to conclude that higher
oxygen transfer rate and higher initial DO level under
pressure are the major reasons leading to the above
observations, it is quite likely that they are part of the
reasons.
Growth Kinetics
The maximum specific growth rate and the Nmax to No ratio
are the major concerns in cell production. Examining Fig.
5 to 7 and Table 1, the maximum specific growth rates, for
the intermittent pressurized aeration modes (2.5, 5 and 8
psi), were all higher than that for the continuous aeration
at atmospheric pressure. In Fig. 4, DO was still rather high
during the growth phase (ca 4 ppm) which implies that the
molecular oxygen in liquid broth was not readily available
to the cells. Possible explanation would be the driving
force for extra- to intra-cellular oxygen transfer was not
high enough to compensate high oxygen consumption
rate during growth phase, and this could be overcome by
increasing pressure. Comparing continuous aeration
modes in Table 1, max for 2.5 and 5 psi were much higher
-1
(0.94 and 0.74 h ) than that for atmospheric aeration
-1
(0.58 h ).
When pressure was further increased from 8 to 10 psi
(intermittent mode), max dropped from 0.69 to 0.54 h
-1
(Table 1), and this was accompanied with a decrease in
Nmax to No ratio. This indicates that although OTR is higher
at 10 psi, during the cycle of cell dilation (pressure
release) and cell contraction (pressurization), possible cell
wall damage may be developing due to pressure
fluctuation, and led to inefficient cell growth and sugar
consumption (2.7% residual glucose compared to 0.6%) .
For continuous aeration operations, as stated above, a
mild increase of pressure to 2.5 psi, max increased
significantly, but decreased when pressure was further
increased to 5.0 psi (Table 1). This agrees with the above
speculation that increased OTR aids cell growth, but only
to some point. However, high cell growth rate was not
accompanied with improved Nmax to No ratio and sugar
uptake.
The specific cell growth rate depends on factors like the
strain used, culturing temperature, medium composition,
the design of the fermenter, as well as operation
conditions. The maximum specific growth rates observed
in this study were comparable to those reported in the
literature for Saccharomyces cerevisiae (Biener et al.,
2012; Blank and Sauer, 2004).
Amount of Oxygen Supplied
The amount of oxygen provided to the bioreactor in a
fixed period of time is another perspective in understanding
 043 Int. J. Chem. Biochem. Eng.

(○) glucose ( % w/v)

( ) Ln ( N / NO )
(  ) DO ( ppm )

time ( h )

Figure 5. Time courses, aeration to 2.5 psi, hold for 2 min, open vent valve for
30 sec., solid line is regression result.
(○) glucose ( % w/v)

( ) Ln ( N / NO )
(  ) DO ( ppm )

time ( h )

Figure 6. Time courses, aeration to 5 psi, stop, hold for 2 min, open vent
valve for 30 sec., solid line is regression result.

the fermentation system. For 5 psi, it is evident that the the latter is higher than the 4 min. intermittent operation.
amount of oxygen supplied in continuous operation is Table 1 showed that higher amount of supplied oxygen
higher than that in the 2 min. intermittent operation, and resulted in higher max (0.74, 0.67 and 0.64 h-1 respectively).
Yang et al. 044

(○) glucose ( % w/v)

( ) Ln ( N / NO )
(  ) DO ( ppm )

time ( h )

Figure 7. Time courses, aeration to 10 psi, hold for 2 min, open vent valve
for 30 sec., solid line is regression result.
(○) glucose ( % w/v)

( ) Ln ( N / NO )
(  ) DO ( ppm )

time ( h )

Figure 8. Time courses, aeration continuously at 0.44 vvm, pressure

maintained at 5 psi, solid line is regression results.

Yet, such comparison was not applicable on the Nmax to No Possible rationale for the above comparisons would be
ratio (80, 316 and 271 respectively) and sugar that for the intermittent operations, cells were experiencing
consumption (3.5, 0.6 and 0.6 % respectively). dilation-contraction (depressurizing-pressurizing) cycles.
 045 Int. J. Chem. Biochem. Eng.

Table 1. Comparison of growth kinetic parameters.

max (h )
-1
pressure aeration mode Nmax / No residual glucose (%)

atmospheric continuous 0.58 102 5.7

2.5 psi 2 min / 30 s 0.60 188 1.4

5 psi 2 min / 30 s 0.67 316 0.6

4 min / 30 s 0.63 271 0.6

8 psi 2 min / 30 s 0.69 260 0.6

10 psi 2 min / 30 s 0.54 201 2.7

2.5 psi continuous 0.94 85 3.3

5 psi continuous 0.74 80 3.5

During dilation, intracellular pressure was higher, and
pressure gradient across the cell wall might have
facilitated (or accelerated) exporting metabolites. During
pressurization, extracellular pressure was higher. This is
helpful in transporting oxygen and nutrients into the cell.
However, cell walls are flexible only to some extent
beyond which damage may develop. As shown in Table 1,
for 2 min. intermittent operation, when pressure was
increased from 5 to 8 psi, although max increased (from
-1
0.67 to 0.69 h ), the Nmax to No ratio decreased (from 316
to 260). This conforms to the above discussion that
increasing the amount of suppled oxygen does not
necessarily benefit the cell growth efficiency.
Growth Efficiency
When discussing aerobic cell production, DO or oxygen
supplement was the most frequent discussed issue, while
the intracellular O2 is actually used by the cell. Therefore,
increasing DO in the fermentation should not be the only
concern. Parameters like max, Nmax to No ratio, and
nutrient utilization are important ones that reflect the
efficiency of oxygen utilization. These fermentation
variables should be considered together, that is the
growth efficiency. In Table-1, if the Nmax to No ratio was
divided by the percent sugar, the quotient will reflect the
growth efficiency. For example, the quotient is 21.94 (per
unit % sugar consumed) for the 5 psi - 2 min operation,
which is higher than that for the 5 psi - 4 min operation
(18.82) and the rest of the tested operational conditions.
After all, the growth efficiency is the question all engineers
must answer. Although no direct proof on a molecular
basis provided in this report, the determination of
operational conditions such as pressure and duration will
certainly depend on the flexibility and permeability of the
cell membrane of the microorganism under consideration.
Whereas no attempt was made to pinpoint an optimal
operation condition for best growth efficiency, this work
merely intends to propose a feasible option to improve
growth efficiency in cell production.
CONCLUSION
This study demonstrated that mild pressure (ca 5 psi)
aeration operated intermittently is a strategy worth
considering for the production of Z. rouxii cells. Instead of
the total amount of supplied oxygen, increased OTR
within tolerable range of pressure gradient across the cell
membrane is helpful in improving cell growth efficiency.
This suggests that pressure gradient during
depressurization and pressurization might have facilitated
exporting metabolites and importing oxygen and nutrients.
For most industrial steam-sterilizing fermenters, the
vessels were designed to withstand a pressure of 20 psi,
and intermittent 5 psi aeration is practically applicable.
Hydraulic pressure was not considered in this research,
but needs to be further studied for large bioreactors.
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