was investigated during batch
cultivations of Pseudomonas aeruginosa on a nutrient media. The effects
of process variables (viz. impeller speed, oxygen flow and geometry of
impeller) on the volumetric mass transfer coefficient of oxygen, k,
in a biocalorimeter (Bio-RC1) was investigated and reported in this research
work. The experimental data have been analyzed employing MATLAB to obtain the
influences of the process parameters on _{L}ak. An attempt was
made to correlate volumetric mass transfer coefficient with metabolic heat
production rate at optimized process conditions. The correlation reported in
this work would be useful to control and scale up of bioprocesses._{L}a
With the recent activities for developing
biological processes on industrial scale, there is a need for evolving robust
design and scale-up methods for bioreactors. Bioreactor design is a complex
engineering task (Garcia-Ochoa and Gomez, 2000; Garcia-Ochoa and Gomez, 2005).
Under optimum conditions the microorganisms or cells will reproduce at an
astounding rate. The vessel's geometry and operating conditions like gas ( k) plays an important role in design, scale-up
and economy of the process._{L}aMost of the volumetric mass transfer
coefficient correlations in the literature belong to two categories. In the
first kind i.e., expressing the
dependence of the Sherwood number as a function of the Reynolds, Weber and
Aeration numbers. Biocalorimeter (RC1) is a bench scale bioreactor widely
applied to investigate several biochemical reactions using the metabolic heat
generated due to metabolic activity (Marison et al. 1998). Though lot of
studies has been dealt on thermodynamics of microbial growth using biocalorimeter,
knowledge of mass transfer occurring simultaneously with metabolic heat
generation is feeble (which is more important when the calorimetric results are
used for bioreactor scale-up work). The present study aims to determine the influence
of process variables such as impeller speed and oxygen flow rate on the gas-liquid
mass transfer of oxygen in a biocalorimeter. Correlations for k are needed to be developed for biocalorimeter, which could immensely help for
efficient scale up of calorimetric results for large scale level. Using a
bench-scale biocalorimeter, with a single impeller and a capacity of 1 l (total volume), the effects of process variables on the _{L}akwere investigated.
Later, _{L}a kvalues were analyzed to establish correlations
with impeller speed and oxygen flow rate. From the established correlations,
the numerical values for the exponents (α, β) were determined. A
correlation was also developed between _{L}a k and metabolic heat
(q) at optimized process conditions._{L}aThe organism used for oxygen transfer studies was
Nutrient broth (a complex growth media)
was used for cultivation of All the experiments have been
carried out in a biological reaction calorimeter (Bio-RC1, Mettler-Toledo, AG, Switzerland) with 1 l of working volume (Internal dia 8.2 cm & Height of reaction broth on reactor 15 cm), equipped with DO (Schott, Oxy12) and temperature probes (Figure 1). Agitation was
provided with a 4-blade turbine impeller (0-1000 rpm) type with a ratio of
stirrer/tank diameters (D/T) of 0.44. Oxygenation was performed by supplying
iolar grade pure oxygen from an oxygen cylinder and the flow was controlled by
a rotameter. The inlet oxygen was sterilized through a membrane filter (0.2 µm)
and sparged in to the bottom of the reactor. In this study, 2 different
stirrers viz. Turbine-impeller type (4-bladed and 4 cm dia) and a Rushton-turbine type (4-bladed and 4 cm dia) of 1 mm of thickness were used to investigate the oxygen mass transfer ‘L-bend’ type gas sparger (4 cm long) with holes of 0.5 mm diameter was employed for oxygen supply. Oxygen uptake studies were
performed at isothermal mode and the reactant temperature ( T). This was done by
circulating at high rate (2 ls_{j}^{-1}) low-viscosity silicone oil through
the reactor jacket. The jacket temperature was carefully controlled by blending
oils from ‘hot’ and ‘cold’ circuits, via an electronically controlled metering
valve. Heat generated from the biological reactions was monitored by WINRC
software (Marison et al. 1998), which acquired the temperatures of both reactor
(T) and jacket (_{r}T) and evaluated the value
for heat production rate._{j}The measured heat production rate (q in W) is given by
T)
[1]_{j}Where U is the global heat
transfer coefficient in (W K T is the jacket temperature in ºC._{j}All the inlet and outlet
streams were completely insulated to avoid heat loss due to heat bridge
phenomenon. Baseline heat signal ( q). After reaching a stable baseline heat signal (_{r}q),
the reaction was initiated by addition of inoculum and similar kind of stable
baseline signal was observed at the end of the experiment (endogenous phase). This
baseline heat value (_{b}q) was carefully eliminated from the
heat evolved due to biological reaction. The value of metabolic heat evolution
during the bioprocess (_{b}q) could simply be determined by
deducting the measured _{r}q (from Equation 1) from that of the base-line
signal. Hence the metabolic heat production rate (q) is
given as follows:_{r}
UA (ΔT - ΔT)
= _{b}q [2]_{r}The UA factor was determined for each run at regular time intervals by means of in situ calibration electrical heater provided in calorimeter (Hariri et al. 1996). The experimental work has been
carried out in a complex growth (NB) media, maintaining optimum operational
conditions (pH 7.0, 37ºC and 2% seed culture conc.). The oxygen flow rate was
varied in the range of 0.5-1.5 l min Biomass concentration was determined by centrifuging the culture sample taken from the calorimeter in an ultracentrifuge (Sigma, Model no. 3K30) at 10000 rpm for 10 min. The harvested biomass was dried in an oven for 24 hrs at 105ºC. The cell dry biomass was estimated gravimetrically. DO was monitored online by means of a polarographic electrode obtained from M/s. Schott, Germany (OXY12 Model).
Dynamic method (Singh, 1996)
was used for measuring OUR (oxygen uptake rate) and The following mass balance equation for the DO in batch reaction can be established:
where the first term on the right hand side of Equation (3) is the oxygen transfer rate (OTR) and the second term is the volumetric OUR of the culture. The measurement of OUR and OTR can be made using the dynamic technique, in two stages (Figure 2). In the first stage, the inlet of airflow to the broth is interrupted, and a decrease of DO concentration due to cellular respiration is observed, which was recorded by a polarographic oxygen probe. The volumetric OUR is determined by noting the changes in the DO concentration when the oxygen flow was stopped. Equation (3) can be simplified to: [4] The DO concentration should be
maintained higher than 10% of saturation value to ensure that the microorganisms
are not damaged due to lack of oxygen. The volumetric OUR is obtained from the
slope of the linear regression of the change in DO concentration vs. time. This
first stage is employed to obtain the value of the specific OUR (
Equation (5) was used to
determine the volumetric mass transfer coefficient. Equation 5 was solved by
plotting ^{-1}) and impeller type (Rushton-Turbine
& Turbine). The measurements have been carried out changing the superficial
gas velocity, V, as 0.00165, 0.0003304 and 0.004976 ms_{s}^{-1},
which corresponding to oxygen flow rates between 0.5, 1 and 1.5 l min^{-1}. The stirrer speed, N, was varied between 50 and 250 rpm and simultaneously
OUR, OTR and k values were determined._{L}aThe oxygen mass transfer rate
in the biocalorimeter with mechanical agitation of broth is a function of many
variables, such as the physical properties of the liquid (viscosity, surface
tension, etc.), the geometry of the vessel and stirrer, the type of sparger and
the operational conditions. In the literature, both dimensional and
dimensionless equations for the volumetric mass transfer coefficient have been
proposed. In this work, the effect of impeller speed (N), superficial gas
velocity ( _{, }oxygen flow) and geometry of stirrer
on oxygen mass transfer were extensively studied. Predictions about the rate of
absorption of a gaseous species in a stirred tank is usually based on
correlations of overall volumetric mass transfer coefficient (k)
with mechanical agitation power per unit volume (P/V_{L}a_{L}) and gas
sparging rate expressed as the superficial velocity (V).
The power input per unit volume (P/V_{s}_{L}) and, superficial gas velocity V are the major correlation coefficients for _{s}k.
Therefore, equations of the following type are frequently found in the
literature (Vant Riet, 1979)._{L}a
where,
AND^{α1,α2} [V]_{s} [7]^{β}Where, N^{3}D^{2}) by
performing multiple regression analysis in MATLAB (Version 7.0).Attempts were made to
correlate the heat production rate (q) with volumetric mass transfer
coefficient (
valuesFigure 3 shows the plot of k value at all the growth phases of the culture and
was fixed as a suitable impeller for oxygen mass transfer experiments. Oxygen
mass transfer in Rushton-turbine type impeller was found to decrease with the
biomass growth. This may be attributed to the increase in viscosity of broth
media due to biomass growth and the inefficiency of Rushton-turbine type to break
the bubbles effectively for enhanced mass transfer. Further, the high shear
force caused a negative effect on growth of culture and made it unsuitable for
further studies. Excessive foaming was also observed when Rushton-Turbine type
impeller was used._{L}a
valuesThe effects of impeller speed (turbine)
on the ^{-1}, temperature of 37ºC and pH 7.0. Experiments were performed at respective impeller speeds starting from lag-phase until the culture reached
its stationary phase. The values of k for different impeller
speeds are plotted in Figure 4. The volumetric mass transfer
coefficient, _{L}ak, increased from 0.1212 to 0.582 min_{L}a^{-1} when the impeller speed was increased from 50 to 200 rpm. It can be also seen
from the Figure 5 that further increase in impeller speed to 250 rpm
showed no significant rise in k value (results not
shown). Hence an optimum stirrer speed of 200 rpm was fixed for efficient
oxygen mass transfer for the cultivation of _{L}aP. aeruginosa. Analysis of
biomass growth of P. aeruginosa at different impeller speeds also proved
that 200 rpm is an optimum value (Figure 5). Further increase in
impeller speed to 250 rpm caused a significant decrease in growth, perhaps due
to inefficient oxygen transfer under high turbulence conditions. Further we
also observed high foaming formation at 250 rpm. In order to prove the decrease
in biomass growth rates at high stirrer speeds, the specific growth rates (μ)
were estimated for all the evaluated conditions and shown in Figure 6. It
can be seen that at 250 rpm the specific growth rate decreases and supports our
decision for fixing the stirrer speed to 200 rpm.
valuesThe effects of oxygen flow
rates on k were plotted in Figure 7. Volumetric
mass transfer coefficient, _{L}ak, increased from 0.181 to 0.41
min_{L}a^{-1} when the oxygen flow rate was increased from 0.5 to 1 l min^{-1}.
Further increase in oxygen flow to 1.5 l min^{-1} show no improvement in mass transfer (results not shown). Cell dry weight was estimated
at regular time intervals and specific OUR (q_{O2}, min^{-1}) was
calculated. Figure 8 shows variation inq_{O2}profiles with change in
oxygen flow rates. From Figure 8, it was found thatq_{O2}results also showing similar
trend like k plot, where 1 l min_{L}a^{-1} was found to be optimum value.Metabolic heat generated due
to the growth of Total metabolic heat generated
(cumulative) till the exponential growth phase at varying impeller speed
conditions were evaluated using WINRC software (Mettler-Toledo,
AG, Switzerland) and compared in the plot shown in Figure 10. Heat
added due to stirring power was systematically eliminated from the cumulative
metabolic heat generated by performing baseline experiments (results not
given). Figure 10 clearly depicts that the metabolic heat production was
increased approximately 4-fold when the impeller speed was raised from 50 to
200 rpm. It can be also noted from the Figure 10, that further increase
in impeller speed (to 250 rpm) caused a considerable fall in heat production. In
the similar way, heat productions at varying oxygen flow rates were analyzed
and results were shown in Figure 11. From the Figure 11, it can
be seen that increasing the oxygen flow from 0.5 l min ^{-1}).
) with impeller speed (N) and Superficial gas velocity (V_{s})It can be found from
experimental data that ^{-1} oxygen flow showed a
maximum k value when compared to other flow rates._{L}aIn order to determine the
influence of operational conditions ( k values have been correlated according to the relationship given below Equation 8,_{L}a
N)^{3}D^{2} (^{α}V)_{s} [8]^{β}In the above equation, the
influence and the relative importance of the considered variables are
determined by the coefficients
N)^{3}D^{2}^{0.24}
[9]The statistical values of The exponential relationship
between V_{s})
for this study is given by Equation (10),
V)_{s}^{0.41}
[10]The statistical values of V_{s})
was compared with the literature values. Previous investigators (Hyman and
Bogaerde, 1960) obtained the values of the exponent on the superficial gas
velocity in the range 0.17 to 0.67, depending on the agitator speed and the
geometry of the equipment. The exponent obtained from this study again well
corroborates to the reported values. A satisfactory linear correlation (Figure
12) was observed between volumetric mass transfer coefficient (k)
and metabolic heat (q) at optimized process conditions (200 rpm & 1 l min_{L}a^{-1}) as follows
q) + 0.3176 [11]
Evaluation of the experimental
data showed that k value
and the increase in oxygen flow rate caused a two-fold rise in _{L}ak values. Turbine-type impeller showed maximum oxygen transfer compared to
Rushton-turbine type. The _{L}ak values obtained from the data
of oxygen absorption were used to establish correlations as a function of the
process variables. The exponents of power number (_{L}aN^{3}D^{2})
and superficial gas velocity (V) found for NB growth
media were 0.24 and 0.41 respectively, are in good agreement with the exponents
given in the literature. Calorimetric experiments revealed that the growth of _{s}P.
aeruginosa in NB media was found to follow the heat generated due to
metabolic activity. Bothq_{O2}and metabolic heat are found
to be influenced greatly by growth phases of P. aeruginosa and attained
their maximum value at the middle of the exponential phase. Heat data (q) were
found to correlate well with volumetric mass transfer coefficient (k)
and a linear correlation was obtained at optimized process conditions. The
correlations obtained in this study clearly indicates that both respirometric
and calorimetric results from biocalorimetric experiments could be used for
effective scale-up and design of biological reactions to large scale._{L}aOne of the authors Mr. S. Senthilkumar is grateful to CSIR, New Delhi for the SRF fellowship. The author thanks the Director, CLRI for his kind permission to publish this work. The author also wishes to express his gratitude to Prof. K.S. Gandhi for his useful suggestion and Prof. N.R. Rajagopal for his constant encouragement. CALIK, Güzide; VURAL,
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