Journal of Biotechnology 63 (1998) 85 – 95

Minireview
Measurement of volumetric (OUR) and determination of
specific (qO2) oxygen uptake rates in animal cell cultures

Pierre-Alain Ruffieux, Urs von Stockar *, Ian William Marison

Institute of Chemical Engineering, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland

Received 17 October 1997; received in revised form 9 March 1998; accepted 13 March 1998

Abstract

Oxygen is a key substrate in animal cell metabolism. It has been reported that the oxygen uptake rate (OUR) is
a good indicator of cellular activity, and even under some conditions, a good indicator of the number of viable cells.
The measurement of OUR is difficult due to many different reasons. In particular, the very low specific consumption
rate (0.2 × 10 − 12 mol cell h − 1), the sensitivity of the cells to variations in dissolved oxygen concentration and the
difficulty to provide oxygen without damaging the cells are problems which must be taken into account for the
development of OUR measurement methods. Different solutions based on an oxygen balance on either the liquid
phase or around the entire reactor, and with a variable or stable concentration of dissolved oxygen have been
reported. The accuracy of the OUR measurements and the required analytical devices are very different from method
to method. © 1998 Elsevier Science B.V. All rights reserved.

Keywords: Oxygen uptake rate; Animal cell culture; Oxygenation

Abbre6iations: kLa, volumetric oxygen transfer coefficient
(h − 1); C, concentration (mol m − 3); G, molar gas flow rate
(mol h − 1); H, Henry’s constant (atm m − 3 mol − 1); OTR,
oxygen transfer rate (mol m − 3 h − 1); OUR, oxygen uptake
rate (mol m − 3 h − 1); P, total pressure (atm); pO2, dissolved
oxygen, % of air saturation; qO2, specific oxygen uptake rate
(mol cell − 1 h − 1); t, time (h); V, volume (m3); X, cell concen-
tration (cell m − 3); y, molar fraction in the gas phase; *,
relative to the equilibrium; G, relative to the gas phase; L,
relative to the liquid phase; max, maximum.
* Corresponding author. Fax: +41 21 6933680.
1. Introduction
All animal cell cultures are aerobic processes in
which the oxygen requirement varies between cell
lines (Spier and Griffiths, 1982). The effects of
oxygen concentration on animal cell cultures has
been studied by many authors (Mizrahi, 1982;
Miller et al., 1987; Phillips et al., 1987; Ozturk
and Palsson, 1990, 1991; Ogawa et al., 1992; Shi
et al., 1993). General trends are difficult to estab-

0168-1656/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved.
PII S0168-1656(98)00046-7
86 P.-A. Ruffieux et al. / Journal of Biotechnology 63 (1998) 85–95
Table 1
Literature qO2 values
qO2×10−12 (mol O2 cell−1 h−1) Cell line
0.15–0.36 KS1/4 (hybridoma)
0.21–0.25 NB1 (hybridoma)
0.2349 0.014 C1a (hybridoma)
0.05 FS-4 (Human diploid cells)
0.19–0.4 AB2-143.2 (hybridoma)
0.023– 0.087 167.4G5.3 (hybridoma)
0.33–0.37 HB-32 (hybridoma)
0.15 Hybridoma
0.219– 0.406 NSO (myeloma)
0.4690.05 MAK (hybridoma)
0.23–0.42 X-D (hybridoma)
lish from these reports. Thus Miller et al. (1987)
reported an increase in total and viable cell num-
ber when the dissolved oxygen concentration
(pO2) was decreased to a critical value of 0.5% air
saturation with continuous cultures of AB2-143.2
(hybridoma), while the optimum pO2 for antibody
production was 50% air saturation. However, an-
other group (Mizrahi, 1982) found exactly the
reverse effect with cultures of human lymphoblas-
toid cells (RPMI c 7430): the highest cell yield
was attained at the highest atmospheric pO2,
while the highest immunoglobulin yield was
achieved at the lowest pO2. They also showed the
importance of a stable pO2 in order to obtain a
constant production of antibody.
Since the solubility of oxygen in water is very
low (7.8 mg l − 1 at 25°C), and even lower in
culture media, depending on the composition
(Quicker et al., 1981; Schumpe et al., 1982; Rey-
nafarje et al., 1984; Schneider and Moser, 1984), it
is necessary to supply the medium continuously
with oxygen. The methods generally used in mi-
crobial culture systems are difficult to apply due
to the physiological characteristics of animal cells.
The absence of cell walls in mammalian cells and
their relatively large size make them particularly
sensitive to shear forces (Abu-Reesh and Kargi,
1991). The cell damage caused by sparging (Gard-
ner et al., 1990), bubble formation (Handa et al.,
1985; Kunas and Papoutsakis, 1990; Jordan et al.,
1994) and foaming (Zhang et al., 1992) has been
extensively reported. Techniques to protect the
Reference
Backer et al., 1988
Boraston et al., 1984
Dorresteijn et al., 1996
Fleischaker and Sinskey, 1981
Miller et al., 1987, 1988a,b
Ozturk and Palsson, 1990
Ramirez and Mutharasan, 1990
Singh, 1996
Yoon and Konstantinov, 1994
Zhou and Hu, 1994
Hiller et al., 1991
cells from these damaging effects include the use
of additives to the culture medium (for a review
see Papoutsakis, 1991) such as Pluronic (Sinskey
et al., 1981; Zhang et al., 1992; Jordan et al.,
1994), the use of gas permeable membranes (Fleis-
chaker and Sinskey, 1981; Wagner and Lehmann,
1988; Beeton et al., 1991; Schneider et al., 1995)
to supply bubble-free aeration, the use of pe-
rfluorocarbons with high oxygen solubility
(Hamamoto et al., 1987) and the application of
airlift (Bugarski et al., 1989; Johnson et al., 1990)
or perfusion loop reactors (extensively reviewed
by Spier and Griffiths, 1982) have been widely
discussed.
In order to supply oxygen to the culture at a
non-limiting rate and to facilitate scale-up it is
therefore essential to have a knowledge of the
oxygen uptake rate, OUR (mol O2 m − 3 h − 1), of
a process. In Table 1, different values from the
literature are listed with an average specific oxy-
gen uptake rate for animal cells of 0.2 × 10 − 12
mol O2 cell − 1 h − 1. The knowledge of the oxygen
uptake rate (OUR) is not only useful for scale-up,
but can also provide considerable information
about cellular activity (Yamada et al., 1990) and
physiology, as well as for the estimation of viable
cell number (Fleischaker and Sinskey, 1981).
Kyung et al. (1994) employed OUR measure-
ments as an on-line metabolic indicator of the
physiological state of the cells in the bioreactor
and to adjust the perfusion rate of a continuous
culture with total cell retention. These authors
 P.-A. Ruffieux et al. / Journal of Biotechnology 63 (1998) 85–95
were able to reach almost 108 cell ml − 1 and
showed that regular measurement of the OUR
could be used as the basis of an automated dy-
namic perfusion system. It is also possible to
combine OUR with other on-line measurements
to provide further information about the culture
through so called ‘software sensors’. A ‘software
sensor’ is a virtual sensor, implemented on a
computer, which uses information available on-
line, through analytical devices or probes, to mon-
itor a parameter which is not directly measurable
(Montague et al., 1992). An example of such a
sensor is presented by Dorresteijn et al. (1996). It
is based on the on-line estimation of the ATP-pro-
duction rate from the oxygen uptake and the
lactic-acid production rates. Similar experiments
were also performed by different groups (Glacken
et al., 1986; Eyer and Heinzle, 1996). Such sensors
allow a good estimation of the amount of biomass
with a sensitivity higher than other methods.
However these techniques were originally devel-
oped for yeasts and yield poor results for animal
cells (Dorresteijn et al., 1996).
In summary, it has now been clearly established
that animal cells are sensitive to the concentration
of dissolved oxygen and that aeration must be
provided in a manner which avoids excessive
shear stress. This has resulted in the development
of a number of different techniques to provide
oxygen as a function of cell line, reactor type,
volume of culture etc. In addition, very sensitive
OUR measurements are also needed for monitor-
ing biomass concentration and cellular activity.
The aim of this review is to present the different
ways of measuring OUR in a range of bioreac-
tors, with special emphasis on the advantages,
disadvantages and precision of each method.
2. Reported methods for the determination of
OUR
A schematic representation of the aeration of a
bioreactor is presented in Fig. 1. A flow Gin with
yin as the molar fraction of oxygen enters the
reactor. Oxygen is transferred to the liquid vol-
ume VL across an exchange surface a. This surface
could be either the culture volume – headspace
87
interface, the bubble surface, a membrane inter-
face or any combination of these. In the liquid
phase only the cells are assumed to consume
oxygen. Finally the flow exits the reactor, Gout,
with a composition yout.
To determine OUR, one of the two following
approaches is generally used. It is possible to
consider the whole reactor (Eq. (1)) or only the
liquid (Eq. (2)) phase to write the oxygen mass
balance (Boundaries 1 or 2 on Fig. 1).
dCL d(CG · VG)
· VL +
dt dt
= Gin · yin − Gout · yout − OUR · VL (1)
dCL Ptot · yG
= kLa − CL − OUR (2)
dt H
The first approach is based on a global mass
balance, while the second, based on a liquid phase
balance, is divided into two sub-groups. A distinc-
tion is made between the methods with a constant
dissolved oxygen (DO) concentration, (stationary
liquid phase methods) and those with a non-con-
stant DO (dynamic methods).
Both approaches have certain advantages and
disadvantages. For methods based on a global
mass balance, the main advantage is that only two
fluxes, two gas compositions and the medium
volume are required for the determination of
Fig. 1. Schematic representation of a generalized aeration
system showing phase boundaries.
88 P.-A. Ruffieux et al. / Journal of Biotechnology 63 (1998) 85–95
OUR. These measurements may be on-line and
are non-intrusive and thus exert no adverse effects
on the culture. The main disadvantage is the very
high accuracy needed with respect to the determi-
nation of fluxes and compositions, resulting in the
need for very expensive equipment operating at
the limits of sensitivity.
On the other hand, the measurements based on
liquid mass balances are possible with very simple
and relatively inexpensive instrumentation. How-
ever, values for kLa and C*L are required, which
are difficult to determine since they vary during
the culture and must be frequently estimated. kLa
changes are mainly due to variations in bubble
size caused by changes in the physical properties
of the media such as density, viscosity, surface
tension, and others. Dynamic methods introduce
perturbations to the culture, resulting in a non-
constant pO2, a situation which may result in
physiological/kinetic changes and is thus not rec-
ommended for all systems.
2.1. Global mass balance
In the case of no oxygen accumulation in the
reactor, Eq. (1) may be simplified and Eq. (3) is
obtained. Thus with a knowledge of the flux and
composition of the aeration gas at the inlet and
outlet of the reactor together with the liquid
volume VL it is possible to determine OUR:
Ginyin − Goutyout
OUR=
VL
The precise determination of yO2 and G with
cell suspension cultures may be relatively difficult
since oxygen consumption is very small. Calcula-
tion of the necessary accuracy for determination
of yO2 may be estimated for a standard bioreac-
tor (1.5 l) with a medium volume of 1 l, and with
a typical oxygen uptake rate by animal cells of
0.2 ×10 − 12 mol O2 cell − 1 h − 1. A reasonable
precision for the estimation of OUR would be
1× 10 − 5 mol O2 h − 1 l − 1: which corresponds to a
cell concentration of 5× 104 cell ml − 1. With a
standard aeration rate of 100 ml min − 1, yO2,out is
equal to 0.20996 assuming that yO2,in is 0.21.
Thus the O2 concentration must be measured with
a precision of 219 0.02% if G is considered con-
stant; if this is not the case, Gout could be deter-
mined using an inert gas balance (Heinzle, 1992):
y0,inert
G1 = G0H (4)
y1,inert
Thus, classical devices such as paramagnetic or
acoustic (oxygen in air 209 1%) gas analysers
usually have sensitivities which limit the use for
developing global mass balances, although they
may be suitable for the determination of yO2 in
the case of a stationary liquid phase balance. Only
mass spectrometers (Heinzle, 1987) are able to
determine pO2 with sufficient precision (oxygen in
air 209 0.01%; Heinzle, 1992), however even in
this case the measurement error is of the same
order as the pO2 signal. A possibility to solve this
problem is to aerate the reactor with a smaller gas
flow rate, such that the difference between yO2,in
and yO2,out increases. However, in this case the
residence time of the gas increases (to approxi-
mately 10 h for low flux) leading to long delays
for gas analysis and problems related to a poorly
mixed gas phase. With a small headspace volume,
it is theoretically possible to avoid a long resi-
dence time, however, clogging of filters by foam-
ing could be a major problem.
It has been proposed (Eyer et al., 1995; Oeg-
gerli et al., 1995) to aerate the culture medium by
sparging with O2 while flushing the headspace
with a much higher flux (100-fold) of an inert gas
(helium). This method results in a relatively large
(3) consumption of the oxygen (due to the low flow
rate), while avoiding long residence times of the
gas exiting the medium in the headspace of the
bioreactor. Such a system has been used (Eyer et
al., 1995) to obtain precise gas-phase mass
balances.
Mass spectrometry, without gas flushing of the
headspace, has also been used by Dorresteijn et
al. (1996) to determine OUR. However, in order
to obtain a difference between yO2,in and yO2,out
detectable by the MS, a gas residence time in the
reactor of 3000 s was required. Furthermore it
was essential to have a stable DO concentration
since, with a drift of 9 0.1% air saturation
around the set-point value, the accumulation term
becomes non-negligible, and it is not possible to
determine the OUR simply from yO2,in and
 P.-A. Ruffieux et al. / Journal of Biotechnology 63 (1998) 85–95
yO2,out. In such a case an error of 9 25% in the
OUR determination is not uncommon. Thus, it is
necessary to keep DO drifts within 90.05%.
Mass spectroscopy has been used with large-
scale cell suspension cultures (1300 l; Backer et
al., 1988) for the on-line monitoring of OUR
using gas sparging combined with headspace
flushing (Eyer et al., 1995; Oeggerli et al., 1995).
The DO was controlled by acting on agitation
and air sparging rates. From a knowledge of the
gas flux and composition, a mass balance was
made from which the OUR was reported to be
reliably and reproducibly determined with no cell
damage, due to agitation or sparging, observed.
The reported advantages of this method were
that no kLa measurements are needed, periodic
re-calibration is possible, there is a low risk of
contamination, since only the inlet and the outlet
gas fluxes are analysed, and no electrodes are
required. Thus the only analytical device suitable
for global mass balances is the mass spectrometer.
The main disadvantages reported for MS gas
analysers are the rather high investment cost and
rare, but expensive, maintenance (Heinzle, 1987).
However it should be made clear that this tech-
nique is only suitable under conditions of stable
DO since a change of 0.5% DO corresponds to an
oxygen concentration of 9.37×10 − 7 mol l − 1, a
value which represents the consumption of O2 for
3 × 105 cell for 1 min.
2.2. Stationary liquid phase balance
In this method, the DO is maintained constant
and therefore, according to Eq. (2), the oxygen
transfer rate must be equal to the oxygen uptake
rate. Consequently it is possible to determine
OUR from the OTR, which is given by Eq. (5):
OTR= kLa(C*L − CL)
From a knowledge of the gas composition and
the kLa, it is possible to calculate the oxygen
transfer rate. The kLa must be known throughout
the culture. As this may vary significantly with
sparged systems, surface aeration is generally
used, although it is also possible to use bubble-
free hydrophobic membrane systems. In order to
maintain a constant kLa, the DO is usually regu-
89
lated by changing the composition of the inlet gas
to the bioreactor while maintaining the total flow
constant using either automatic or manual control
(Miller et al., 1988b).
Since the OUR determination methods require
a knowledge of kLa and O2 solubility, it is neces-
sary to measure these values experimentally. A
large number of publications have considered how
these parameters may be determined (Linek et al.,
1973; Linek and Vacek, 1981; Slininger et al.,
1989; Linek and Sinkule, 1990; Gauthier et al.,
1991). The techniques used are based upon sta-
tionary or dynamic methods. More recently a
static method for the simultaneous determination
of kLa and the Henry coefficient based on mass
spectrometry was proposed (Dorresteijn et al.,
1994). This involved circulating the culture
medium from the bioreactor, via silicone tubing,
through a stripping vessel which was continuously
flushed with nitrogen (van Sonsbeek et al., 1991).
From four mass balances (oxygen transfer from
the headspace of the bioreactor to the liquid
phase, two equations for oxygen transport be-
tween the bioreactor and stripping vessel and for
oxygen transfer from the liquid-phase to the
headspace of the stripping vessel), a knowledge of
the composition of the four gas fluxes (bioreactor
inlet and outlet, stripping vessel inlet and outlet)
and of the DO in the reactor and at the end of the
silicon tubing, it was possible to calculate kLa and
H.
Measurement of OUR based on stationary liq-
uid phase methods has been reported by many
authors (Miller et al., 1987, 1988a,b; Ramirez and
Mutharasan, 1990; Bonarius et al., 1995). To ap-
ply Eq. (5), yO2 must be known in order to
calculate C*L from Henry’s law. The simplest way
to obtain the molar fraction is by calculation from
the flows measured with the mass flowmeters used
(5)
for the manual regulation of the DO (Miller et al.,
1987, 1988b; Ozturk and Palsson, 1990; Lin et al.,
1993). It is important to use a mass flowmeter
with good accuracy (1% for the oxygen fraction).
Generally such devices are, or should be, com-
bined with a pressure regulation system for avoid-
ing flow variations, and should be regularly
calibrated. If the DO is regulated automatically,
using a controlled mass flowmeter, the molar frac-
90 P.-A. Ruffieux et al. / Journal of Biotechnology 63 (1998) 85–95
tion can be calculated from the set point of each
gas (Ramirez and Mutharasan, 1990). This
method has the advantage that yO2 may be con-
tinuously determined throughout the culture ex-
periment. It is also possible to use an analytical
device such as infra-red and paramagnetic analy-
sers or mass spectrometry.
Using surface aeration, the kLa at the gas – liq-
uid interface is small, therefore silicone, or some
other hydrophobic membrane or tubing may be
used, the choice of the appropriate length or
surface of which enables a chosen kLa to be
supplied. With this appropriate kLa, it is then
possible to aerate with a gas flow, in which the O2
fraction may be varied from 0.2 at inoculation to
1 at Xmax. A precision of 1% on the molar fraction
is common and allows measurement of the con-
sumption of 104 cells ml − 1, assuming that the
maximum cell density reached in the culture,
Xmax, is equal to 106 ml − 1. In the case where it is
desired to measure the OUR by maintaining a
fixed DO and, maintaining a constant gas flow
rate, determining the mole fraction of oxygen
required to maintain this DO e.g. by the use of
mass flowmeters, the flow of gas through the
lumen of the tubular membrane should be rela-
tively high in order that there is no appreciable
difference between the inlet and outlet gas compo-
sition, i.e. that there are no gradients along the
length of the membrane. Clearly this means that it
is impossible to measure the oxygen consumption
by means of gas analyzers since there is no differ-
ence in inlet and outlet concentrations. However,
if a global mass balance is desired then instead of
measuring the mole fraction of oxygen, a much
lower flow rate must be chosen to ensure sufficient
oxygen transfer to the medium while permitting a
sufficiently significant difference in inlet and out-
let gas composition for detection by gas analyzers.
Both techniques may be used with cell concentra-
tions as low as 104 cells ml − 1 or up to 50 × 106
cells ml − 1 providing a suitable membrane surface
area, i.e. length of membrane tubing, is chosen.
2.3. Dynamic method
The most straightforward means to determine
the oxygen uptake rate is the so called ‘dynamic
method’. The general principle is to interrupt the
supply of oxygen and to measure the decrease of
the dissolved oxygen concentration with time. If
there is no further transfer from the gas to liquid
phase, the decrease in oxygen concentration is
only due to consumption by the cells, conse-
quently a linear decrease over the time interval is
observed and the oxygen consumption rate is
proportional to the slope of the curve. Upon
resumption of aeration, it is also possible to deter-
mine the kLa from the rate of re-saturation of the
dCL
medium. A plot of versus CL yields a slope
dt
equivalent to the kLa. Thus in order to determine
dCL
kLa it is only necessary to measure CL and
dt
(Eq. (6)):
dCL Ptot · yG
= kLa − CL − OUR (6)
dt H
This method was first reported (Taguchi and
Humphrey, 1966) for microbial fermentation sys-
tems and has since been widely used, with or
without minor modifications.
The dynamic measurement has been success-
fully undertaken with reactors up to 300-l scale
(Singh, 1996; Preissmann et al., 1997). However,
in standard laboratory bioreactor cultures, it is
necessary to take into account the contribution of
surface aeration during the measurement period.
In order to overcome this surface effect, an off-
line determination of qO2 was performed (Frame
and Hu, 1985) using a 50-ml flask with magnetic
agitation, completely filled with media containing
cells, that is with no headspace, in which the rate
of decrease of oxygen concentration was assumed
to represent the oxygen uptake rate. A similar
procedure was employed (Yamada et al., 1990) to
measure the OUR with a very small number of
cells (0.5–10 × 106 cells) using a completely filled
5-ml glass bottle. In order to overcome the
headspace effects using bioreactor systems, it was
proposed (Zhou and Hu, 1994) to flush the reac-
tor headspace with nitrogen and, from a knowl-
edge of the mass transfer coefficient at the surface,
to correct the OUR for the rate of O2 diffusion to
the headspace. Other groups (Fleischaker and
Sinskey, 1981; Glacken et al., 1986) simply mea-
 P.-A. Ruffieux et al. / Journal of Biotechnology 63 (1998) 85–95
sured the OUR during a small change of dissolved
oxygen concentration e.g. from 20 to 10% air
saturation, assuming that under these conditions
the effect due to the headspace is negligible.
An on-line method based upon the dynamic
method has also been reported (Singh, 1996) in
which the dissolved oxygen response to intermit-
tent sparging was used to determine the OUR.
The pO2 values are continuously monitored and
stored on a computer and the OUR determined
and corrected for the constant contribution of the
surface, determined from a separate experiment.
The contribution of the surface is assumed to be
constant due to the high flow of inert gas (1000
times larger than the flow of sparged air). As a
result an OUR value could be obtained every
10 – 15 min. This method of intermittent sparging
causes swings in the pO2 from 30 to 55%, which
may have adverse effects on some cell lines. The
limit to OUR measurements by this method is
0.05 mM O2 l − 1 h − 1, corresponding to an ap-
proximate cell concentration of 105 cell ml − 1.
Zhou and Hu (1994) also used the dynamic
method to determine OUR using surface aeration
only. In this case when the pO2 reached 65% of
air saturation the flux of air was replaced by
nitrogen. The rate of decrease of pO2 between
50% and 30% air saturation was then used to
determine OUR. Such measurements were per-
formed every 1–2 h. The same frequency of OUR
measurements was also obtained by Oh et al.
(1996) who, in order to minimise signal noise
from the pO2 probe, averaged the pO2 measure-
ment over a period of 10 min. Thus the dynamic
method is relatively inexpensive and straightfor-
ward, the only requirement being a short response
time of the pO2 electrode. A considerable amount
of literature discusses methods for calculation of
the response time of the electrode and for the
correction of the pO2 signal (Linek et al., 1973;
Linek and Sinkule, 1990; Merchuk et al., 1990).
Some authors (Yoon and Konstantinov, 1994)
have proposed a modification of the dynamic
method based on the fact that when a cell culture
flows through a tube at a constant rate, the pO2
decreases linearly along the length of the tubing.
Thus by placing pO2 electrodes at the entrance
and exit of a tube forming an external loop to the
91
reactor it is possible to determine OUR from Eq.
(7).
1
OUR= · (C iO2 − C Of 2) (7)
Dt
This method has the advantage that no kLa
value and expensive gas analysers are required,
that the pO2 within the reactor remains constant
while only a small fraction of the culture volume
is subjected to a decreasing pO2 for a relatively
short period of time. The only major difficulty is
the requirement for careful calibration of the two
pO2 electrodes, since the OUR determination is
based on the difference between the signals of the
two electrodes. The latter might necessitate a sys-
tem for the sterile removal, washing, calibration,
re-sterilisation and re-insertion of the probes
from/to the bioreactor at intervals during the
culture. Such a technique has also been applied to
OUR measurements in fluidized-bed and hollow-
fibre reactors. However, in these cases, due to the
high cell concentration, the external loop is un-
necessary and it is possible to simply measure the
difference in pO2 between the top and bottom of
the bioreactor.
Table 2 presents a rapid overview of the differ-
ent methods for determination of OUR with the
main advantages and disadvantages.
3. Conclusions
The supply of oxygen to cell culture bioreactors
is an extremely important parameter which can
lead to rapid changes in cell physiology,
metabolism, productivity, product quality and cell
death if not adequately controlled. The supply of
oxygen can be achieved by surface aeration,
sparging or bubble-free membrane systems, each
of which has problems associated with either
foaming, shear stresses on the cells or scalability.
To determine OUR, mass balance techniques
are used. However, these involve precise measure-
ments of dissolved oxygen concentrations, gas
flows, gas compositions, kLa and Henry’s coeffi-
cient values.
Dynamic measurements are the more easy to
perform, although they introduce perturbations to
Table 2
Review of methods for the determination of OUR
Method Description of aeration system
Global mass bal- Surface aeration. yO2 deter-
ance mined by mass spectrometer
Bubble aeration, flushing of
the surface yO2 determined by
mass spectrometer
Bubble aeration, flushing of
surface, yO2 determined by
mass spectrometer
Surface aeration, small aera-
tion flow, yO2 determined by
mass spectrometer
Stationary liquid Surface aeration. yO2 deter-
phase mined by mass spectrometer
yO2 calculated from set point
of each gas flow rate (O2, N2,
CO2) set manually to control
DO
yO2 calculated from set point
of each gas flow rate (O2, N2,
CO2) set automatically to con-
trol DO
yO2 calculated from set point
of each gas flow rate (O2, N2,
CO2) set manually to control
DO
Dynamic method Automatic method based on
measurement of decrease in
DO after sparge pulse
Gas flow switched off, linear
decrease of DO monitored
Off-line measurement of DO
decrease in bottle completely
filled with medium and agi-
tated with magnetic agitator
Off-line measurement of DO
decrease in a bottle completely
filled with medium and agi-
tated with magnetic agitator
Aeration performed with sili-
cone tubing. Gas mixture
varied and OUR determined as
function of the variation of
DO and kLa
92
Type of reactor Advantages Disadvantages Reference
1.5 l STR No kLa needed Noise (200 mmol O2 h−1 l−1) Eyer et al., 1995; Oeg-
is equal to half of the ex- gerli et al., 1995
pected signal
1.5 l STR Short residence time for the Expensive gas analyser Eyer et al., 1995; Oeg-
gas. yin/yout high. Good accu- gerli et al., 1995
racy
1300 l STR Backer et al., 1988
3 l STR Accuracy for OUR determina- Long residence time for oxy- Dorresteijn et al., 1996
tion = 8% gen. Very sensitive to fluctua-
tions of DO
1.5 l STR Low noise (5 mmol O2 h−1 l−1) Expensive gas analyser. kLa Eyer et al., 1995; Oeg-
must be constant gerli et al., 1995
1l Very simple system Non-continuous signal, one Miller et al., 1987, 1988
operator required to log and
adjust flows. Approximate es-
timation of OUR
STR 600 ml Continuous signal, kLa con- Ramirez and Mutha-
stant, low cost determination rasan, 1990
of yO2
CSTR 0.5 l Ozturk and Palsson,
1990
200 l CSTR Automatic, no kLa needed for Non-continuous signal (mea- Singh, 1996
sparging. High sensitivity (0.05 surement every 10–15 min)
mmol l−1 h−1)
300 l air lift One measurement per 2 h Preissmann et al., 1997
P.-A. Ruffieux et al. / Journal of Biotechnology 63 (1998) 85–95
50 ml STR Fast measurement, small vol- Culture conditions different Frame and Hu, 1985
ume of cells required than in a bioreactor
5 ml STR Fast measurement, small vol- Culture conditions not con- Yamada et al., 1990
ume of cells required trolled
14 l STR One measurement per hour Fleischaker and Sinskey,
1981
 P.-A. Ruffieux et al. / Journal of Biotechnology 63 (1998) 85–95
the culture resulting in a non-constant pO2, a
situation which is not recommended. While meth-
ods based on a global mass balance are limited by
analyser sensitivity and cost. Oxygen electrode-
based methods are limited by stability, the re-
quirement for probe calibration during the culture
and position of the probes within the bioreactor.
Methods which require a knowledge of the kLa
are subject to variations since this may vary dur-
ing the culture. There is no simple solution to this
problem other than to use bubble-free systems or
surface aeration, neither of which are easy to
scale-up. On-line calibration is generally difficult
to perform and may introduce perturbations in
the physiology of the cells.
The optimal method for the determination of
OUR is the stationary liquid phase balance, which
is both very accurate and involves a constant DO.
The molar fraction of O2 can be determined by
many methods, since a precision of 1% is gener-
ally accurate enough.
If a sufficiently good determination of the OUR
is available then this can be used to both control
the OTR and to provide an on-line method for
the determination of biomass concentration and/
or the qO2, as well as for the control of the
physiological state of the cells.
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