Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 75:469±474 (2000)

Improving kLa determination in fungal

fermentation, taking into account electrode
response time
Alberto C Badino Jr, M Cândida R Facciotti and Willibaldo Schmidell*
Escola Politécnica da USP, Departamento de Engenharia Quı́mica, PO Box 61548, 05424-970 São Paulo SP, Brazil

Abstract: An alternative way for determining the oxygen mass transfer coef®cient, kLa, based upon the
traditional dynamic method, is proposed. The oxygen material balance equation in the liquid phase is
integrated after insertion of the oxygen probe response time (®rst order type), and kLa values are
determined by employing Marquardt's algorithm, considering as a weighting factor the model's
sensitivity with respect to kLa. Bench-scale fermentations of Aspergillus awamori, performed under
different agitation (300±700 rpm) and aeration conditions (0.2±0.6 vvm), were utilized for calculating
kLa values (0.0283±0.0874 sÿ1), employing three methods: two so-called traditional, the gas balancing
and the dynamic methods, and the one proposed here. The latter method is shown to be as reliable as
the aforementioned methods but is easier to apply when the oxygen level in the reactor is above the
critical value.
# 2000 Society of Chemical Industry

Keywords: oxygen mass transfer coef®cient; oxygen uptake rate; electrode response time; dynamic method

NOTATION QO2X Molar microbial oxygen uptake rate

C Dissolved oxygen concentration (mol mÿ3) (mol mÿ3 sÿ1)
C(ts) Dissolved oxygen concentration at instant rpm Revolutions per minute (minÿ1)
t = ts in eqn (16) (mol mÿ3) R2 Correlation coef®cient
C* Dissolved oxygen saturation concentration SSR Sum of squares of residuals
in liquid phase at gas±liquid interface t Time (s)
(mol mÿ3) t0 Initial time for integration of eqn (10) (s)
C0 Dissolved oxygen concentration at t = t0 ts Instant in which the aeration is re-started
(mol mÿ3) (eqn (16)) (s)
Ccrit Critical dissolved oxygen concentration vvm Air volume per liquid volume per minute
(mol mÿ3) (m3 mÿ3 minÿ1)
Ce Electrode signal (mol mÿ3) V Broth fermentation volume (m3)
Ce0 Electrode signal at t = t0 (mol mÿ3) YOin Oxygen molar fraction in the inlet gas
Ces Electrode signal on quasi steady state stream
(mol mÿ3) YOout Oxygen molar fraction in the outlet gas
CÃei ith experimental value of dependent stream
variable Ce (mol mÿ3)
Cei ith calculated value of dependent variable te Electrode response time (sÿ1)
Ce obtained from eqns (9) and (10) f Speci®c air ¯ow rate (vvm or m3 mÿ3
(mol mÿ3) minÿ1)
Cs Dissolved oxygen concentration for quasi o Weighting factor
steady state (mol mÿ3) oi Weighting factor corresponding to ith
ke Electrode's sensitivity (sÿ1) dependent variable Ce
kLa Oxygen mass transfer coef®cient (sÿ1)
n Number of experimental values
nin Inlet dry molar ¯ow rate (mol sÿ1)
nout Outlet dry molar ¯ow rate (mol sÿ1) 1 INTRODUCTION
N Stirrer speed (rpm) Various methods for determining the oxygen mass
QO 2 Speci®c respiration rate (mol kgÿ1 sÿ1) transfer coef®cient (kLa) have been proposed in the

* Correspondence to: Willibaldo Schmidell, Escola Politécnica da USP, Departmento de Engenharia Quı́mica, PO Box 61548, 05424-970 São
Paulo SP, Brazil
Contract/grant sponsor: Secretaria da Ciência, Tecnologia e Desenvolvimento Econômico do Estado de São Pauls – SCTDE; contract/grant
number: 00775/93
(Received 6 September 1999; accepted 16 January 2000)

# 2000 Society of Chemical Industry. J Chem Technol Biotechnol 0268±2575/2000/$17.50 469

AC Badino Jr, MCR Facciotti, W Schmidell
literature. The traditional ones employ either a gas
phase oxygen mass balance (steady state or gas
balancing method), or a liquid phase oxygen mass
balance (dynamic method). Heinzle and Dunn1
pointed out the potential dif®culties which arise from
their utilization. For instance, the gas balancing
method demands a large number of accurate measure-
ments (gas ¯ow rates, oxygen molar fractions in both
inlet and outlet gas streams, and dissolved oxygen
concentration), in particular with reference to the
initial and ®nal cultivation stages, when the inlet and
the outlet gas stream compositions are very similar.
The dynamic method (`gas-in±gas-out' method)2
requires a short gas-out period, in order to avoid the
condition of the dissolved oxygen concentration
dropping below the critical value (Ccrit). This time
condition will ensure a constant molar microbial
oxygen uptake rate (QO2X) during the dynamic
method application. For processes in which the
dissolved oxygen falls below critical levels, Kim and
Chang3 proposed that the inlet air be enriched with
oxygen in order to make the dynamic method possible.
Koizumi and Aiba4 presented a reassessment of the
dynamic method by means of a transfer function
focusing on the gas-in period during Bacillus stearo-
thermophilus cultivation. Although reliable, such a
method demands a very exhaustive calculation routine
for kLa determination. Yang et al 5 proposed a method
based upon the molar microbial oxygen uptake rate
(QO2X) which takes into account the electrode
response time. This method could be employed in
process stages where the dissolved oxygen concentra-
tion (C) is slightly above the critical oxygen concen-
tration (Ccrit), again not permitting a long gas-out
period. Mignone6 presented a rapid method for kLa
estimation under growth conditions based on `®rst
moment analysis', obtaining the pro®les of dissolved
oxygen concentration by sudden changes in the stirrer
speed, while the aeration rate was held constant.
The majority of physical methods for kLa estimation
are based on changes of dissolved oxygen concentra-
tion with time. Since the probe response is not
instantaneous, such methods can become inadequate
if this delay is neglected. For commercial sterilizable
electrodes, the response time can be approximated by
a ®rst order equation,3,7,8 hence de®ning a constant
response time (te) to characterize these electrodes.9
For reasonably accurate measurements, the criterion
te = 1/ke1/kLa is recommended.10 Commercial
sterilizable dissolved oxygen electrodes have response
time constants of 10±100 s, but since this value may
change with membrane age and sterilization cycles,11
te should be determined after each cultivation.
Tribe et al 9 introduced the electrode response time
in the liquid phase oxygen balance equations for both
gas-out and gas-in periods of the dynamic process.
They simulated pro®les of the electrode signal and of
the real dissolved oxygen concentration, detecting
large errors on both QO2X and kLa estimations, when
the electrode response time was neglected or even
470
when the standard criterion of te = 1/ke1/kLa was
taken into account. However, their main concern was
the mathematical simulations, without further experi-
mental tests for model validation.
The present article presents an analogous approach
for QO2X and kLa estimations, but by introducing a
®rst-order electrode response time in the liquid phase
oxygen balance, this method goes beyond the afore-
mentioned work of Tribe et al,9 due to the experi-
mental features as well as to the appropriate choice of
the instant in which the integration should be initiated.
Experimental data obtained during Aspergillus awamori
cultivations were utilized for QO2X and kLa estimates,
through two traditional methods (gas balancing and
dynamic method) and the one proposed, here.
2 THEORETICAL ASPECTS
Assuming complete mixing, the mass balance for
dissolved oxygen in the liquid phase during an aerobic
batch cultivation can be expressed as:2
dC
ˆ kL a…C  ÿ C† ÿ QO2 X …1†
dt
As previously stated, the dynamic method is a
classical methodology for both QO2X and kLa deter-
mination during a fermentation process in the pre-
sence of viable microbial mass. It has to be executed in
a very short period of time (some seconds or a few
minutes), in order to not interfere with the process
kinetics. The dissolved oxygen concentration (C)
usually shows a slow variation during a typical batch
fermentation, because it varies from saturation with
air, ie, about 0.20 mol mÿ3 at 30 °C, to about 10% of
saturation, ie, about 0.02 mol mÿ3, during a period of
several hours (100 h in some processes). Thus, if we
consider a very short period of time, we could assume
that the dissolved oxygen concentration is effectively
constant. In this situation, if we have C = Cs at the
beginning of the dynamic method, then C will return
to the same previous value (Cs), after the application of
the method, in spite of the forced variation that it will
present during the dynamic method. Other process
variables, such as QO2, X and kLa, can also be assumed
as constants during the application of this method.
Hence, considering a short time period just before
the dynamic method is started, the assumption is made
that a quasi steady state condition, ie, dC/dt = 0 exists.
In this condition, C = Cs, and eqn (1) becomes:
QO2 X ˆ kL a…C  ÿ Cs † …2†
So, eqn (2) gives the molar microbial oxygen uptake
rate (QO2X), at the beginning of the dynamic method.
During the gas-out period (when the aeration is
suddenly stopped), the variation of the dissolved
oxygen concentration can be expressed by the follow-
ing equation:2
dC
ˆ ÿQO2 X …3†
dt
J Chem Technol Biotechnol 75:469±474 (2000)

When the condition C > Ccrit is obeyed, Ccrit being
the critical dissolved oxygen concentration just suf®-
cient to maintain the speci®c oxygen uptake rate (QO2)
constant, the integration of eqn (3), with the initial
condition C = Cs at t = 0 (instant in which the aeration
was interrupted), yields the following linear relation-
ship:
C ˆ Cs ÿ QO2 Xt
For the gas-in period (after the aeration is re-
started), substituting eqn (2) into eqn (1) gives:
dC
ˆ kL a…C  ÿ C† ÿ kL a…C  ÿ Cs † ˆ kL a…Cs ÿ C†
dt
…5†
Equation (5) can be integrated for the initial
condition C = C0 at t = t0 (initial time for integration),
which yields:
 
Cs ÿ C
ln ˆ ÿkL a…t ÿ t0 † …6†
Cs ÿ C0
or,
C ˆ Cs ÿ …Cs ÿ C0 †eÿkL a…tÿt0 †
Thus, the molar microbial oxygen uptake rate
(QO2X) and the oxygen mass transfer coef®cient
(kLa) can be determined, for a speci®c instant of the
process, as the slopes of the straight lines described by
eqns (4) and (6), respectively.
Nevertheless, estimation of these parameters would
be strictly correct only if the electrode signals (Ce) of
dissolved oxygen concentration were real. In reality
this does not occur due to the delay in electrode
response. As previously mentioned, the response time
of modern sterilizable electrodes can be described as a
®rst-order equation, as follows:12
dCe
ˆ ke …C ÿ Ce †
dt
where ke (=1/te) is the electrode's sensitivity. Equation
(8) relates the probe signal (Ce) with the real dissolved
oxygen concentration (C). During the gas-out period,
introducing eqn (4) into the response delay equation
(eqn (8), yields after integration:
 
1 eÿke t
Ce ˆ Ces ÿ QO2 X t ÿ ‡ …9†
ke ke
where Ces is the dissolved oxygen concentration in the
quasi steady state, before the gas-out period. In this
case Ces = Cs, or by eqn (9), Ce = Ces (t = 0).
For the gas-in period, the integration of the equation
derived by substitution of eqn (7) into eqn (8), with
the initial condition Ce = Ce0 (t = t0), gives:
Ce ˆ Ce0 eÿke …tÿt0 † ‡ Ces …1 ÿ eÿke …tÿt0 † †
ke …Ces ÿ C0 † ÿke …tÿt0 †
‡ …e ÿ eÿkL a…tÿt0 † † …
ke ÿ kL a
where Ces is the electrode signal in the quasi steady
J Chem Technol Biotechnol 75:469±474 (2000)
Improving kLa determination in fungal fermentation
state period after aeration is re-started and C0 is the
real dissolved oxygen concentration at t = t0. It should
be noted that for a fast response time electrode
(ke  kLa), eqns (9) and (10) reduce to eqns (4) and
(7), respectively.
The parameters QO2X, C0 and kLa are then
estimated through Marquardt's procedure13 which
utilizes the least squares nonlinear regression by
…4†
utilizing eqns (9) and (10), respectively. The criterion
for the best ®tting and parameter optimization is the
minimization of the sum of the squares of residuals
(SSR) given by the equation:
X
n
SSR ˆ !i …Cei ÿ C^ei †2 …11†
i
where CÃei is the ith experimental value of dependent
variable Ce, Cei is the ith calculated value of dependent
variable Ce obtained from eqns (9) and (10), oi is the
weighting factor corresponding to ith variable Ce, and
n is the number of experimental values.
The model's sensitivity, resulting from the model's
derivative with respect to the parameter to be
…7† estimated, was considered as the weighting factor (o)
(Giudici R, private communication). Following Yang
et al,5 to estimate QO2X by eqn (9), the set of
experimental values between interruption and re-start
of aeration was chosen. In this case, the weighting
factor is given by the following equation:
 
@Ce 1 eÿke t
ˆÿ tÿ ‡ …12†
@…QO2 X† ke ke
For eqn (10), the sensitivities of the model in
relation to parameters kLa and C0, @Ce /@kLa and
@Ce /@C0 respectively, presented similar pro®les over
time. In this case, the model's sensitivity in relation to
parameter kLa (@Ce /@kLa) was chosen as the weighting
factor (o), as expressed by eqn (13):
…8†
@Ce ke …Ces ÿ C0 † ÿke …tÿt0 †
ˆÿ …e ÿ eÿkL a…tÿt0 † †
@…kL a† …ke ÿ kL a†2
ke …Ces ÿ C0 † ÿkL a…tÿt0 †
‡ e …t ÿ t0 † …13†
…ke ÿ kL a†
10†
Figure 1. Profiles of electrode signal (Ce) and the model’s sensitivity
(@ Ce /@ kLa) during the dynamic method.
471
AC Badino Jr, MCR Facciotti, W Schmidell
Figure 1 depicts both electrode signal (Ce) and the
model's sensivity pro®les with respect to kLa, obtained
through the dynamic method. The weighting factor
(o) is observed to be higher in the initial region of the
Ce curve, where Ce varies strongly with time. Never-
theless, the values for the weighting factor are smaller
in the terminal region of the curve, where Ce tends
asymptotically towards Ces. In this region, experimen-
tal values of Ce have little in¯uence on model ®tting.
Thus, C0 and kLa estimates are more accurate.
An estimate of kLa by eqn (10) requires the choice of
an appropriate initial condition, t = t0, from which a set
of experimental values of Ce is de®ned for the
nonlinear regression. In the present work, t0 was
chosen, by visual inspection, as the instant at which an
in¯exion point in the electrode signal (Ce) curve
occurs, from which the signal (Ce) and the real
dissolved oxygen concentration (C) pro®les present
the same shape (Fig 2). This choice has the purpose of
excluding the real transient period of the phenomenon
from the set of experimental values, which occurs after
the aeration is re-started.
3 MATERIALS AND METHODS
3.1 Microorganism and culture medium
Aspergillus awamori strain NRRL 3112 was grown in a
culture medium with the following composition, in
kg mÿ3: cassava ¯our syrup, 20.0 (total reducing
sugars); yeast extract, 0.20; MgSO4.7H2O, 1.0;
(NH4)2SO4, 10.0; Na2HPO4.12H2O, 7.56; KH2PO4,
7.0.
3.2 Experimental procedure
Experiments were conducted at 35 °C and pH 5.0 in a
10 dm3 Microferm MF-14 fermenter (New Brunswick
Sci Co Inc, USA), provided with two ¯at blade turbine
disk impellers, with four blades each.
Dissolved oxygen concentration was measured by a
Figure 2. QO2X and kLa estimates by the proposed method (N = 700 rpm,
f = 0.6 vvm)–QO2X = 3.1 mmol mÿ3 sÿ1 (eqn (9), kLa = 0.079sÿ1 (eqn (10)),
C0 = 0.091mol mÿ3 (eqn (10)). (*) Experimental values, (—) adjusted
curve (electrode signal), (---) real dissolved oxygen concentration (eqns (4)
and (7)).
472
sterilizable galvanic electrode (New BruÈnswick Sci Co
Inc) bearing an FEP (¯uorinated ethylene propylene)
membrane, 0.0254 mm thick. The electrode response
time (te = 1/ke), de®ned as the time interval in which
the electrode reaches 63.2% of its ®nal value when
submitted to a stepwise change in oxygen concentra-
tion in a ¯uid similar to the fermentation broth, was
previously determined. Xantham gum solution (0.4%
w/v at 35 °C) was employed as the similar ¯uid,
yielding an electrode sensitivity (ke) of 0.215 sÿ1 in
triplicate assays.
Measurements of oxygen molar fraction in the outlet
gas (YOout) were performed through a polarographic
electrode (Digimed Instr Anal Ltda, Brazil). As the
inlet gas was air, the oxygen molar fraction was
considered to be YOin = 0.21. The oxygen saturation
concentration in the broth was determined according
to Schumpe et al. 14 Employing the gas balancing
method, the oxygen mass transfer coef®cient (kLa) was
assessed by eqn (2), and the molar microbial oxygen
uptake rate (QO2X) was determined through the
following expression:
QO2 X ˆ …nin 0:21 ÿ nout YOout †=V …14†
Three batch experiments were carried out at
700 rpm, each one at 0.2, 0.4 and 0.6 vvm, respec-
tively. Pro®les of the electrode signal (Ce), recorded
over time, allowed the values of QO2X and kLa to be
calculated through the dynamic method utilizing the
gas-out period (eqns (2) and (9), and the value of kLa
to be determined through the modi®ed proposed
method (eqn (10)). The measurement routine was
initiated by turning off the aeration (`gas-out period'),
keeping, however, the stirrer speed (N) at 200 rpm in
order to avoid surface aeration. After the electrode
signal dropped to approximately 30% of saturation,
aeration was restarted, (`gas-in period'), keeping the
stirrer speed at 300, 500 or 700 rpm, respectively. The
experimental apparatus is shown schematically in
Figure 3.
4 RESULTS AND DISCUSSION
Figure 2 shows a typical electrode signal (Ce) pro®le
obtained when the stirrer speed was set at 700 rpm and
the speci®c air ¯ow rate at 0.6 vvm. The models
expressed by eqns (9) and (10) are observed to ®t
accurately to the experimental values. Other experi-
mental curves, under different operational conditions,
presented similar behavior, allowing accurate QO2X
and kLa estimates.
It should be emphasized that the successful ®tting
obtained, particularly regarding eqn (10), is due to the
adequate choice of the initial instant for the integration
(in¯exion point in the electrode signal (Ce)), as well as
to the strategy adopted for the weighting factor,
considering the model's sensitivity.
The estimated value of QO2X by regression of eqn
(9) was slightly lower than the value obtained through
the slope of the straight line in the experimental curve
J Chem Technol Biotechnol 75:469±474 (2000)
 Improving kLa determination in fungal fermentation

Figure 3. Experimental apparatus (dimensions

in ‘m’).

(Fig 2). This result was considered to be a conse- instant in which the aeration was re-started (ts) (Fig 4).
quence of a very high electrode sensitivity and in this According to these considerations, the following
case, after a short period, the curves of electrode signal equations were obtained:
(Ce) and dissolved oxygen concentration (C) became h  i
parallel, a fact already mentioned by Yang et al. 5 Ce ˆ Cs ÿ QO2 X t ÿ te 1 ÿ eÿt=te …for t < ts †
Values of kLa determined at different agitation (N)
and aeration (f) conditions are shown in Table 1. …15†
Regression coef®cient (R2), as well as the relative  
deviations in each case, are also shown. The high Ce ˆ Cs ‡ QO2 Xte eÿ…tÿts †=te ÿ eÿt=te
correlation (R2) obtained through the proposed
method (A) con®rm the good ®tting of eqn (10) to ÿ QO2 Xts eÿ…tÿts †=te
the experimental data. A maximal relative deviation of
Cs ÿ C…ts †  ÿ…tÿts †=te 
21.8% was observed between the distinct methodol- ‡ e ÿ eÿkL a…tÿts † …for t > ts †
ogies employed. Furthermore, kLa estimated values 1 ÿ kL ate
seem quite coherent with reference to the different …16†
agitation and aeration conditions examined. From
these results, the conclusion was made that the
proposed method is consistent and reliable. Considering the experimental results from the
As previously stated, Tribe et al. 9 also introduced present work, Fig 4 shows that there is a good ®tting
the electrode response time in the liquid phase oxygen of eqn (15) for the gas-out period, utilizing data
balance equations for both gas-out and gas-in periods obtained at 500 rpm and 0.4 vvm. However, for the
of the dynamic method. For the gas-out period, when gas-in period, eqn (16) does not ®t properly to the
t = 0, the authors established that Ce = Cs, as con- experimental Ce values. This fact can be explained,
sidered in eqn (9). However, for the gas-in period, they remembering that during ®lamentous microorganism
adopted as an initial condition for the integration, the cultivations, which frequently generate non-

Table 1. Comparison between kLa values obtained during Aspergillus awamori cultivation in a 10 dm3 bioreactor

kLa (sÿ1) Relative deviation

N f
2
(rpm) (vvm) Proposed method (A) R Gas balancing method (B) Dynamic method (C) (A-B)/A  100 (%) (A-C)/A  100 (%)
700 0.2 0.0670 0.998 0.0742 0.0816 ÿ10.7 ÿ21.8
500 0.2 0.0479 0.997 0.0530 0.0551 ÿ10.6 ÿ15.0
300 0.2 0.0314 0.996 0.0380 0.0304 ÿ20.8 3.4
700 0.4 0.0801 0.994 0.0669 0.0810 16.4 ÿ1.1
500 0.4 0.0601 0.997 0.0620 0.0598 ÿ3.2 0.6
300 0.4 0.0363 0.998 0.0296 0.0284 18.4 21.6
700 0.6 0.0790 0.998 0.0874 0.0801 ÿ10.7 1.3
500 0.6 0.0731 0.997 0.0623 0.0734 14.8 ÿ0.4
300 0.6 0.0294 0.995 0.0309 0.0283 ÿ5.2 3.6

J Chem Technol Biotechnol 75:469±474 (2000) 473

AC Badino Jr, MCR Facciotti, W Schmidell
Figure 4. QO2X and kLa estimations as proposed by Tribe et al 9
(N = 500rpm, f = 0.4 vvm), ts = 51 s, QO2X = 1.83 mmol mÿ3 sÿ1 (eqn (15),
kLa = 0.0448sÿ1 (eqn (16). (*) Experimental values, (—) adjusted curve
(electrode signal), (--) dissolved oxygen concentration (eqns (4) and (7).
Newtonian broths, a transient mixing state, character-
ized by a non-homogeneous bubble arrangement in
the bulk ¯uid, is observed after aeration is re-started.
5 CONCLUSIONS
The method proposed here, by taking into account the
electrode response time, is suitable for the determina-
tion of both QO2X and kLa in non-Newtonian broths,
typically found in Aspergillus awamori fermentations.
Accordingly, the method is easy to apply and very
reliable, particularly at the beginning of the fermenta-
tion, when differences in oxygen composition between
inlet and outlet gas are small and the levels of dissolved
oxygen concentration are high; a situation for which
the other methodologies are inadequate. The pro-
posed method will be useful only when the dissolved
oxygen concentration is signi®cantly above the critical
value (Ccrit); for lower values, the gas balancing
method would be more appropriate.
ACKNOWLEDGEMENTS
This study was supported by `Secretaria da CieÃncia,
474
Tecnologia e Desenvolvimento EconoÃmico do Estado
de SaÄo Paulo ± SCTDE' (Project 00775/93).
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