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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
* 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)
literature. The traditional ones employ either a gas when the standard criterion of te = 1/ke1/kLa was
phase oxygen mass balance (steady state or gas taken into account. However, their main concern was
balancing method), or a liquid phase oxygen mass the mathematical simulations, without further experi-
balance (dynamic method). Heinzle and Dunn1 mental tests for model validation.
pointed out the potential dif®culties which arise from The present article presents an analogous approach
their utilization. For instance, the gas balancing for QO2X and kLa estimations, but by introducing a
method demands a large number of accurate measure- ®rst-order electrode response time in the liquid phase
ments (gas ¯ow rates, oxygen molar fractions in both oxygen balance, this method goes beyond the afore-
inlet and outlet gas streams, and dissolved oxygen mentioned work of Tribe et al,9 due to the experi-
concentration), in particular with reference to the mental features as well as to the appropriate choice of
initial and ®nal cultivation stages, when the inlet and the instant in which the integration should be initiated.
the outlet gas stream compositions are very similar. Experimental data obtained during Aspergillus awamori
The dynamic method (`gas-in±gas-out' method)2 cultivations were utilized for QO2X and kLa estimates,
requires a short gas-out period, in order to avoid the through two traditional methods (gas balancing and
condition of the dissolved oxygen concentration dynamic method) and the one proposed, here.
dropping below the critical value (Ccrit). This time
condition will ensure a constant molar microbial
oxygen uptake rate (QO2X) during the dynamic 2 THEORETICAL ASPECTS
method application. For processes in which the Assuming complete mixing, the mass balance for
dissolved oxygen falls below critical levels, Kim and dissolved oxygen in the liquid phase during an aerobic
Chang3 proposed that the inlet air be enriched with batch cultivation can be expressed as:2
oxygen in order to make the dynamic method possible.
Koizumi and Aiba4 presented a reassessment of the dC
kL a
C ÿ C ÿ QO2 X
1
dynamic method by means of a transfer function dt
focusing on the gas-in period during Bacillus stearo- As previously stated, the dynamic method is a
thermophilus cultivation. Although reliable, such a classical methodology for both QO2X and kLa deter-
method demands a very exhaustive calculation routine mination during a fermentation process in the pre-
for kLa determination. Yang et al 5 proposed a method sence of viable microbial mass. It has to be executed in
based upon the molar microbial oxygen uptake rate a very short period of time (some seconds or a few
(QO2X) which takes into account the electrode minutes), in order to not interfere with the process
response time. This method could be employed in kinetics. The dissolved oxygen concentration (C)
process stages where the dissolved oxygen concentra- usually shows a slow variation during a typical batch
tion (C) is slightly above the critical oxygen concen- fermentation, because it varies from saturation with
tration (Ccrit), again not permitting a long gas-out air, ie, about 0.20 mol mÿ3 at 30 °C, to about 10% of
period. Mignone6 presented a rapid method for kLa saturation, ie, about 0.02 mol mÿ3, during a period of
estimation under growth conditions based on `®rst several hours (100 h in some processes). Thus, if we
moment analysis', obtaining the pro®les of dissolved consider a very short period of time, we could assume
oxygen concentration by sudden changes in the stirrer that the dissolved oxygen concentration is effectively
speed, while the aeration rate was held constant. constant. In this situation, if we have C = Cs at the
The majority of physical methods for kLa estimation beginning of the dynamic method, then C will return
are based on changes of dissolved oxygen concentra- to the same previous value (Cs), after the application of
tion with time. Since the probe response is not the method, in spite of the forced variation that it will
instantaneous, such methods can become inadequate present during the dynamic method. Other process
if this delay is neglected. For commercial sterilizable variables, such as QO2, X and kLa, can also be assumed
electrodes, the response time can be approximated by as constants during the application of this method.
a ®rst order equation,3,7,8 hence de®ning a constant Hence, considering a short time period just before
response time (te) to characterize these electrodes.9 the dynamic method is started, the assumption is made
For reasonably accurate measurements, the criterion that a quasi steady state condition, ie, dC/dt = 0 exists.
te = 1/ke1/kLa is recommended.10 Commercial In this condition, C = Cs, and eqn (1) becomes:
sterilizable dissolved oxygen electrodes have response
time constants of 10±100 s, but since this value may QO2 X kL a
C ÿ Cs
2
change with membrane age and sterilization cycles,11
So, eqn (2) gives the molar microbial oxygen uptake
te should be determined after each cultivation.
rate (QO2X), at the beginning of the dynamic method.
Tribe et al 9 introduced the electrode response time
During the gas-out period (when the aeration is
in the liquid phase oxygen balance equations for both
suddenly stopped), the variation of the dissolved
gas-out and gas-in periods of the dynamic process.
oxygen concentration can be expressed by the follow-
They simulated pro®les of the electrode signal and of
ing equation:2
the real dissolved oxygen concentration, detecting
large errors on both QO2X and kLa estimations, when dC
ÿQO2 X
3
the electrode response time was neglected or even dt
When the condition C > Ccrit is obeyed, Ccrit being state period after aeration is re-started and C0 is the
the critical dissolved oxygen concentration just suf®- real dissolved oxygen concentration at t = t0. It should
cient to maintain the speci®c oxygen uptake rate (QO2) be noted that for a fast response time electrode
constant, the integration of eqn (3), with the initial (ke kLa), eqns (9) and (10) reduce to eqns (4) and
condition C = Cs at t = 0 (instant in which the aeration (7), respectively.
was interrupted), yields the following linear relation- The parameters QO2X, C0 and kLa are then
ship: estimated through Marquardt's procedure13 which
utilizes the least squares nonlinear regression by
C Cs ÿ QO2 Xt
4
utilizing eqns (9) and (10), respectively. The criterion
For the gas-in period (after the aeration is re- for the best ®tting and parameter optimization is the
started), substituting eqn (2) into eqn (1) gives: minimization of the sum of the squares of residuals
(SSR) given by the equation:
dC
kL a
C ÿ C ÿ kL a
C ÿ Cs kL a
Cs ÿ C X
n
dt
SSR !i
Cei ÿ C^ei 2
11
5
i
Equation (5) can be integrated for the initial
where CÃei is the ith experimental value of dependent
condition C = C0 at t = t0 (initial time for integration),
variable Ce, Cei is the ith calculated value of dependent
which yields:
variable Ce obtained from eqns (9) and (10), oi is the
Cs ÿ C weighting factor corresponding to ith variable Ce, and
ln ÿkL a
t ÿ t0
6
Cs ÿ C0 n is the number of experimental values.
The model's sensitivity, resulting from the model's
or, derivative with respect to the parameter to be
C Cs ÿ
Cs ÿ C0 eÿkL a
tÿt0
7 estimated, was considered as the weighting factor (o)
(Giudici R, private communication). Following Yang
Thus, the molar microbial oxygen uptake rate et al,5 to estimate QO2X by eqn (9), the set of
(QO2X) and the oxygen mass transfer coef®cient experimental values between interruption and re-start
(kLa) can be determined, for a speci®c instant of the of aeration was chosen. In this case, the weighting
process, as the slopes of the straight lines described by factor is given by the following equation:
eqns (4) and (6), respectively.
Nevertheless, estimation of these parameters would @Ce 1 eÿke t
ÿ tÿ
12
be strictly correct only if the electrode signals (Ce) of @
QO2 X ke ke
dissolved oxygen concentration were real. In reality
For eqn (10), the sensitivities of the model in
this does not occur due to the delay in electrode
relation to parameters kLa and C0, @Ce /@kLa and
response. As previously mentioned, the response time
@Ce /@C0 respectively, presented similar pro®les over
of modern sterilizable electrodes can be described as a
time. In this case, the model's sensitivity in relation to
®rst-order equation, as follows:12
parameter kLa (@Ce /@kLa) was chosen as the weighting
dCe factor (o), as expressed by eqn (13):
ke
C ÿ Ce
8
dt
@Ce ke
Ces ÿ C0 ÿke
tÿt0
where ke (=1/te) is the electrode's sensitivity. Equation ÿ
e ÿ eÿkL a
tÿt0
@
kL a
ke ÿ kL a2
(8) relates the probe signal (Ce) with the real dissolved
oxygen concentration (C). During the gas-out period, ke
Ces ÿ C0 ÿkL a
tÿt0
e
t ÿ t0
13
introducing eqn (4) into the response delay equation
ke ÿ kL a
(eqn (8), yields after integration:
1 eÿke t
Ce 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 Ce0 eÿke
tÿt0 Ces
1 ÿ eÿke
tÿt0
ke
Ces ÿ C0 ÿke
tÿt0
e ÿ eÿkL a
tÿt0
10
ke ÿ kL a
Figure 1. Profiles of electrode signal (Ce) and the model’s sensitivity
where Ces is the electrode signal in the quasi steady (@ Ce /@ kLa) during the dynamic method.
Figure 1 depicts both electrode signal (Ce) and the sterilizable galvanic electrode (New BruÈnswick Sci Co
model's sensivity pro®les with respect to kLa, obtained Inc) bearing an FEP (¯uorinated ethylene propylene)
through the dynamic method. The weighting factor membrane, 0.0254 mm thick. The electrode response
(o) is observed to be higher in the initial region of the time (te = 1/ke), de®ned as the time interval in which
Ce curve, where Ce varies strongly with time. Never- the electrode reaches 63.2% of its ®nal value when
theless, the values for the weighting factor are smaller submitted to a stepwise change in oxygen concentra-
in the terminal region of the curve, where Ce tends tion in a ¯uid similar to the fermentation broth, was
asymptotically towards Ces. In this region, experimen- previously determined. Xantham gum solution (0.4%
tal values of Ce have little in¯uence on model ®tting. w/v at 35 °C) was employed as the similar ¯uid,
Thus, C0 and kLa estimates are more accurate. yielding an electrode sensitivity (ke) of 0.215 sÿ1 in
An estimate of kLa by eqn (10) requires the choice of triplicate assays.
an appropriate initial condition, t = t0, from which a set Measurements of oxygen molar fraction in the outlet
of experimental values of Ce is de®ned for the gas (YOout) were performed through a polarographic
nonlinear regression. In the present work, t0 was electrode (Digimed Instr Anal Ltda, Brazil). As the
chosen, by visual inspection, as the instant at which an inlet gas was air, the oxygen molar fraction was
in¯exion point in the electrode signal (Ce) curve considered to be YOin = 0.21. The oxygen saturation
occurs, from which the signal (Ce) and the real concentration in the broth was determined according
dissolved oxygen concentration (C) pro®les present to Schumpe et al. 14 Employing the gas balancing
the same shape (Fig 2). This choice has the purpose of method, the oxygen mass transfer coef®cient (kLa) was
excluding the real transient period of the phenomenon assessed by eqn (2), and the molar microbial oxygen
from the set of experimental values, which occurs after uptake rate (QO2X) was determined through the
the aeration is re-started. following expression:
QO2 X
nin 0:21 ÿ nout YOout =V
14
3 MATERIALS AND METHODS Three batch experiments were carried out at
3.1 Microorganism and culture medium 700 rpm, each one at 0.2, 0.4 and 0.6 vvm, respec-
Aspergillus awamori strain NRRL 3112 was grown in a tively. Pro®les of the electrode signal (Ce), recorded
culture medium with the following composition, in over time, allowed the values of QO2X and kLa to be
kg mÿ3: cassava ¯our syrup, 20.0 (total reducing calculated through the dynamic method utilizing the
sugars); yeast extract, 0.20; MgSO4.7H2O, 1.0; gas-out period (eqns (2) and (9), and the value of kLa
(NH4)2SO4, 10.0; Na2HPO4.12H2O, 7.56; KH2PO4, to be determined through the modi®ed proposed
7.0. method (eqn (10)). The measurement routine was
initiated by turning off the aeration (`gas-out period'),
3.2 Experimental procedure keeping, however, the stirrer speed (N) at 200 rpm in
Experiments were conducted at 35 °C and pH 5.0 in a order to avoid surface aeration. After the electrode
10 dm3 Microferm MF-14 fermenter (New Brunswick signal dropped to approximately 30% of saturation,
Sci Co Inc, USA), provided with two ¯at blade turbine aeration was restarted, (`gas-in period'), keeping the
disk impellers, with four blades each. stirrer speed at 300, 500 or 700 rpm, respectively. The
Dissolved oxygen concentration was measured by a experimental apparatus is shown schematically in
Figure 3.
(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 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 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
REFERENCES
1 Heinzle E and Dunn IJ, Methods and instruments in fermenta-
tion gas analysis, In BiotechnologyÐA Comprehensive
Treatise, Vol 4, ed by Rehm H-J and Reed G VCH, Weinheim.
pp 27±74 (1986).
2 Taguchi H and Humphrey AE, Dynamic measurement of the
volumetric oxygen transfer coef®cient in fermentation systems.
J Ferment Technol 44:881±889 (1966).
3 Kim D-J and Chang H-N, Dynamic measurements of kLa with
Figure 4. QO2X and kLa estimations as proposed by Tribe et al 9 oxygen-enriched air during fermentation. J Chem Technol
(N = 500rpm, f = 0.4 vvm), ts = 51 s, QO2X = 1.83 mmol mÿ3 sÿ1 (eqn (15), Biotechnol 45:39±44 (1989).
kLa = 0.0448sÿ1 (eqn (16). (*) Experimental values, (—) adjusted curve 4 Koizumi J and Aiba S, Reassessment of the dynamic kLa method.
(electrode signal), (--) dissolved oxygen concentration (eqns (4) and (7). Biotechnol Bioeng 26:1131±1133 (1984).
5 Yang X-M, Mao Z-X and Yang S-Z, An improved method for
determination of the volumetric oxygen transfer coef®cient in
fermentation processes. Biotechnol Bioeng 31:1006±1009
Newtonian broths, a transient mixing state, character- (1988).
ized by a non-homogeneous bubble arrangement in 6 Mignone CF, The agitation-step method for kLa measurement.
the bulk ¯uid, is observed after aeration is re-started. Chem Eng Sci 45:1583±1587 (1990).
7 Linek V and Sinkule J, Comments on validity of dynamic
measuring methods of oxygen diffusion coef®cients in fermen-
tation media with polarographic electrodes. Biotechnol Bioeng
5 CONCLUSIONS 35:1034±1041 (1990).
The method proposed here, by taking into account the 8 Robinson CW and Wilke CR, Oxygen absorption in stirred
electrode response time, is suitable for the determina- tanks: a correlation for ionic strength effects. Biotechnol Bioeng
tion of both QO2X and kLa in non-Newtonian broths, 15:755±782 (1973).
9 Tribe LA, Briens CL and Margaritis A, Determination of the
typically found in Aspergillus awamori fermentations.
volumetric mass transfer coef®cient (kLa) using the dynamic
Accordingly, the method is easy to apply and very `gas out±gas in' method: analysis of errors caused by dissolved
reliable, particularly at the beginning of the fermenta- oxygen probes. Biotechnol Bioeng 46:388±392 (1995).
tion, when differences in oxygen composition between 10 Ruchti G, Dunn IJ and Bourne JR, Comparison of dynamic
inlet and outlet gas are small and the levels of dissolved oxygen electrode methods for the measurements of kLa.
Biotechnol Bioeng 23:277±290 (1991).
oxygen concentration are high; a situation for which
11 Van't Riet K and Tramper J, Basic Bioreactor Design, Marcel
the other methodologies are inadequate. The pro- Dekker, New York (1991).
posed method will be useful only when the dissolved 12 Aiba S and Huang SY, Oxygen permeability and diffusivity in
oxygen concentration is signi®cantly above the critical polymer membranes in immersed liquids. Chem Eng Sci
value (Ccrit); for lower values, the gas balancing 24:1149±1159 (1969).
13 Marquardt DW, An algorithm for least square estimation of non-
method would be more appropriate.
linear parameters. J Soc Ind Appl Math 11:431±441 (1963).
14 Schumpe A, Quicker G and Deckwer WD, Gas solubilities in
microbial culture media, in Adv Biochem Eng, Vol 24, Ed by
ACKNOWLEDGEMENTS Ghose TK, Fiechter A and Blakebrough N, Springer-Verlag,
This study was supported by `Secretaria da CieÃncia, Berlin. pp 1±38 (1982).