Communication to the Editor

Determination of the Volumetric Mass Transfer Coefficient (k,a) Using the Dynamic Gas Out-Gas In Method: Analysis of Errors Caused by Dissolved Oxygen Probes
L. A. Tribe, C. L. Briens, and A. Margaritis Department of Chemical and Biochemical Engineering, University of Western Ontario, London, Ontario, Canada N6A 5B9
Received June 28, 1994lAccepted December 13, 1994
There are many dynamic methods for measuring the volumetric mass transfer coefficient. The gas out-gas in method can directly determine the volumetric m a s s transfer coefficient in a bioreactor system and provide estimates of the volumetric microbial oxygen uptake rate and the average oxygen saturation concentration at the gas-liquid interface. The errors on these parameters are large if the dissolved oxygen probe response time is not considered. For reliable measurements, deconvolution of the oxygen probe measurements must be made. o
1995 John Wiley & Sons, Inc.

Key words: volumetric mass transfer coefficient oxygen uptake rate probe response time dynamic gas out-gas in method.

INTRODUCTION
In aerobic bioreactors, sufficient levels of oxygen must be supplied to the liquid broth to establish optimum conditions for microbial growth. Often, the oxygen transfer rate is the limiting factor of a bioreactor system and an important parameter for scale-up. The oxygen transfer rate is dependent upon the volumetric mass transfer coefficient, k,a, and the driving force, C; - C, (where C, is the dissolved oxygen concentration and C: is the oxygen saturation concentration in the liquid phase at the gas-liquid interface).*18 The oxygen transfer rate can be measured by chemical or physical techniques. The application of a chemical technique requires accurate reaction kinetics which can be difficult to determine.20 Moreover, the chemicals (e.g., sodium sulfite) cannot be used in bioreactor systems. A physical technique usually involves performing an upstep or downstep in the oxygen concentration of the bioreactor inlet gas and measuring the dissolved oxygen concentration. In simulated broths without viable microorganisms, the volumetric mass transfer coefficient can be determined by either absorption or desorption. Steady state is first established with either air or nitrogen sparging and either oxygen or nitrogen is then substituted for the air or nitrogen .4,5,7,10,16,18,20 As pointed out by Kim and Chang,

* To whom all correspondence should be

addressed

performing an upstep with oxygen-enriched air is preferable to a downstep with nitrogen since it eliminates the risk of going below the critical dissolved oxygen concentration in actual microbial broths. A variant of this technique is the dynamic pressure method, where the pressure inside the bioreactor is increased causing an instantaneous increase in the interfacial oxygen concentration. 2-14 Although these physical methods can be applied to bioreactor systems, they require either an independent measurement of the volumetric oxygen uptake rate by the microorganisms in the bioreactor4 or an accurate determination of the interfacial oxygen concentration, Ct2 The oxygen uptake rate measurement requires taking a sample and performing an experiment in a separate laboratory unit. The interfacial oxygen concentration, C;, cannot be easily determined since it depends on the evolving broth composition, the local pressure, and the oxygen concentration in the gas, which varies as the gas bubbles rise through the broth. The physical gas out-gas in method avoids these problems by determining directly the volumetric mass transfer coefficient in the actual microbial broth.3 The dissolved oxygen concentration is monitored during a short period of non-aeration (gas out) and subsequent re-aeration (gas in). The gas-out period must be short and the dissolved oxygen concentration, C,, kept above the critical oxygen concentration, Ccrit,to ensure that the volumetric oxygen uptake rate is approximately constant during the gas out-gas in experiment; the respiration rate coefficient is constant at dissolved oxygen concentrations above the critical value and the biomass concentration does not significantly change over short periods of time. The gas out-gas in method can also provide estimates of both the microbial oxygen uptake rate and the average oxygen interfacial concentration. For all the physical methods the determination of the oxygen mass transfer coefficient becomes inaccurate when the response time constant, T ,of the dissolved oxygen probe is large. The response time constant is defined as the time at which the probe reaches 63.2% of its final value when exposed to a step change in concentration. For reasonably accurate values, a criterion of T + (l/k,a) is usually recommended. 19-21 The commercially available sterilizable

Biotechnology and Bioengineering, Vol. 46, Pp. 388-392 (1995) 0 1995 John Wiley & Sons, Inc.

CCC 0006-35921951040388-05

dissolved oxygen probes which are used for measurements in bioreactor systems have large response time constants of lCL100 s.21Several models of the probe response have been developed,'1318 most modem sterilizable probes can be but approximated by a first-order hence the general use of the response time constant, 7 , to characterize dissolved oxygen probes. This article quantifies the errors caused by neglecting the probe response time when interpreting the results of gas out-gas in experiments. As shown below, previous attempts have made assumptions that do not correspond to realistic conditions.

Equation (3) can be rearranged to

3317

Therefore, k,a can be determined from the slope of C12 versus dC,/dt. Using the value of the microbial oxygen uptake rate obtained from the gas-out results, the oxygen interfacial concentration can be obtained from the intercept, which is (Cf - Qo2X/k,a). Alternately, since initially, under steady-state conditions,'

METHOD
Figure 1 shows an example of the results from a typical gas out-gas in experiment. For the gas-out period, the change in dissolved oxygen concentration equals the volumetric microbial oxygen uptake rate, given by the equation. Equation (3) can be integrated from the time, t,, at which the air flow is re-started to any subsequent time, t , to give'X2'

which yields, by integration,

The volumetric microbial oxygen uptake rate, Q,,X, can thus be obtained from the variation with time of the dissolved oxygen concentration, c,, during the gas-out period. For the gas-in period, the rate at which the dissolved oxygen concentration changes is given by the
(3)

.......

yt2 .......

Thus, k,a can be determined from the slope of ln(C,, C,) versus t. If required, the oxygen interfacial concentration can then be obtained from Equation ( 5 ) and the value of the volumetric microbial oxygen uptake rate obtained from the gas-out results. Data from Bambot et a1.2 for the response of a sterilizable Clark-type electrode from Ingold Electronics to a step in dissolved oxygen concentration verified the applicability of the first-order system to the response of such a probe. This type of probe is commonly used to measure dissolved oxygen concentrations in bioprocesses. Their data gave a probe response time of approximately 3 1 s. Ingold lists 98% response in 60 s , which, for a first-order system, corre~ sponds to a response time constant of 15 s based on 63.2% response. The discrepancy may be due to the aging of the membrane. A first-order system with a response time of 15 s was assumed to minimize the errors caused by the probe response. Other probes may have different response characteristics, but errors will be of the same order of magnitude as predicted with a first-order probe. The probe signal, at time t, was obtained by convolution of the concentration history from the start of the experiment until time t with the probe response characteristics. For t < t,,
y(r) = CLO- Qo,X [f
- T

(1 - e-"")]

(7)

and for t >

cs
0
20

ts,

40

60

80

100

120

I 140

Time (s)

Figure 1. Difference between the actual, C,, and the measured probe signal, y, for dissolved oxygen concentration changes in a bioreactor during a gas out-gas in experiment. Assumptions: C,,,, = 0.1 mg OJL, k,a = 0.07 s I , and Po, X = 0.25 mg O,/L/s. () Actual concentration. (---) Probe signal.
~

Determined values for the oxygen uptake rate and the volumetric mass transfer coefficient may vary depending on

COMMUNICATION TO THE EDITOR

389

the experimental data selected for the calculations. Therefore the following boundaries were set (see Fig. 1): The aeration should be re-started when the dissolved oxygen concentration reaches 1.5 times the critical concentration. That is, C, = 1.5Cc~t. volumetric miThe crobial oxygen uptake rate should remain approximately constant over a gas out-gas in experiment. The initial probe signal, yo, corresponds to the actual initial dissolved oxygen concentration in the bioreactor, Co (i.e., yo = Co). Experimental data selected for analysis for the gas-out and gas-in periods are within the range defined by ytl and yt2 (see Fig. 1):
yfl
=

Percent error on Qo,X and

=
\

Table I lists the range of values of the oxygen uptake rate, the critical dissolved oxygen concentration, and the volumetric mass transfer coefficient used in this article. This range of values was derived from Atkinson and Mavituna' and represents typical aerobic fermentation conditions.

yo - 5 Ayl

where Ayl

0.01~~

yt2 = ymini + 5 Ayz where Ay2 = 0.Olymini

RESULTS AND DISCUSSION

Only the error from neglecting the probe response is evaluated here. All other sources of error are not included. For example, this article does not consider the assumption of rapid bubble disengagement during the gas-out period, which is not verified for tall vessels. Other authors have proposed solutions to this problem.2o321 However, the determined value of the volumetric mass transfer coefficient will still be inaccurate unless the probe response time is properly considered. Figure 1 illustrates the large difference between the actual and measured concentration profiles in a bioreactor during a typical gas out-gas in experiment. The boundaries used for the evaluation of the oxygen uptake rate and the volumetric mass transfer coefficient from the probe response are indicated. Figure 1 shows that the probe indicated a dissolved oxygen concentration of over 2.6 mg O,/L when the actual concentration was 0.15 mg 02/L. Neglecting the probe response time may therefore lead to oxygen starvation. Figure 2 shows that the oxygen uptake rate may be greatly underestimated by neglecting the probe response time. The error increased when either the oxygen uptake rate or the volumetric mass transfer coefficient was increased. Such errors would lead to a defective scale-up. Neglecting the probe response time caused large errors on the volumetric mass transfer coefficient. A comparison of Figures 3 and 4 show that, for most cases, larger errors resulted using method 1. With either method, the error was almost independent of the volumetric oxygen uptake rate (Figs. 3 and 4). A reliable estimate of the error on the mass transfer coefficient can thus be made even when the oxygen

and yminiis the minimum probe signal value obtained from a gas out-gas in experiment. These boundaries were followed in evaluating the errors caused by neglecting the probe response time. In practice, the probe response time is often neglected and the volumetric microbial oxygen uptake rate and the volumetric mass transfer coefficient are obtained directly from the probe signal. Linear regression of the decreasing probe signal versus time is performed between ytl and yf2. The negative of the slope yields an estimate of the volumetric microbial oxygen uptake rate, (Q,, as indicated by Equation (9):

me,

(9)

There are two methods to obtain estimates of the volumetric mass transfer coefficient, &a),. Method 1 . For the increasing probe signal,

The estimated volumetric mass transfer coefficient is the negative of the inverse of the slope of y versus dyldt evaluated between the boundaries ytl and yf2. Method 2. For the increasing probe signal,

The estimated mass transfer coefficient is the negative of the slope of lnb, - y) versus t evaluated between the boundaries ytl and yr2. The percent error on the volumetric microbial oxygenuptake rate and the volumetric mass transfer coefficient were evaluated by

Table I. Range of values of volumetric microbial oxygen uptake rate, critical oxygen concentration, and volumetric oxygen mass transfer coefficient. Parameter Volumetric oxygen uptake rate Critical oxygen concentration Volumetric mass transfer coefficient Range 0.05-1.0 0-2 0.014.15 Unit rng O,/L/s rng O,/L
S-'

390

BIOTECHNOLOGY AND BIOENGINEERING, VOL. 46, NO. 4, MAY 20, 1995

0 -1 0

-20

a 4 ,

2
Y W
c Q

-20

m
C

b
-40
m ,
Y

3
W

-30
-40 -50
-60

:
W

c 0

-60

b 2
c

5
c
0

-80

b c
c

-100 -70 -120 00

-80
10

02 04 06 08 Oxygen Uptake Rate, Qo2X (rng0,lLls)

00

02

0.4

06

08

10

Oxygen Uptake Rate, Qo2X (rng0,lLls)

Figure 2. Error on the volumetric microbial oxygen uptake rate caused by neglecting the probe response time. Assumptions: C,,, = 0.1 mg 0,IL and the valuesof k,aare 0.03 s - ' (a), 0.05 s - ' (b), 0.07 s - ' (c), 0.09 s - ' (d), 0.11 s-' (e), 0.13 s C ' (9, and 0.15 s C 1 (8).

Figure 4. Error on the volumetric mass transfer coefficient (method 2) caused by neglecting the probe response time. Assumptions: C,,,, = 0.1 mg0,iLand thevaluesofkLaare0.03 s - ' (a), 0.05 s C ' (b), 0.07 s C 1(c), 0.09 s - ' (d), 0.11 s - l (e), 0.13 s C 1(f). and 0.15 (8).
s C '

uptake rate is not known. This relative error increased as the mass transfer coefficient increased. According to van't Riet,20 the error on the mass transfer coefficient caused by neglecting the probe response time is less than 6% when the mass transfer coefficient is smaller than the inverse of the probe response time constant (k,a C 1 / = 0.07 s-'). This criterion assumes that the probe has ~ equilibrated with the bioreactor broth before an upstep in
0

-10
-20

3
c
0 L

-30
-40

b c
c

-50
-60

-70
-80

the oxygen concentration of the bioreactor inlet gas is performed. Figure 4 shows that for a mass transfer coefficient less than 0.07 s-' the error in bioreactors could be as high as 37%. Linek et a1.'' also assume that the probe has equilibrated with the bioreactor broth when the gas is turned back on at time ts, as shown in Figure 1. This approximation can only be valid if the critical dissolved oxygen concentration and the microbial volumetric oxygen uptake rate are very small. Such conditions are rarely encountered in practice. Neglecting the probe response time and then underestimating the required volumetric mass transfer coefficient would lead to a defective scale-up. Figure 5 shows that the error on the volumetric mass transfer coefficient caused by neglecting the probe response time is practically independent of the critical oxygen concentration. In practice, however, the effect of probe calibration errors may become significant as the critical oxygen concentration becomes larger. If the interfacial oxygen concentration, C,*, is known, the volumetric mass transfer coefficient, k,a, could theoretically be obtained from Equation (5) and the value of the oxygen uptake rate estimated from the gas-out results. However, this would lead, in most cases, to larger errors than obtained by using the gas-in results.

0.0

0.2

0.4

0.6

0.8

10

CONCLUSIONS
Neglecting the probe response time causes large errors on the oxygen uptake rate and on the volumetric mass transfer coefficient even when the standard criterion that the probe response time constant be much smaller than the inverse of the volumetric mass transfer coefficient is verified. The

Oxygen Uptake Rate, Qo2X (rng0,ILls)

Figure 3. Error on the volumetric mass transfer coefficient (method 1) caused by neglecting the probe response time. Assumptions: C,,, = 0.1 mg0,/LandthevaluesofkLaare0.03sC' (a),0.05s-'(b),0.07sC'(c), 0.09 s C ' (d), 0.11 s-' (e), 0.13 s-' (0, and 0.15 s - ' (g).

COMMUNICATION TO THE EDITOR

391

Y11

YO

-I0 -20

1 i

y,,,,
a -. -.

\.\

A,, A,,

upper boundary for analysis (mg 0,iL) lower boundary for analysis (mg O,/L) minimum probe signal value (mg 0,iL) parameter used to calculate y r , (mg O,/L) parameter used to calculate yr2 (mg 02/L)

References
I . Atkinson, B., Mavituna, F. 1991. Biochemical engineering and biotechnology handbook, 2nd edition. MacMillan, London. 2. Bambot, S. B., Holavanahali, R., Lakowicz, J. R., Carter, G . M., Rao, G . 1994. Phase fluorometric sterilizable optical oxygen sensor. Biotechnol. Bioeng. 43: 1139-1 145. 3. Bandyopadhyay, B., Humphrey, A. E., Taguchi, H. 1967. Dynamic measurement of the volumetric oxygen transfer coefficient in fermentation systems. Biotechnol. Bioeng. 9: 533-544. 4. Bartholomew, W. H., Karow, E. O., Sfat, M. R., Wilhelm, R. H. 1950. Oxygen transfer and agitation in submerged fermentations. Mass transfer of oxygen in submerged fermentation of Streptomyces griseus. Ind. Eng. Chem. 42: 1801-1809. 5. Chang, H. N., Halard, B., Moo-Young, M. 1989. Measurement of KLa by a gassing-in method with oxygen-enriched air. Biotechnol. Bioeng. 34:1147-1157. 6. Cole-Parmer Instrument Company Catalogue. 1993-94. Niles, IL. 7. Dang, N. D. P., Karrer, D. A , , Dunn, I. J. 1977. Oxygen transfer coefficients by dynamic model moment analysis. Biotechnol. Bioeng. 19: 853-865. 8. Dunn, I. J., Einsele, A. 1975. Oxygen transfer coefficients by the dynamic method. J. Appl. Chem. Biotechnol. 25: 707-720. 9. Gauthier, L., Thibault, J., LeDuy, A. 1991. Measuring k,a with randomly pulsed dynamic method. Biotechnol. Bioeng. 37:889-893. 10. Kim, D.-J., Chang, H.-N. 1989. Dynamic measurement of KLa with oxygen-enriched air during fermentation. J. Chem. Tech. Biotechnol. 45: 39-14. 11. Linek, V., Sinkule, J . 1990. Comments on validity of dynamic measuring methods of oxygen diffusion coefficients in fermentation media with polarographic oxygen electrodes. Biotechnol. Bioeng. 35: 1034-1041. 12. Linek, V., Benes, P., Vacek, V. 1989. Dynamic pressure method for kLa measurement in large-scale bioreactors. Biotechnol. Bioeng. 33: 1 4 0 6 1 4 12. 13. Linek, V . , Moucha, T . , Dousova, M., Sinkule, J. 1994. Measurement of kLa by dynamic pressure method in pilot-plant fermentor. Biotechnol. Bioeng. 43: 4 7 7 4 8 2 . 14. Linek, V., Sinkule, J., Benes, P. 1991. Critical assessment of gassing-in methods for measuring k,a in fermentors. Biotechnol. Bioeng. 38: 323-330. 15. Linek, V., Sobotka, M., Prokop, A. 1973. Measurement of aeration capacity of fermentors by rapidly responding oxygen probes. Biotechnol. Bioeng. Symp. 4: 429453. 16. Nakanoh, M . , Yoshida, F. 1980. Gas absorption by Newtonian and non-Newtonian liquids in a bubble column. Ind. Eng. Chem. Process Des. Dev. 19: 19C-195. 17. Rane, K. D., Sims, K . A. 1994. Oxygen uptake and citric acid production by Candida lipolytica Y 1095. Biotechnol. Bioeng. 43: I3 1-1 37. 18. Robinson, C. W., Wilke, C. R. 1973. Oxygen absorption in stirred tanks: A correlation for ionic strength effects. Biotechnol. Bioeng. 15: 755-782. 19. Ruchti, G . , Dunn, I. J., Bourne, J. R. 1981. Comparison of dynamic oxygen electrode methods for the measurement of KLa. Biotechnol. Bioeng. 23: 277-290. 20. van't Riet, K . 1979. Review of measuring methods and results in nonviscous gas-liquid mass transfer in stirred vessels. Ind. Eng. Chem. Process Des. Dev. 18(3): 357-364. 21. van't Riet, K . , Tramper, J. 1991. Basic bioreactor design. Marcel Dekker, New York.

00

0.2

0.4

0.6

08

10

Oxygen Uptake Rate, QO2X(mgO,/L/s)

Figure 5. Comparison of the error on the volumetric mass transfer coefficient, caused by neglecting the probe response time for various values of kLa and C,,,,. The values of kLa are 0.03 s - (a), 0.05 s C ' (b), 0.07 s ( ~ ) , 0 . 0 9 s - ~ ( d ) , O s l I ( e ) , 0 . 1 3 s C ' ( f ) , a n d 0 . 1 5 s ~ ' ( g )Valuesof . -' . C,,,,: (-.-) 0 mg 0,iL; (--) 0.5 mg 0,iL; (--) 1 mg 0,iL; (. . . . .) 1.5 mg 0,iL; () 2 mg 0,iL.

'

calculation method which uses the derivative of the measured probe response to determine the mass transfer coefficient should be avoided. The alternate (method 2) yields less inaccurate estimates. For reliable measurements and scale-up, deconvolution of the oxygen probe measurements should be performed.
The authors thank the Natural Sciences and Engineering Research Council (NSERC) of Canada for their financial support.

NOMENCLATURE
dissolved oxygen concentration (mg O,/L) dissolved oxygen saturation concentration in liquid phase at gas-liquid interface (mg O,/L) estimated dissolved oxygen saturation concentration in liquid phase at gas-liquid interface (mg 02/L) critical dissolved oxygen concentration (mg 0,iL) initial dissolved oxygen concentration (mg 0,iL) dissolved oxygen concentration at t = t, (mg 0 /L volumetric oxygen mass transfer coefficient (s ?) ) estimated volumetric oxygen mass transfer coefficient (s I) respiration rate coefficient (mg 02/g dry biomass weighus) volumetric microbial oxygen uptake rate (mg O,/L/s) estimated volumetric microbial oxygen uptake rate (mg 0,i L/s) time (s) time corresponding to minimum probe signal value (s) time at which gas flow is re-stafied (s) characteristic probe response time constant (s) biomass concentration (g dry weight/L) initial probe signal (mg O,/L) probe signal at time t (mg O,/L)

392

BIOTECHNOLOGY AND BIOENGINEERING, VOL. 46, NO. 4, M A Y 20, 1995