Agitation-Aeration in Submerged Fermentation

I. A Comparative Study of the Sulfite and Polarographic Methods for Measuring

Oxygen Solution Rates in a Fermentor
R. STEEL1 AND M. R. BRIERLEY2
Indu4strial Biochemistry Section, College of Science and Technology, M1anchester, England
Received for puiblication July 21, 1958

Although the submerged aerobic cultivation of 6-in. diameter glass tank fitted with 4 diametrically
microorganisms has become an established feature spaced baffles. An open-pipe sparger was located
of the present-day fermentation industry, equipment centrally below an 8-bladed turbine impeller. The
design and operation have been based largely uponi ratio of the tank diameter, impeller diameter, blade
empirical studies. Attention has been directed to the length, and blade width was 15:5:1.25: 1. The impeller
problem of supplying sufficient oxygen to microbial was situated one impeller diameter from the base of
cultures and to methods for defining aeration efficiency the tank; the operating liquid volume was 2.5 L.
in fermentors (reviewed by Finn, 1954). The sulfite The fermentor was kept in a temperature controlled
oxidation method for determining oxygen solution water bath (25 ± 1 C). Air flow rates were measured
rates in a fermentor has been used to test the efficiency with capillary orifices calibrated at 25 C and atmos-
of equipment design (Chain and Gualandi, 1954; pheric pressure. Agitator speed was determined from
Roxburgh et al., 1954; Bowers, 1955) as a parameter the current produced by a tachometer-generator driven
for increase in scale (Cooper et al., 1944; Karow et al., by the impeller shaft, the ammeter scale-deflection
1953) or as a measure of the actual amount of oxygen having been calibrated against impeller speed with a
transferred to a microbial culture (Maxon anid Johnson, stroboscopic flash unit.
1953; Pirt, 1957). Sulfite-oxidation experiments. The method of Cooper
In certain cases, measurements of oxygen solution et al. (1944) was used. The recommendations of Finn
rate determined by the sulfite method have agreed (1954) have been followed with regard to the use of
with those obtained by the polarographic technique units; the absorption coefficients (KLa values) are
(Bartholomew et al., 1950; Chain and Gualandi, -1954) expressed as mmoles of oxygen per L per hr (unit
whereas in others (Wise, 1950), a large disparity has concentration difference), or hr-1.
been noted between the results obtained by the two Polarographic equipment and techniques. A manual
methods. Because of the lack of agreement between polarograph3 was used in all experiments. Preliminary
the results of different investigators, the present work studies dealt with the design and operating char-
was undertaken to compare the sulfite and polarographic acteristics of "axial" and "radial" rotating platinum
techniques. Oxygen absorption-coefficients were meas- 3 Electrochemical Laboratories, Mlanchester, England.
ured over a fairly wide range of agitator speeds and
air flow rates and correlations between absorption CONTACT WIRE
coefficients and operating variables were determined. -RUBBER SLNG
The results indicate the limitations of the sulfite oxi-
PVC BE-ARING
dation technique as applied to fermentations.
LEADPGM
TO BRASS CYLINDER 3OD
-GLASS CUP
MATERIALS AND METHODS 1 -RUBBER BUNG
Theoretical. The theory of oxygen absorption in a K- t MERCURY -WIRE LEAD
fermentor liquid has been adequately discussed by PVC BEARINGSA'KC
Bartholomew et al. (1950), Wise (1951), Finni (1954), --GLASS TUBE
-RUBBER STOP MERCURY
and Elsworth et al. (1957).
Fermentor and accessories. Fermentor design was of
GLASS CAPILLARY
LIOD d'
A R S
UBRBN
SNG
-z- -IIIGLASS CASING
the conventional type (Finn, 1954) consistinig of a
PLATINUM
BEAD 14
'-SILVER FOIL
1 Present address: The Upjohn Company, Kalamazoo, U SINTERED DISC
Michigan. RADIAL ROTATING
PLATINUM CATHODE
AXIAL
CATHODE
SILVER-SILVER CHLORIDE
ANODE
2 Present address: Department of Biological Sciences, Wye
College, near Ashford, Kent, England. Figllre 1. Construction of electrodes
51
52 R. STEEL A'ND R. BRIERLEY
R. [VOL. 7I
cathodes used in conjunction with saturated calomel azide. From these data the value of Qo2 was calculated.
and silver/silver chloride reference electrodes. The The Warburg experiments were conducted by the
design and construction of electrodes are shown in method of Umbreit et al. (1949) with undiluted yeast
figure 1. Satisfactory operation of the electrode system suspension (3 ml) removed from the fermentor. Other
was found to be related to the tip velocity of the cathode experiments with yeast showed that the oxygen supply
(see later); rotation at 1500 rpm, using a synchronous rate to the Warburg vessels was not limited by in-
speed motor4 (150 hp), was adopted in the final experi- adequate shaking.
ments. The silver/silver chloride anodes were made Scope of the work. With the sulfite system, oxygen
from discs of silver foil (assay grade) 0.25 in diameter solution rates were measured over a range of air flow
which were silver soldered to silver wire. This was then rates from 0.5 to 4.5 L per min and agitator speeds
chloridized in 0.1 N hydrochloric acid by passing a from 385 to 1150 rpm. Because of differences in the
current of 2 ma for 30 min. The electrode was removed results obtained by the two methods, the polarographic
from the bath, and rubbed with fine emery paper and work was extended to cover a slightly wider range of
then chloridized for a further 30 min. During this time these variables.
the silver became a purplish-brown color. The assembly RESULTS
of this electrode is shown in figure 1. Under actual
conditions of operation, the electrodes entered the Sulfite experiments. Oxygen absorption-coefficients
fermentor liquid from the upper surface and were obtained for all conditions of agitation and aeration
inserted to a depth of about 2 in. The peak of the oxygen TABLE 1
wave was found to be at -0.75 and at -0.90 v using Absorption coefficients obtained by the sulfite method
standard calomel and silver/silver chloride reference for various conditions of agitation and aeration
electrodes, respectively, as reported previously by KLa, hr-'
Chain and Gualandi (1954).
Oxygen absorption coefficients were determined by Air Flow, Agitator speed, rpm
the gassing-out method of Bartholomew et al. (1950) 385 600 800 925 1150
and Wise (1951), the polarograph having been cali- 0.5 34 75 192 288 360
brated with salt solutions containing a known oxygen 1.0 32 85 252 432 490
concentration (Hixson and Gaden, 1950). Briefly, 1.5 33 105 300 608 735
the salt solution in the fermentor (0.5 M KCl) was 2.5 39 131 376 638 950
gassed-out with oxygen-free nitrogen and the residual 3.5 36 128 385 708 1032
4.5 44 140 386 726 1030
current reading noted; then the agitation and aeration
were started at constant levels and galvanometer
readings were taken every 5 sec until saturation was AGITATOR SPEED RPM
000
reached. The results of duplicate determinations were
almost identical when the experiments were conducted 600
within a short period of time (see remarks on vari-
ability given later). 600
To check the operation of the electrode system,
the rate of oxygen uptake by a yeast suspension (wet .1

op
brewers' yeast, 300 g; Na2HPO4, 4.75 g; KH2PO4, 4001 S

18.22 g; glucose, 25 g; KCl, 37 g; final volume 2.5 L; T-

pH 6.2; olive oil added as required to combat foaming),
in the fermentor was compared with that in a Warburg Y.
-J

apparatus. Using the fermentor, the values of KLa, 200-

C and CL were determined for the relationship.
KLa =
Q..2
(Q0- (1) 6OE
0

where KLa is the oxygen absorption coefficient, Qo, 1001_

0 a . . X. 0
is the oxygen uptake rate in mmoles per L per hr, C
is the saturation concentration of dissolved oxygen3
OA 1.0 2.0 3.0 40 0
and CL is the actual concentration of dissolved oxygen AIR FLOW RATE £G] L/MIN
in the fermentor liquid. Values of K,a were determined
for the fermentor after poisoniing the yeast with sodium Figure 2. Effect of air flow rate on oxygen absorption by
sodium sulfite solution. Agitator speed as parameter, logarith-
4Metropolitan Vickers Electrical Company, Manchester, mic plot; the value of X in the relationship KLa K Gx is =

England. given where G is air flow rate L per min.

1959] AGITATION-AERATION IN SUBMIERGED FERAIENTATION. I la"3

are given in table 1. Over the range of variables ex- efficients increased with increase in air flow rate up
amined, the absorption coefficients varied from 32 to to the point at which the impeller became "loaded,"
1032. At all air flow rates tested the absorption co- that is, the rotational speed of the impeller was in-
efficients increased with increase in agitator speed sufficient to completely disperse the air supplied. At
whereas at constant agitator speed the absorption co- the loading point a change was observed visually in
the character of the air dispersion. In addition to small
AIR RATE L/MIN 4.5
bubbles circulating with the bulk flow-pattern, a
complement of large bubbles was produced and these
4000 escaped around the impeller and passed through the
liquid at a relatively high velocity. This effect was
more pronounced as the air flow rate was further
600 -
increased beyond the loading point. At the lowest
agitator speed (385 rpm) tested, the magnitude of
Y=3.2 increase of absorption rate over the range of air flow
400- 05 tested was slight, thus indicating that loading occurred
under all conditions of operation. This was also sub-
stantiated by visual observations of bubble dispersion.
When absorption coefficients were plotted against
5
air flow rate (G), on logarithmic scales, a linear re-
lationship was noted below the loading point for each
So -
impeller speed (figure 2). However, the value of the
100
exponent on G was greater with higher agitator speeds.
The correlation between absorption coefficient and
agitator speed (N) chaniged slightly as the air flow
800
rate was varied (figure 3) but averaged about 3.0.

60-
Polarographic Experiments
Performance of the electrode systems. The performance
40-
of the electrode systems was found to be affected by
the turbulence of the liquid in the fermentor (table 2).
When the tip velocity of the cathode was increased
400 600 K=
AGITATOR SPEED [N] RPM from 1.5 to 9.1 feet per second, the magnitude of the
Figure 3. Effect of agitator speed on oxygen absorption by
stirring effect brought about by turbulence of the fer-
sodium sulfite solution. Air flow as parameter, logarithmic mentor liquid was decreased considerably. Further,
plot; the value of Y in the relatonship KLa = K N" is given the silver/silver chloride reference electrode appeared to
where N is agitator speed, rpm. be more stable than the calomel electrode. Con-
sequently, all subsequent experiments were made with
TABLE 2 the "radial" platinum cathode (tip velocity 9.1 feet
Effect of impeller speed (liquid turbulence) on the per second) and silver/silver chloride reference elec-
galvanometer deflection using various electrode trode.
arrangements
Variability of absorption coefficients. It was observed
Galvanometer that the maximum galvanometer deflection, cor-
deflection, mm
Electrode System Impeller
responding to an oxygen-saturated solution, tended to
speed, rpm in deflec- decrease over a period of hr. This did not seem to affect
500 1500 tion, % the calculated values of the absorption coefficients
Axial platinum cathode rotated at 1100
determined under similar conditions of agitation and
rpm (1.5 ft per sec), saturated cal- aeration, the largest difference being about 2 per cent.
omel anode; applied potential,
-0.75 v .... ... ..... ...... 87 111 25.7 TABLE 3
Radial platinum cathode rotated at Oxygen uptake by a yeast suispension in the fermentor
1500 rpm (9.1 ft per sec), saturated Air Flow
Agitator C CL K La, hr-' Qo 2
calomel anode; applied potential, Speed Rate
-0.75 v .......... 92 96 4.3
Radial platinum cathode rotated at rpm L/min mtmttoles/L/hr
1500 rpm (9.1 ft per sec), Ag/AgCl 605 1.3 0.155 64 9.9
anode; applied potential, -0.90 v 87 89 2.3 635 0.7 0.143 86 12.3
54 R. STEEL AND M. R. BRIERLEY[ [VOL. 7
Further, the silver/silver chloride electrode decom-
posed with extended use which necessitated a replace-
ment every 3 to 4 weeks. For this reason, absorption
coefficients were determined under standard operating
conditions (air flow rate, 2.5 L per min; agitator speed,
800 rpm) at the start of daily operations. The absorp-
tion coefficients determined for 30 runs yielded a mean
value of 305 with a coefficient of variation of 5.6 per
cent. It should be noted that this variability arose from
three main sources, (a) the use of different "radial"
< 200 platinum cathodes, (b) the use of different silver/silver
chloride anodes, and (c) variations in the performance
of these electrodes with age.
Check on electrode operation. Determinations of oxygen
100 uptake by a yeast suspension in the fermentor yielded
80 the data as given in table 3. The average value of the
two determinations of oxygen uptake was 11.1 mmoles
60 per L per hr which compared with a value of 10.8
obtained by the Warburg method.
Correlation of absorption coefficients with operating
40
variables. The data obtained in this part of the work
covered air flow rates from 0.5 to 5.5 L per min and
AIR FLOW RATE (GI L/MIN agitator speeds from 500 to 1500 rpm. The results are
given in the form of appropriate plots. Figure 4 shows
Figure 4. Effect of air flow rate on oxygen absorption by the correlation of absorption coefficient with gas flow
potassium chloride solution. Agitator speed as parameter, rate; betw een 600 and 1500 rpm the data follow the
logarithmic plot; the value of X in the relationship KLa =
K Gx is given where G is air flow rate, L per min. relationship, KLa = K G053. A logarithmic plot of
absorption coefficient against agitator speed with air
flow! rate as parameter (figure 5) followed the relation-
8000
AIR RATE,L,M4IIN
ship KLa = K N' ° for agitator speeds above about 800
rpm. Below 800 rpm the exponent on N varied with
air flow rate from 4.7 at 0.5 L per min to 8.1 at 5.5
YI-lO" L per miii. The marked change in the correlation at
600
agitator speed 800 rpm shown in figure 5 probably
reflects the change from laminar to turbulent flow
400- in the fermentor liquid.
DISCUSSION
For the sulfite system, the correlations between
Il
oxygen absorption coefficients and operating variables
200
.
(KLa = K G0 40-0.70 and KLa = K N3 0) agree reason-
ably wA-ell wvith those reported by previous investigators.
Further, the correlation between absorption coefficient
and air flow rate in the polarographic system is in
100 general agreement writh that found by the sulfite
80
method. However, the correlation, KLa = K N' 0,
obtained polarographically, does not seem to have
60
been.reported previously. The different values obtained
for the exponent on N in the two systems probably
reflects differences in the oxygen transfer mechanism.
400 600 800 1000 2000
It is noteworthy that the sulfite technique may
AGITATOR SPEED tN) RPM give a false impression of equipment performance,
a fact which has been noted previously by Wise (1950),
Figure 5. Effect of agitator speed on oxygen absorption by
potassium chloride solution. Air flow as parameter, logarithmic Schultz and Gaden (1956), and Carpani and Roxburgh
plot; the value of Y in the relationship KLa = K N' is given (1958). In the present work, the results obtained by
where N is agitator speed, rpm. the two methods agreed reasonably well at 800 rpm
1959] AGITATION-AERATION IN SUBMERGED FERAIENTATION. 1
at air flow rates between 0.5 and 3.5 L per min (figure
6) whereas agreement outside this narrow range of
operating conditions was rather poor.
From a comparison of the results (compare figures
2 and 4 and figures 3 and 5, also arithmetic plots in
figures 6 and 7), it may be noted that the polarographic
coefficients exceeded the sulfite values under conditions
of equipment operation where the impeller speed was
insufficient to disperse completely all the air supplied,
that is, "loading conditions." Under these conditions,
bubbles escaped around the impeller, their velocity
relative to an element of fluid was high (as indicated
by "boiling" at the liquid surface), and flow around
these bubbles was probably turbulent. Examples of
these operating conditions are as follows: in figure 6,
at 600 rpm at all gas flow rates; at 800 rpm at air
flow rates above 3 L per min; in figure 7, below about
800 rpm at air flow 1.0 L per min and below about
850 rpm at air flow 4.5 L per min. Since visual ob-
servations indicated no marked differences in bubble
dispersion between the two systems, it seemed that
the explanation for this difference in results was related
to the oxygen absorption mechanism.
It is noteworthy in this regard that an increase in
bubble velocity relative to an element of fluid (that is,
a change in flow conditions around bubbles, either
from laminar to turbulent flow or increase in the degree
of turbulence) in the two systems may influence the
liquid-film oxygen-transfer coefficient (KL) in a differ-
ent manner in each case. Coppock and Mleiklejohn
(1951) reported that an increase in bubble velocity in
water resulted in a corresponding increase in K,
presumably because of the increase in turbulence
adjacent to the liquid film and a consequent reduction
in the liquid-film resistance (thickniess). In contrast,
the results of Schultz and Gaden (1956) showed that
o SULPH IT E AGITATOR SPEED,RPM
1000[
800a
Ii
-Z 6001
-i
400
200
0 1 2 3 4 5
AIR FLOW RATE [ L/M IN
Figutre 6. Effect of air flow rate on oxygen absorption;
agitator speed as parameter, arithmetic plot.
55
KL for sulfite solution decreased with increase in inter-
facial turbulence, presumably because of a reduction
in the "time average" oxygen concentration and its
effect on the oxidation of S03= to S04=. Accordingly,
it would appear that the loading air flow rate in the
sulfite system (see figure 6) is a function of the chem-
istry of the reaction rather than of equipment per-
formance.
Similarly, the higher sulfite coefficients obtained
at agitator speeds above 800 rpm (figure 6) may also be
indicative of changes in boundary or film conditionis.
As the agitator speed is increased beyond 800 rpm
(figure 5) turbulent flow is produced in the liqjuid bulk,
a greater bubble area is incorporated within the bulk
flow-pattern and these bubbles tend to follow the path
of fluid flow. Consequently, there is less bubble area
with motion relative to an element of fluid. Under
these conditions the increase in absorption coefficient
in the sulfite system measures the increase in surface
area plus any increase in apparent KL brought about
by the increased stirring rate and the corresponding
increase in the "time average" oxygen concentration.
The increase in absorption coefficient in the polaro-
graphic system measures the increase in transfer area
minus the effect brought about by the faster stirring
speed and the corresponding increase in liquid-film
resistance. Hence with increase in agitator speed the
sulfite coefficients increase at a faster rate than the
polarographic values (figure 7). It should be noted,
however, that this explanation may in fact be an over-
simplification because the transfer area was not meas-
ured in the two systems.
Differences between oxygen absorption by sulfite
solution and by oxygen-free water have been noted
by other workers considering temperature effects.
For example, Schultz and Gaden (1956) calculated
from the data of other workers that the activation
energy for the sulfite reaction was characteristic of
6
500 700 900 1100 1300 1500
AGITATOR SPEED (N) RPM
Figulre 7. Effect of agitator speed on oxygen absorption; air
flow rate as parameter, arithmetic plot.
56 5R. STEEL AND Mi. R. BRIERLEY
control by a chemical reactioni, whereas Bartholomew
et al. (1950) reported that oxygen absorption into
water was controlled by diffusioni through a liquid
film.
The present results indicate that absorption coeffi-
cients obtained by the sulfite method should not be
used as a measure of the actual amount of oxygen
transferred to a microbial culture. Nevertheless, they
are useful for assessing improvements in equipment
design as long as the impeller speed is sufficient to
disperse the air supplied. For the equipment used in
the present work the highest air flow rate which could
be used at any particular agitator speed was expressed
by the relationship G = K N0O83 where G was the air
flow rate at which the loaded condition occurred
(results not given in the text).
ACKNOWALEDGMENTS
The authors are grateful to Professor T. K. Walker
for his interest in this work. One of us (R. S.) acknowl-
edges receipt of an Imperial Chemical Industries Re-
search Fellowship, and the other (M. R. B.) thanks
the Manchester Education Committee for the grant
of funds.
SUMMAIRY
Mleasurements were made of oxygen absorption rates
by catalyzed (Cu++) sodium sulfite solution and by
potassium chloride solution (polarographic method)
using a fermentor of conventional design. Correlations
obtained between oxygen absorption-coefficients (KLa,
hr-1) and operating variables were as follows: for the
sulfite system, KLa = K G0 40-0.70 and KLa = K
N2 5-3 2 where G and N are air flow rate and agitator
speed, respectively; for the polarographic system,
KLa = G K0 53 and KLa = K N' °. Absorption coeffi-
cients obtained by the two methods were essentially
similar only over a very narrow range of operating
conditions, namely, at agitator speed of 800 rpm with
air flow rates between 0.5 and 3.5 L per min. At low
agitator speeds (below 800 rpm) the sulfite coefficients
were lower than the polarographic values at all air
flow rates tested whereas at high agitator speeds
(above 800 rpm) the sulfite coefficients were higher
than the polarographic values. At constant agitator
speed (for example 600 rpm) the "loading" air flow
rate was lower for the sulfite system (2.0 L per min)
[VOL. 7
than for the polarographic system (4.5 L per min).
The discrepancies between the results obtained by the
two methods are explained in the terms of the differ-
ences in oxygen absorption mechanism in the two
systems.
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