FERMENTATION RESEARCH & ENGINEERING Dissolved Oxygen Measurement
. . . in
Yeast Propagation
I
JACK A. STROHMl and ROBERT F. DALE Red Star Yeast & Products Co., Milwaukee 8, Wis.
The polyethylene-covered platinum electrode can be used to monitor production and control aeration of yeast propagation in large and small vessels IMPORTANCE OF AERATION in many industrial fermentations is well known and has been the subject of a number of excellent studies and reviews ( I , 6, 73). T h e rate of oxygen transfer to aerobic antibiotic or vitamin €ermentations may affect the direction of metabolism of the culLivated microorganisni as well as the rate of product formation. Inadequate aeration in bakers’ yeast propagations (Saccharomyces cerezlisiae) results in the production of ethyl alcohol rather than cell substance, which is reflected as a loss in cellular yield. Overaeration, however, serves no useful purpose and results in sizable added expenses for chemical antifoam agents and air compressor operation. Commercial bakers’ yeast propagations are usually made batchwise, follow nonuniform molasses-substrate feed schedules, and produce a several-fold increase in yeast concentration. Optimal air requirements, therefore, vary during different stages of these propagations and cannot be estimated from the yield values of the entire propagation. General expressions of aeration, such as the cubic feet of air sparged per minute or the volume of air introduced per volume of fermentor liquor in unit time, have little import, since only dissolved oxygen is available to the yeast cell. T h e solubility of oxygen in aqueous solutions is very low, and only a small fraction of the oxygen sparged into commercial €ermentors is utilized by the organism. Techniques are available for measuring the oxygen transfer rate into K a & 0 3 solutions or fermentation broths which are useful in the study of fermentor design and aeration efficiency, but the most direct and meaningful index of optimal aeration during fermentation is the measurement of the dissolved oxygen present in the broth. Polarographic techniques have been T H E
1
Present address, Froedtert M a l t Gorp.,
Milwaukee, Wis.
760
successfully adapted to submerged fermentations (7, 3, 8, 73) and are preferred because of their high semitivity, speed, and applicability to continuous operation. Dropping mercury, rotated platinum, or stationary platinum electrodes have usually been used in these polarographic studies. Several conditions were found previously (72) in commercial bakers’ yeast propagations which complicated the application of polarographic techniques : 0
0
Very low dissolved oxygen concentrations were found to be adequate for maximum yield and yeast quality. Oxygen uptake rate was so rapid that measurements had to be made with the electrodes directly inside the fermentor. T h e low p H of the medium, often below 5.0, and the presence of other electroreducible substances caused a large and variable residual current to flow, even when no dissolved oxygen was present.
Large residual currents were observed with either a dropping mercury, stationary platinum, or stationary gold electrode. I n most periods of fermentation, the residual current was greater than the oxygen diffusion current being measured. The magnitude of the residual current bore a general inverse relationship to pH, but could not be accurately predicted and had to be measured whenever a dissolved oxygen reading was taken. I n small vessels, this was accomplished by flushing with nitrogen, but no arrangement was feasible for production sized yeast fermentors. Laboratory tests were recently begun using a polyethylene-covered platinum electrode, and negligible residual currentsbvere observed in yeast fermentations. The polyethylene-platinum electrode was found to be a simple, rugged, and useful device and was successfully adapted 10 monitor dissolved oxygen content in production-sized baker’s yeast and food yeast
INDUSTRIAL AND ENGINEERING CHEMISTRY
(Candidautilis) fermentors. The dissolved oxygen activity registered by the electrode system was shown to respond to changes in aeration and substrate feed rate. Correlation was found between average dissolved oxygen activity and yield of bakers‘ yeast propagations. The dissolved oxygen activity of bakers’ yeast fermentor liquor increased when the p H was increased by about 0.4 units in the p H range of 5.0 to 6.5 following the addition of NHdOH. This increase in dissolved oxygen activity was attributed to a reduction in the respiraton rate of the yeast. Experimen ta I
Equipment. The polyethylenecovered platinum electrode (Type OM2) used in these experiments was obtained from the Beckman Instrument Co., Fullerton, Calif. A diagrammatic sketch of this electrode and of the electrical circuit used is shown (p. 761.) I n operation, oxygen diffused through the outer polyethylene membrane to reach the platinum cathode which was charged by the battery circuit at -0.6 volt against the Ag-AgC1 reference electrode which is also contained in the probe. Ions and large molecules cannot diffuse through the polyethylene, so the residual currents obtained in yeast fermentation broth were so small that they could be considered negligible. The diffusion rate of oxygen through the polyethylene membrane must reach equilibrium before a measurement can be taken, so the response of the electrode is slow. About 5 minutes were required to obtain a precise dissolved oxygen measurement when changing the aeration conditions in yeast propagations. The electrode readings are understandably quite sensitive to changes in temperature. When using 2-mil polyethylene, the dissolved oxygen reading increase by 10% as the temperature of air-saturated fermentation broth was raised from 90’ to 95’ F. This effect was
was able to autoclave a ‘Teflon-covered silver electrode which he used for dissolved oxygen measurements. Measurements. Finn (7) reported the polyethylene-platinum electrode could not be calibrated by observing diffusion current in various air-saturated salt solutions and concluded the electrode measured oxygen activity rather than oxygen concentration. Finn’s findings were confirmed by the data of Figure 1 which show simultaneous dissolved oxygen measurements made with the dropping mercury and polyethyleneplatinum electrodes in air-saturated NaCl and sucrose solutions of various concentrations. A range of dissolved oxygen concentration (4.20 to 5.52 cc. per liter) ( I I ) was obtained by changing the oxygen solubility with the addition of salt or sucrose. The dropping mercury electrode measurements were directly proportional to the dissolved oxygen concentration, while those of the membrane-covered electrode were independent of the oxygen concentration, as governed by the solubility. When various mixtures of air and nitrogen were sparged through salt or sucrose solution, the readings of both electrodes were directly proportional to the dissolved oxygen concentration. A schematic representation of the polyethylene covered platinum electrode and the oxygen concentrations occurring during its use is shown (p. 762). Oxygen from the air is moving in series through the aqueous solution and poly-
unimportant in the work, since the fermentations took place a t a constant temperature, Carritt (2) has described a temperature compensator which can be used to nullify the temperature sensitivity of an electrode of this type. A continuous recording of the dissolved oxygen measurements was obtained with a Bristol recorder. The dissolved oxygen content of air-saturated 90’ F. broth registered full scale on the recorder when using the resistances shown on the diagram. The recorder reading could be multiplied by a factor of 8 to 10 when measuring low oxygen concentrations by switching to the high position of the sensitivity switch. The linearity of the system was unaffected by this change. If desired, intermittent oxygen measurements can also be made using the millivolt scale of a pH meter and a slightly modified resistor circuit. Replacement of the polyethylene film was sometimes necessary after the probe had been used in several fermentations. Following film changes, the electrode v a s standardized in 90’ F. air-saturated salt solution by adjusting the 100-ohm variable resistor to obtain a full scale recorder reading. The polyethylene-platinum probe was used to monitor the dissolved oxygen activity of more than 50 yeast propagations and was found to be a very convenient, rugged, and dependable device. The probe was sterilized chemically before use because it could not withstand steaming. Phillips (70), however,
DISK or MIL ER PAPER
where
No, = moles of Os discharged per
D C,
d
unit time and per unit electrode area = diffusivity of 0 2 in polyethylene, sq. cm./sec. = 0 2 saturation concentration in the polyethylene a t polyethylene-solution interface, moles/cc. = membrane thickness, cm.
For oxygen to move through the air and aqueous solution, concentration gradients must exist in these phases. T h e bulk of the resistance to oxygen transfer, however, takes place in the polyethylene, and the concentration changes in the air and aqueous solution can be considered negligible. Under these circumstances, all of the aqueous solution is in equilibrium with the surrounding air, and the polyethylene surface is in equilibrium with the solution. I t then follows that the polyethylene surface is in equilibrium with the air and that the aqueous solution is serving solely as a transfer medium. T h e solubility of oxygen in the aqueous solution then becomes unimportant.
SOLUTION
NaCI I
ELECTRODE LEAD WIRES
ethylene membrane to the platinum electrode surface which is maintained a t a n oxygen concentration of zero. Because the concentration is very small, the rate a t which oxygen reaches a unit area of the electrode may be expressed as :
-4
17.5
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PROBE LENGTH, 5 IN. DIAMETER.1 IN. ELECT RODE CONNECT IONS
HIGH 5000-
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SWITCH
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SBRISTOL J,
DY NAMASTER 1OMv. D.C. RECORDER
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drawing of polyethylene-platinum electrode and circuit shows important parts and gives requirements for electrode-recorder system
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:
16.5
4.5 5.0 5.5 60 DISSOLVED OXYGEN CONC., CC/LlTER
4.0
Figure 1 , Relative response of dropping mercury and polyethylene-platinum electrode to changes in 0 2 solubility Polyethylene-platinum electrode measured 0 2 activity rather than concentration
VOL. 53, NO. 9
SEPTEMBER 1961
761
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PLAT1 NUM
OXYGEN CONCENTRATION
Oxygen gradients occur when using the polyethylene-platinum electrode under ideal conditions
8
0 With the dropping mercury electrode, the place of the membrane is taken by a "film" of aqueous solution: NO,
k(Cb
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% O X Y G E N I N SATURATING GAS Figure 2 . Polyethylene-platinum electrode measurements were proportional to dissolved 0 2 activity in '/& salt solution
(2)
\\>here
k
mass transfer coefficient for "stagnant solution film:" moles/(sec.) (sq. cm.) (concn. diff.) C; = 0 2 concentration in bulk of' solution which is approximately equal to that a t saturation, cc./liter =
'The solubility of oxygen in the stagnant film is the same as that in the bulk of the liquid. Clearly C,? and thus the oxygen transfer rate, is a function of the salt content of the solution. Changing
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the salt content of an aqueous solution affects the dropping mercury electrode in the same way that changing the chemical nature of the membrane affects the polyethylene-platinum electrode-- -that is, by changing solubility of oxygen in the region of diffusional resistance. *Measurements obtained with the polyethylene-platinum electrode are therefore related to the partial pressure or activity of the dissolved oxygen. I n microbiological systems, i t seems likely
6
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24 d
that these quantities are more significant than the actual concentration of dissolved oxygen present, because a cellular membrane undoubtedly offers a high diffusional resistance and acts somewhat like the membrane-film of the oxygen eleclrode. Throughout the remainder of this report the measureinenis obtained with the polyethylenccovered platinum electrode are referred to as dissolved oxygen activity, realizing, however, that it measures oxygen partial pressure andlor oxygen activity. Calibration. T h e polyethylene-platinvm electrode was calibrated by sparging various air and nitrogen mixtures through salt solution and yeast-free fermentation broth. Figure 2 shows the electrode measurements were directly proportional to the dissolved oxygen activity of a l,'pA4; hTaCl solution. Similar results were obtained in yeast-free fermentation broth below pW 5.0, but a t higher pH, a nonlinear response was obtained a t low oxygen concentrations. Figure 3 presents the calibration results in molasses fermentation broth. The cause of the flattened response at pH 6.0 is unknown.
W
n d
0 2 2 d
%
OXYGEN I N SATURATING GAS
Figure 3. Response of polyethylene-platinum electrode in molasses fermentation broth at low 0 2 activity levels is linear at p H 4.5and nonlinear at pH 6.0
762
INDUSTRIAL AND ENGINEERINGCHEMISTRY
Results Food Yeast Propagations. Dissolved oxygen measurements made with the polyethylene-platinum probe in a conimercial food yeast (C. utzlis) propagation a t several levels of aeration and substrate feed rate are shown in Figure 4. This propagation ( 5 ) , in large, agitated tanks of the modified Waldhof type, was continuous. Sulfite waste liquor substrate was added to the fermentor a t a constant rate with the simultaneous withdraival of beer and yeast. T h e pH of the broth was held constant a t about 5.0. For the measurements reported
O X Y G E N MEASUREMENT here, the electrode was mounted in 0.75-inch pipe, sealed with rubber tubing, and lowered to a position of several feet below the liquid surface. T h e first 15-minute portion of the graph shows the dissolved oxygen activity during normal operating conditions. This was about '/loth of that present when the broth was saturated with air (CJ. At a time of 15 minutes on Figure 4, the air supply to the fermentor was turned off, and the dissolved oxygen activity of the broth decreased to a low value. Even though the external air supply was off, some atmospheric oxygen was still drawn into the fermentor by the draft tube and agitator. At 30 minutes, normal air flow to the fermentor was resumed, and the oxygen activity increased to a high level before returning to the previous value for normal operation. There are some indications that this peak was caused by a lag period in yeast metabolism. At 60 minutes, a 20% reduction was made in the air flow rate, and a t 75 minutes a 50% reduction occurred; both air reductions are clearly reflected in the dissolved oxygen activity. When the normal air flow was resumed a t 90 minutes, a peak in oxygen activity was again observed before a n equilibrium value was obtained. When the sulfite liquor feed to the fermentor was stopped at 120 minutes, the dissolved oxygen activity of the broth rapidly increased to approach the
0
15
30
60
75
165
I20
90
MINUTES Figure 4. Recorder tracing from production-size C. utilis propagation shows influence o f imposed variables on dissolved 0 2 activity
saturation level. When the normal feed rate was restored, the oxygen activity quickly decreased. At 165 minutes, the feed rate was increased 10% above the normal value; this caused the dissolved oxygen activity to fall to a level of about '/S"th of that present a t saturation. Restoring the normal feed rate a t
180 minutes resulted in a gradual increase in dissolved oxygen activity. The electrode was also successfully used to investigate the uniformity of aeration within the fermentor and as a tool to monitor continuously the dissolved oxygen activity during production. The electrode proved itself reliable and
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OFF-SCALE
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HOURS OF FERMENTATION
Figure 5. Superimposed plots show relation o f raw material feed, yeast concentration, pH, and dissolved plant special S. cerevisiae propagation VOL. 53, NO. 9
0 2
activity in pilot
SEPTEMBER 1961
763
gen measurements made in a 4-hour portion of another type of experimental bakers’ yeast propagation. The molasses feed rate during this period was constant; pH adjustments were made at half-hour intervals by the addition of relatively large amounts of T\-H40H. Large increases in the dissolved oxygen activity of the medium followed the NHdOH additions. When the metabolism of the yeast was inhibited by the addition of iodoacetic acid, NHhOH additions comparable to those of Figure 6 had no effect on the dissolved oxygen content of the medium. Under the conditions of the experiments, it appeared that the rate of sugar assirnilation by bakers’ yeasts was inversely related to p H in the range of p H 5.0 to 6.0 (9). Therefore, these increases in dissolved oxygen are attributed to a reduction in the respiration rate of the yeast resulting from the addition of large quantities of hTHdOH.
d z
a
>--
40
k
2-
-I
V
a
5 W
20
> 0
X
0
HOURS
-
Figure 6. Recorder tracings show effect of NH40H additions ond issolved activity in S. cerevisiae propagations
02
When actively growing yeast cells a r e chemically inactivated in same media, lower tracing will b e straight line during similar pH changes
rugged. No poisoning occurred, even though the free SO2 content of the broth was usually about 0.5 mg. per ml. Bakers’ Yeast Propagations. Dissolved oxygen measurements made with the polyethylene-platinum electrode in an experimental bakers’ (5’. cereuisiee) yeast propagation are shown in Figure 5. T h e vessel was nonagitated and was aerated through spargers a t a level equivalent to an oxygen transfer rate of 150 millimoles of oxygen per liter-hour in NazSOs solution. The fermentation was pitched with a portion of the molasses and yeast solids shortly before the propagation was begun. T h e remaining molasses was fed incrementally to the propagation following the schedule as shown. The pH of the medium \vas regulated by frequent additions of
NHIOH. T h e dissolved oxygen measurements were made using the high sensitivity setting of the circuit, so the full-scale reading was equivalent to one fifth of the oxygen activity which would be present when the broth was saturated with air. T h e air supply to the vessel was shut off for 5 minutes after 10.5 hours of fermentation, and the dissolved
oxygen activity dropped from about 1% of that a t saturation to almost zero. When all the molasses was fed a t 11 hours, the dissolved oxygen activity present in the medium rapidly increased to the saturation level, which on Figure 5 is off-scale. Dissolved oxygen measurements were also made in a series of bakers’ yeast propagations like that shown in Figure 5, but over a range of aeration levels. Sulfite oxidation values a t all aeration levels were obtained in KasSOs solution by the method of Cooper ( 4 ) . Table I compares the average amount of dissolved oxygen activity present between 4 to 11 hours of fermentation. the sulfite oxidation value at that air rate, and the molasses yield for each propagation. Good correlation was obtained between the average dissolved oxygen activity of the medium and the molasses yield. The sulfite oxidation values for the agitated propagations correlated with the average dissolved oxygen activity, but more oxygen activity was found in the nonagitated propagation than would be expected from the sulfite oxidation determination. Figure 6 shows p H and dissolved oxy-
Acknowledgment T h e authors express their appreciation to E. N. Lightfoot, University of Wisconsin, Madison, Wis., for his assistance in explaining the diffusional behavior of the polyethylene-platinum electrode and to M. W. McNaughton, Red Star Yeast & Products Co., and W. B. Jacobs, Charmin Paper Products Co., Green Bay, Wis., for help in procuring the experimental data. Literature Cited
(1) Bartholemew, W. H., Karow, E. O., Sfat, M. R., Wilhelm, R. H., IND. ENG.CHEM.42, 1801-9 (1950). (2) Carritt, D. E., Kanwisher, J. W., Anal. Chem. 31, 5-9 (1959). (3) Chain, E. B., Gualandi, G., Rend. ist. super. sanitci 17, 5-60 (1954). (4) Cooper, C. M., Fernstrom, G. A., Miller, S. A., IND. END. CHEM. 36, 504-9 (1944). (5) Dyck, A. W. J., Paper Ind. 39, 26-8 (April 1957). (6) Finn, R. K., Bacteriol. Rev. 18, 254-74 (1954). (7) Finn, R. K., 136th Meeting, ACS, Atlantic City, N. J., September 1959. (8) Hixon, A., Gaden, E. L., Jr., IXD. END.CHEM. 42, 1792-1800 (1950). (9) Mueller, R. L., Red Star Yeast Pr Products Co., Milwaukee, Wis., unpublished work, 1958. (10) Phillips, D. H., Univ. Wisconsin Antibiotics Research Rept. No. 31, June 1, 1959.
Table 1.
Comparison of Oxygen Measurements and Yield Shows Good Correlation
Vessel
Type Agitated Agitated Nonagitated Agitated Agitated
Capacity, gal.
Average 0 2 Present during 4-11 Hr. Period, yo Present at Oz/Liter-Hr. Saturation Sulfite Oxidation Value, mM
5 5
120 160
70
5
135 200
70
350
yo of optimum growth.
764
INDUSTRIAL AND ENGINEERING CHEMISTRY
1.1 1.2 1.4 1.6 23.0
(11) Seidell, A., “Solubilities of Inorganic and Metal-Organic Compounds,” Vol. I, 3rd ed., Van Nostrand, New York,
__
I94n
(12) Strohm, J.,
Relative Yield,” yo 75.0
78.0 83.7 85.0 100
Dale, R. F., Peppler, H. J., A@$. Microbiol. 7, 235-8 (1959). (13) Wise, W. S., J . Gen. Microbid. 5 , 167-71 (1951).
RECEIVED for review December 30, 1961 ACCEPTEDMarch 31, 1961
Division of Agricultural and Food Chemistry, 138th Meeting, ACS, New York, September 1960.
