yjj+ SWITCH 2 .
-P O S I T I O N I N G \ V A R I A C MOTOR
-
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CONTROLLED MOTOR
t
S W I T C H I.
W-I RELAY
t. HUMIDIFIER
M E T E R 2.
AIR
Figure 1.
Schematic diagram
of oxygen-uptake program control system
PING SHU Prairie Regional laboratory, National Research Council, Saskatoon, Saskatchewan, Canada
Control of Oxygen Uptake in Deep Tank Fermentations Oxygen uptake in deep tank fermentations may be controlled aulomaticasly to follow a simple and flexible predetermined program
b A control system based on the analysis and control of oxygen concentration in effluent gas makes it possible to control oxygen utilization automatically to follow a predetermined program. The apparatus offers a direct and simple means for transferring aeration data between different fermentors.
Ax
APPARATUS for recording oxygen uptake in shake flask fermentations has been described (3),with which the pattern of oxygen utilization during the entire period of fermentation may be observed, and the optimum rate of oxygen utilization under shake flask conditions determined. Later, a coupling method (2) was developed with which the oxygen-uptake pattern of a fermentation in one fermentor may be duplicated in an
Present address, Lederle Laboratories, American Cyanamid Co., Pearl River, N. Y .
2204
INDUSTRIAL AND ENGINEERING CHEMISTRY
agitated tank. it'ith a fixed rate of air flow, the agitator speed or power demand for such duplication may be determined. The power data thus obtained may be used for further scaling-up purposes by the conventional method. As yet no suitable method for direct transfer of the oxygen-uptake pattern to a large scale fermentation has been described. The rate of oxygen utilization in a fermentor may be directed to follow a predetermined schedule by maintaining a steady flow of air while varying the agitator speed, so that the oxygen concentration in the ferrentor effluent gas is made to follow a prescribed program drawn on the recording chart.
IMPROVED FERMENTATION E Q U I P M E N T & D E S I G N
Figure 2.
Contact block
Apparatus Figure 1 shows the schematic diagram of the device for control of the oxygenuptake rate for a stirred tank fermentation. A steady flow of air passing through the fermentor is obtained with the help of a Cartesian manostat. The output end of the flowmeter is kept at a constant pressure (4 pounds per square inch), so that any pressure fluctuation in the fermentor does not affect the rate of air flow. Air is filtered and humidified before entering the fermentor. A small portion (120 to 100 ml.) of the effluent gas is tapped, freed from carbon dioxide by bubbling through 6 N sodium hydroxide, and dried by passing through a watercooled condenser, anhydrous calcium chloride, and Anhydrone. The dried gas is led into a magnetic oxygen analyzer (Model F-3, Arnold 0. Beckman, Inc., South Pasadena, Calif.) and the oxygen concentration of the effluent gas is recorded on a strip chart recording potentiometer. In general, the ratio between rate of air flow and air space above the liquid level in the fermentor is so large as compared with the rate of change of oxygen uptake rate in any conventional deep tank fermentation that the collected sample of the effluent gas may be considered as representative of the whole. The rate of oxygen utilization is then expressed in terms of its concentration difference between air and the effluent gas. Hoover, Jasewicz, and Porges (7) first used this type of, oxygen analyzer for the analysis of effluent gas from a fermentor. A line, outlining the proposed rate of oxygen uptake, is drawn on the recording chart with metallic paint, and a set of relay contacts molded in a Plexiglas block is attached to the pen carriage of the recorder. When the oxygen concentration in the effluent gas deviates from the line, relay 1 is activated and the positioning motor turns the Variac dial in such a
Figure 3.
Contact block assembly
direction that it changes the stirring speed to correct the deviation. Under normal load conditions the operating time of the positioning motor is limited by timer switch 1. This switch is used to compensate the time lag in the control loop. If the overshooting of oxygen uptake rate exceeds a given limit (auxiliary line), relay 2 is activated and the circuit between relay 1 and the positioning motor is closed through timer switch 2. The duration of the circuit-on time of this switch is relatively larger than that of switch 1. This arrangement increases the rate of adjustment and corrects overshooting in a relatively short time. With this closed loop control system the oxygen-uptake rate of a fermentation may be regulated to follow a given program by varying the agitator speed. Contact Block and Program Line. The contact block is a piece of rectangular Plexiglas embedded with copper relay contacts in the shape of thin rod and points arranged in the order as shown in Figure 2. The contact rod is connected to contact C of relay 1 (this relay motor circuit is shown in Figure 6). The point closer to the rod is connected to contact A of the same relay. The other point, designated as D, is connected to the grid contact of electronic relay 2. The contacts are fixed in one plane by milling and polishing, and elevated by filing off the neighboring plastic. The exploded view of the assembly is shown in Figure 3. This whole assembly is attached to the pen carriage of the recorder (Figure 4), so that the contacts under the weight of the copper block may slide freely along in the direction of the pen movement and make positive contact with the chart. There should be very little or no upward thrust against the pen carriage. The program line is drawn on both sides of the recording chart with silver conductive paint (General CementManufacturing Co., Rockford, Ill.) and joined
Figure 4. Attachment of contact block to recorder pen carriage
together over the edge of the paper. This line is drawn offset from the intended program by a distance between pen and contact A . The chart drum is also coated with the paint and grounded to the recorder case and the cathode contact of relay 1, B, is also connected to the case. Thus, the program line becomes the cathode contact of relay 1. Regulation of Agitator Speed. The mechanism for regulating the agitator speed by the interaction between contacts and the program line is demonstrated in Figure 5. I n examples 1 and 2, with contact point A off the program line, the rotational direction of the positioning motor depends upon whether contact rod C is on or off the line. When contact C is off the line-Le., too low a n oxygen concentration in the effluent gas-the positioning motor turns in a direction to reduce the agitator speed and this results in a reduction of the rate of oxygen utilization. When contact C is on the line-Le., too high an oxygen concentration-the action of the positioning motor is reversed. I n examples 3 and 4,contact A is on the program line-Le., the oxygen concentration in effluent gas coincides with that desired within the
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M
Figure 5. Interaction between contact block and program line VOL. 48, NO. 12
DECEMBER 1956
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Experimental and Results
T o observe the performance of this program control device, fermentation tests were made by growing Xunthomonas uredouorui. PRL F21 and Aspergillus niger PRL 558. The fermentation media have the following composition : For X.
Substances
Figure 6.
Relay motor circuit
error of the thickness of the line drawn on the chart. Then, the positioning motor is inactivated regardless of the status of contact C. Thus, during the fermentation, the oxygen concentration in the effluent gas from the fermentor is continuously checked against the desired concentration. and the device adjusts the agitator speed so that the distance between contact A and the program line is kept at a minimum. There is a long time lag in the control system due to gas space in the fermentor and sampling line. T o reduce the undesirable oscillation, a timer switch was therefore inserted in the circuit joining relay 1 and the positioning motor. If an overshooting of oxygen-uptake rate is anticipated, a n auxiliary line, constructed similar to the program line, is drawn on the chart and connected to the cathode contact of relay 2 through the chart drum and recorder case. As shown in example 5 (Figure 5), when point D makes contact with the auxiliary line, relay 2 is activated and the circuit between relay 1 and the positioning motor is closed through timer switch 2. This added shunt circuit increases the rate of adjustment of the agitator speed and thus brings down the oxygen-uptake rate rapidly. As soon as contact D is off the auxiliary line, agitator speed is adjusted only through timer switch 1. Flexpulses (Fisher Scientific Co.. Kew York, N. Y . ) were employed as the switches. The relay positioning motor circuit shown in Figure 6 is similar to that described in a previous paper (2). Fermentor and Other Accessories. The fermentor (Figure 7) is a tall glass cylinder (13-liter capacity) equipped with two four-bladed turbine impellers, four removable baffle plates, a sampling device, and a hypodermic syringe for adding antifoaming agent. The materials of construction of all parts in
2206
contact with the culture are stainless steel, glass, and rubber. The agitator is rotated by a I/a-hp. variable-speed direct current motor (General Radio Co., Cambridge, Mass.) through a gear reducer (3 to 1). The stirring speed is adjusted by turning the Variac speedcontrol dial with the positioning motor. For fine adjustment the coupling is made through friction disks of large diameter ratio (1 to 17). The positioning motor is a 1-r.p.m. Bodine motor such as that employed in previous studies (2, 3 ) . Carbon dioxide in the effluent gas is removed by bubbling through 6 N sodium hydroxide in a small scrubber. The latter is provided with a hole covered with a serum bottle cap for replacing sodium hydroxide solution without interruption by a hypodermic syringe. Duplicate sets of dryers are installed (Figure 1); and tubes A and A’ contain coarse (mesh 4) anhydrous calcium chloride and tubes B and B ‘ contain .4nhydrone. In actual operation only one set of dryers ( A and B. or A’ and B‘) is used. The set in operation (solid line) may be replaced by the spare set (dotted line) without interruption. The replacement may be done one at a t i m e 4 . e . . A by A’ and B by B’. The change of tl to A’ may be done by first turning V I in position such that both A and A’ ate connected to the gas inlet. This is rollowed by turning vent Vd to open position and letting a portion of the gas leak out to the atmosphere while increasing the flow rate to maintain the same floic ol‘ gas into the oxygen analyzer. After 10 to 15 minutes the vent valve is closed, and V I and V Bare turned in position to close off dryer tube A and let all the sample flow through A’. The rate of gas flon- is then readjusted to normal and tube A may be removed for renewal of the drying agent. Likewise B may be replaced by B ’.
INDUSTRIAL AND ENGINEERING CHEMISTRY
Glucose Starch Peptone Yeast extract Ammonium phosphate, monobasic Potassium phosphate, dibasic Magnesium sulfate, heptahydrate Calcium carbonate
For
uredo-
A.
vorus, G./L.
niger,
20
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13
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2.5
1 0.25 10
Ten liters of sterile medium were transferred aseptically into the sterile fermentor and inoculated with 1% (for bacteria) or 57, (for fungi) of a 16-hour shake flask culture. Air was passed through the medium at a rate of 2060 ml. per minute. The frrmentor temperature was kept a t 25’ C. Samples were withdrawn at intervals and analyzed for residual carbohydrate and products. The range of the oxygen analyzer, set at 11 to 21g-/, oxygen, was standardized and checked a t the beginning and end of each fermentation. I n experiments for duplication of oxygen-uptake rate: the on times of timer switches 1 and 2 u e r e set for 5 seconds per
Figure 7.
Fermentor
I M P R O V E D FERMENTATION E Q U I P M E N T & D E S I G N l
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TIME, (hours)
Figure 8.
5.5 minutes and 1 second per 25 seconds, respectively. Fermentations were first run in a baffleless fermentor agitated at constant speed (pattern fermentation) and the oxygen-uptake rate curves were then used as a program for reproduction in the same fermentor with baffles inserted (reproduced fermentation). I n the fermentation with A. niger the agitator speed for the pattern fermentation was set at 500 r.p.m. Two sets of experiments were made with X. uredovorus and the agitator speeds for these pattern fermentations were set at 315 and 550 r.p.m., respectively. Figures 8 and 9 are the actual recordings of the oxygen-uptake rate curves of the pattern and the reproduced fermentations. I n the neighborhood of peak oxygen-uptake rate relatively poor duplication of the pattern occurred. This is probably of inherent nature. Average rates of carbohydrate utilization, oxygen consumption, cell produc-
-
1
1
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i
I
1
At low aeration level,
tion, and enzyme formation for intervals of 2 hours were calculated and these as well as the agitator speeds of the pattern and reproduced fermentations were compared (see Figures 10 and 11). I n general, the rate processes of the reproduced fermentations followed those of the patterns; however, some irregularities and phase shifting occurred. The total yields of products and consumption of substrates were close to those of the pattern fermentations within experimental error.
Discussion The utilization of oxygen in a batch fermentation with constant rate of air flow and fixed agitator speed may generally be divided into three phases. I n the first phase, the rate of oxygen uptake is limited by cell population; in the second, by oxygen supply; and in the third, by the substrate concentration. Duration of the second phase depends I
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Recordings of oxygen-uptake rate of pattern and reproduced fermentations with Xanthomonas uredovorus left.
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upon the intensity of aeration and the concentration of substrate. In reproduced fermentations, during the first phase of the pattern, the agitator speed is increased to satisfy the oxygen demand as cell population increases. If however, for some reason other than the oxygen supply, the growth lags behind that in the pattern fermentation, the rate of oxygen uptake also lags behind. The device then increases the agitator speed to correct this deviation, but in vain. Such false adjustment continues until the rate of oxygen uptake reaches that demanded by the program. As the fermentation progresses into the second phase, the excess aeration cannot be eliminated in a short time; thus the overshooting phenomenon appears in the curve and an oscillation of high amplitude occurs. This type of disturbance may be reduced by using either the auxiliary time switch system used in the present device or a more complicated instrument with derivative control action. I n the second phase, when the supply of oxygen is a limiting factor, the present simple device seems to offer adequate regulation. I n the third phase, the agitator speed is adjusted to satisfy the oxygen demand of the culture. If there is a difference in the initial substrate concentration or a poor program reproduction in the previous two phases, the third phase may not coincide with that of the pattern fermentation. If it commences ahead of the program, the oxygen-uptake rate control becomes ineffective. However, this does not have a serious effect, as this phase normally is very short. Judging from the results obtained, general trends of the rate processes such as growth, substrate utilization, and enzyme production seems to follow those of the pattern fermentations. However, it is premature to conclude, until sufficient VOL. 48,,NO. 12
DECEMBER 1956
2207
161
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Figure 10.
Agitator speed and chemical changes in pattern and reproduced fermentations with Xanthomonas uredovorus Left.
data are available, whether details of a fermentation may be reproduced simply by duplicating the pattern of the rate of oxygen consumption in fermentors of different sizes and geometry. Aeration gradient zones generally exist in fermentors agitated by rotors. This phenomenon is more pronounced in
TOTAL-0-199
At low oerotion level.
Right.
At high oeration level
fermentations producing heavy mycelial suspension. If the fermentor design does not render adequate broth circulation to offer an even exposure of all cells to these various aeration fields. then the geometry of tha fermentor must be taken into consideration. Under this condition, the duplication of oxygen-uptake rate pat-
3
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c w
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Figure 11. Agitator speed and chemical changes in pattern and reproduced fermentations with Aspergillus niger
2208
INDUSTRIAL AND ENGINEERING CHEMISTRY
tern would bear little significance for the reproduction of fermentation detail in a fermentor of different design. if the optimum rate of oxvgen utilization is lower than the maximum rate. For best resuits, therefore, the use of the present device should be confined to fermentors which offer good circulation of the culture broth. M'ith the use of the present or a similar type of program control system, a given pattern of oxygen utilization may automatically be reproduced in different fermentors and the equivalent agitator speeds may be measured. In other words, a n equivalent aeration condition for a particular fermentation at a fixed rate of air flo~vmay be obtained. Because the oxygen concentration in the effluent gas is considered as a measure of the average rate of oxygen utilization, the geometry of the fermentor is no longer a limiting factor for aeration data translation by- this system as it is for other known methods. Scale-up to Production scale could possibly be done with relative ease. Furthermore, the use of this type of control may also be extended for comparing power efficiency of different aerating systems under actual fermentation conditions. Literature Cited (1) Hoover, S . R., Jasewicz, V., Porges, K.,Instruments and Automation 27, 774
(1954). ( 2 ) Shu, P., Can. J . Teciinol. 33, 279 (1955). P., J . Agr. Food Chem. 1, 1119 ( 3 ) sh$ 5 3 ).
RECEIVED for review October 4, 1955 ACCEPTED May 21, 1956
