potassium chloride solution. Thus, a complete breakage of the electrode, permitting entry of the entire contents of the electrode into the fermentor, still will not sterilize the batch. Principal Difficulties Encountered by Users. Those surveyed were asked what basic obstacles still exist that should be corrected. The responses varied considerably, except for consist-
Principal Difficulties Encountered No. of Specific Problem Responses Short l i e of electrodes 28 Inadequate mechanical seal around electrodes 2 Reference electrode filling solution 4 Subjecting complete electrode assembly to steam sterilization 1 Mechanical breakage of electrodes within fermentor 1 Need for longer electrodes for 1 small-scale fermentors Need for very small electrodes for laboratory scale fermentors 1 Reducing electrode coating prob1 lem _. 39 Total
I
ent complaint regarding short electrode life. No one stated that reference electrode fouling was a problem. On the other hand, only four replied that they employ air pressure to the electrode to oppose steam sterilization pressures, while nine stated that they have never used opposing pressure. Entry of foreign solution into the reference electrode often causes ultimate measurement failure. I t is possible that some lack of success in this application was primarily due to failure of the reference electrode, although other reasons were attributed. Conclusion
The response to this survey, with observation of the activities of various groups, indicates that considerable progress has been made by the industry in employing continuous pH to fermentation processes. On the other hand, considerable effort must still be spent in improving techniques and p H components and in settling questions such as the acceptability of sterilized potassium chloride solution, before this application can be universally adopted.
This review only reports the findings of the survey. Other recent articles have described highly successful pH systems where the difficulties mentioned above have been overcome. The practical experience gained and techniques employed by such successful installations should be thoroughly studied to gain the benefit of their approach. Because of the widely different policies that exist between different organizations, it would be most difficult to describe a universally reliable pH system acceptable to all. On the other hand, each successful installation can offer methods or procedures that can be incorporated into various approaches in the establishment of a specific type of pH system for other installations. Acknowledgment
Sincere appreciation is expressed to the many individuals throughout this industry who contributed their opinions, comments, and suggestions by means of the questionnaire and by personal contact. Their interest and cooperation will greatly facilitate progress in improving and simplifying this application.
FRANK W. DENISON, Jr., IRVING C. WEST, MERLIN H. PETERSON, and JOHN C. SYLVESTER Abbott Laboratories, North Chicago, 111.
Large-Scale Fermentations
A PracticaI System for pH Control This electrode assembly for controlling pH in large-scale fermentations can withstand steam sterilization
OPTIMUM
pH RANGES have been determined for most industrially significant fermentations (7-4). Chemical buffers such as calcium carbonate, phosphates, and citrates, used to maintain such ranges, have narrow limits of application. For submerged pure-culture fermentations, nutrients are frequently added which on utilization produce the desired pH. However, adding acid or base during fermentation is more desirable, because nutrients can then be selected for maximum product yield. Control on a continuous automatic basis requires that the electrodes be in
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constant contact with the beer. Chemical sterilization (3, 4) prior to aseptic insertion of the electrodes into the fermentor is considered too hazardous because of the danger of contaminating high value production batches. Attempts have been made to sterilize electrodes in place with steam (3, 6, 7) but repeated failure of the glass electrode makes this procedure impractical for routine operation. This report describes a method for controlling pH of submerged fermentations, using steam sterilization of the electrodes.
INDUSTRIAL AND ENGINEERING CHEMISTRY
Design and Operation
The 50-gallon stainless steel fermentor with a 30-gallon operating volume is similar to larger scale equipment, but has the flexibility essential for this type of study. In the bell-shaped p H electrode chamber (Figure l), similar to one previously described (6), were mounted at hightemperature glass electrode (Beckman No. 8990-90) a temperature compensator, and a reference electrode. The chamber was placed in the fermentor and lead wires were brought out through
BH CONTROL IN FLRMENTATION j
a stainless steel pipe fitted to a hole in the side of the vessel. The reference electrode, similar to that designed by M. J. Johnson (5) has a silver chlorideplated silver wire fused to the platinum wire. For pressure equalization, a rubber stopper with a glass tube was inserted into the potassium chloride reservoir. An inverted test tube on the end of the glass tube was filled with glass wool to minimize entrance of the fermentor contents. Although it required special attention, the reference electrode was practical, but repeated failures of the glass electrodes were caused by moisture entering the top of the electrodes. This moisture did not enter from the fermentor through the packing glands in the chamber but apparently was vapor which entered the chamber through the opening for the lead wires and condensed while the fermentor was cooled. To resolve this problem of steam sterilization of p H control electrodes, the equipment now in use was designed (Figure 2). The electrodes are mounted in a Beckman No. 4210 p H flow assembly. The glass electrode is a Beckman No. 8990-90 and the reference electrode is a Beckman No. 19700 reservoir reference electrode. A positive flow of potassium chloride from the reference electrode is assured by an air supply to the top of this electrode. In practice pressure at the top is held 7 pounds higher than the pressure to which the bottom portion is subjected. Sterility of the potassium chloride is maintained with 1% benzyl alcohol, and the maximum flow of liquid from the reference electrode is 2 ml. per day. Benzyl alcohol in concentrations 100 times greater had no effect on the fermentations studied. The flow assembly, also containing a temperature compensator, is mounted in an external leg from the 50-gallon pilot plant fermentor. The beer flows by gravity out of the pipe in the bottom of the 'fermentor and is returned through the side, above the liquid level, with the aid of a simple air-lift pump. The static head places the liquid level approximately 60% of the way up the return leg. The flow for the remaining distance is created by sterile air introduced through 50 holes, '/a% inch in diameter, bored in the upper half of the inner pipe. The flow rate is approximately one gallon per minute and the capacity of the external leg is 0.33 gallon (Figure 3). The external leg can be sterilized with the fermentor or it can be isolated and sterilized separately. This allows replacement of electrodes during the course of a run without endangering the fermentor contents. p H is measured by connecting the electrodes to a Beckmain Model R p H
f /4=
OLASS TEST TUBE
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GLASS TUBE RUBBER STOPPER
REFERENCE ELECTRODE
-
HEAT RESISTANT OLASS ELECTROD
SILVER SILVER CHLORIDE PLATED WIRE
LIQUID LEVEL
THERMAL COMPENSATOR Figure 1. Electrode chamber. Failure was caused b y moisture which entered the chamber through openings for the lead wires
indicator. This, in turn, is connected to a Brown Electronic recorder-controller. Two Minneapolis - Honeywell pressure switches connected to the Brown Electronik actuate an external Dower source and turn on a rotarv Dositive displacement pump which delivers either acid or base to the fermentor. This pump is almost identical with that developed by M. J. Johnson (5). A I
timing device on the pump allows reagent delivery for any fraction of a minute desired during the actuation period. This minimizes overshooting and desired pH.
I
Performance and With the electrodes mounted in the flow assembly, a Beckman 8990-90
BLEEDER
50-GALLON
FERMENTOR SIGHT GLASS BECKMAN 4210 FLOW ASSEMBLY
BROWN "ELECTRONIK" RECORDER
,
BECKMAN MODEL
---
jnj
REDUCING VALVE
II PRESSURE REDUCING VALVE
AIR F I L T E R
BLEEDER
Figure 2. This pilot plant fermentor pH system has operated for 11/2 years without glass electrode failure VOL. 50,
NO. 9
e
SEPTEMBER 1958
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Figure 4. Unless controlled, foaming causes erratic behavior on the pH recorder chart Figure 3. The fermentor with external leg and related equipment. The external leg can be sterilized with the fermentor, or it can be isolated and sterilized separately
4
glass electrode has been sterilized 40 times for 45 minutes at 122' C. without failure. This was over a period of 6 months, and each sterilization was followed by continuous p H measurements for 5 days. At the end of this 6-month period, the reference electrode failed for reasons not yet determined. During 11/2 years operation, no glass electrode has failed because of sterilization; however, two have been broken in handling. This system has operated successfully with very thin water-like culture broths as well as extremely heavy, viscous culture broths. Some of the media have contained as much as 5% oil with no fouling of the electrodes. As noted by other workers ( 3 ) ,foaming caused erratic behavior of the p H indicator unless properly controlled (Figure 4). Some batches have been controlled as closely as 3 ~ 0 . 0 5p H units, while others were more difficult. Regulation of the pressure switches, the concentration of
reagents, the interval of reagent addition as controlled by the timer on the positive displacement pump, and the character of the fermentation all contribute to the sensitivity of the control. Response of the system was tested by adding a quantity of sodium hydroxide to a weak buffer in the fermentor. Twelve seconds after addition, the recorder reacted and in 17 seconds the new p H was recorded exactly. Charts were made by adding small quantities of acid to the fermentor a t 60-second intervals and controlling the p H with sodium hydroxide. Charts also were made by adding small quantities of base in the same manner and controlling the p H with sulfuric acid. I n both cases control was within rtO.05 p H units of the setting. The p H and yield data for two penicillin fermentations are shown in Figure 5. The fermentation for which the data are shown by solid lines had automatic p H control of the type described above. The fermentation without p H control, for which the data are shown
8.5
0
F
100
8,O
-I
80
P
7.5 I
60
7.0%
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a w 40
1
5
Acknowledgment
The authors wish to acknowledge the assistance of many members of the engineering department, Abbott Laboratories, in developing this p H control system. Literature Cited
(1) Barham, N.H., Smits, B. L., Trans. Kansas Acad. Sci. 37, 91 (1934). (2) Brown. W. E.. Peterson. W. H.. IND.ENG. CHEM.42,1769 (1950). (3) Deindoerfer, F. H.,Wilker, B. L., Zbid., 49, 1223 (1957). (4) Dworschack, R. G., Lagoda, A. A . , Jackson, R . W.,. Abbl. Microbiol. 2, \
-I W
bv broken lines, was set under otherwise identical conditions. The medium used in both fermentations normally causes a rapid rise in p H after 68 to 92 hours, with concurrent lysis and a drop in yield. The penicillin yield with pH control actually rises after the point at which the yield for the uncontrolled run drops. I n addition, even though lysis occurred in the pH-controlled run. product stability was maintained. The control was within k 0 . 2 p H units of the desired pH, and even greater sensitivity can now be accomplished routinely with this p H control system. I n addition to the penicillin fermentation and the artificial conditions noted above, erythromycin (Streptoqces erythreus), ristocetin (Nocardza lurzda), and gibberellin (Gzbberella fujikuroi) fermentations have been carried out successfully using this system. Satisfactory control has been obtained a t several p H levels between 4.5 and 7.5.
6.5
\ '\
20
YlELCi
5 .5 _
40
80
120
TIME I N HOURS
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INDUSTRIAL AND ENGINEERING CHEMISTRY
__
190 (1954). 15'1 \ , Johnson, M. J.. Univ. of Wisconsin,
Madison, 'Wis., private communication: (6) .Nelson, H. A., Maxon, W. D., Elferdink, T. H., IND. ENG. CHEM.48, 2183
(1956). (7) Wheat, J. A., Can. J . Technol. 31, 73
6.0
W
a .-
F i g u r e 5. P e n i c i l l i n yield with pH control actually rises after yield for uncontrolled runs drops
,
.
(1953). RECEIVEDfor review October 31, 1957 ACCEPTED June 9, 1958
