F. H. DEINDOERFER' and B. L. WILKER Merck Sharp & Dohme Research Laboratories, Division of Merck & Co., Inc., Rahway, N. J.
pH Control in Submerged Pure Culture Fermentations Bench -Scale Fermentor Studies Pure-culture fermentations of widely different composition and consistency have been carried put successfully at controlled pH levels
THE
numerous industrially significant fermentation processes in operation today require consideration of the common factors that affect product yield. The most apparent of these factors are temperature, oxygen supply, turbulence and mixing, nutrient concentration, and pH. All are interrelated to some degree: pH, however, often is dependent upon the others. I n many instances under optimum physical conditions and nutrient concentrations the resulting pH may adversely affect product yield. T o obtain maximum production, p H must be controlled during part, if not all, of the fermentation, usually by use of chemical buffers; choice of nutrients which, upon utilization, produce acidic or basic conditions; or addition of acid or base during the fermentation. One of the chemicals most frequently used for buffering fermentation media is calcium carbonate. I t has, however, a very limited p H range and is used only to avoid extreme acid conditions, such as those encountered in organic acid fermentations (77). Phosphates are often used, but the amounts required are generally too great where 4
must be controlled a t a constant level before a n attempt to maintain continuous fermentation can be made. A number of designs for controlling p H in fermentations have been reported (5, 7, 70-74, 76, 79). Some are capable of controlling pH in one direction only; others are limited to a nonviscous, slowly agitated fermentation. Most are unsuitable for industrial fermentations requiring rugged performance under violently agitated conditions and strict maintenance of a pure culture environment. The objective of this design, therefore, was a bench-scale apparatus which
sons,
Equipment
would permit a strict pure-culture environment, operate under mixing conditions encountered in many modernday fermentation processes, control the
.
rapid and extreme changes are occurring. An excellent example of nutrient utilization to produce desired p H conditions is addition of oils to penicillin fermentations (7). Addition of acids or bases during fermentation to maintain optimum p H has been reported by several authors. Thus, p H control over a wide range of p H levels is important in a number of fermentation processes employing bacterial, yeast, and mold cultures. The effect of p H on the rate of growth of Pseudomonasjuorescens and Saccharomyces cereuisiae was illustrated recently by Finn and Wilson ( 9 ) in a discussion of continuous fermentation. Because of its effect on the growth rate and enzymatic activity of microorganisms, p H 1 Present address, E. R. Squibb & New Brunswick, N. J.
process regardless of the direction of p H potential, and operate equally well for fermentation broths of various degrees of consistency.
30-Liter fermentor with electrodes in place
Vessel. A 30-liter stainless steel and glass fermentor, similar in design to the 5-liter fermentors described by Bartholomew, Karow, and Sfat (2) was used. The head was large enough to facilitate installation of special tapered adapters to hold the p H electrodes rigidly. A 6-inch turbine impeller was used to agitate 2o liters of fermentation broth, usually a t a speed between 250 and 500 r.p.m. This operating volume fills the fermentor to two thirds of its height, covering the bottom inches of the glass electrode and salt bridge under unsparged conditions. Air was introduced through a constricted-pipe sparger 11/2 inches below the center of the impeller disk at superficial air velocities of 30 to 90 feet per hour. pH Electrodes. A Beckman Type 42 glass electrode was sealed into the end of a length of glass tubing with Gelva V 2.5 resin (Shawinigan Chemicals, Ltd., Montreal, Can.). This permitted its extension into the fermentation broth. The tip of the tube was sealed to the glass stem of the electrode with Sauereisen cement (Sauereisen Cements Co., Pittsburgh 15, Pa.). T h e glass tubing was seated rigidly in the head of the vessel in a standard taper adapter and sealed in place with sterile paraftin wax. The calomel reference electrode made electrical contact with the fermenting broth through a specially constructed salt bridge, which was seated and sealed in an adapter similar to that used for the glass electrode. T h e salt bridgefermentation broth liquid junction was designed as shown, because preliminary experiments with yeast fermentations indicated this to be the most critical point in the pH-indicating function. The use of asbestos wicks sealed in glass, as VOL. 49, NO. 8
AUGUST 1957
1223
BOK
GELVA REBlM
SAUEREISEN SEAL
G L A S S ELECPROBE
Glass electrode design
in normal p H salt bridges, resulted in clogging of the junction by yeast cells and debris. The grind of the glass joint is important, because it must permit the required flow of salt solution, so that a good electrical junction exists. The joint was held in place firmly by a spring load. The salt bridge also contained a port for addition of saturated potassium chloride solution and a connection for an equalizer line to compensate for back pressure and avoid subsequent stoppage of flow through the joint. At high aeration rates, back pressures of 2 to 3 pounds per square inch gage developed in the fermentor. K GI PORT
INE
KCI I
The calomel reference electrode was seated in the top of the salt bridge. pH Indicator and Recorder-Controller. The indicating, recording, and controlling apparatus is illustrated diagrammatically with the fermentor and acid and base systems. The electrodes were connected via a terminal box and shielded cable to a Beckman Model R p H indicator. A temperature compensator located in the fermentor water bath was connected to the indicator in the same manner. The indicator was standardized by checking the p H of a sample lrom the vessel on another meter and adjusting the indicator to read at this pH. The indicator was connected to a Brown Electronik recorder-controller which continuously recorded pH and governed entry of acid and base to the vessel as required. Control was accomplished by two Mercoid switches located on the recorder-controller drum. One of these switches closed when the pH moved outside a preset control hand, and thus actuated one of two solenoidcontrolled hose clamps, permitting flow of either acid or base into the vessel to bring the pH back within the desired band. Control on one side or the other of the desired pH level resulted, depending on the pH potential of the fermentation at the time. As the Mercoid switches are set independently of one another, the control level could be allowed a wider band where close control was not critical. When fermentations caused intolerable pH levels in one direction only, pH was maintained above or below certain limits by use of only one of the Mercoid switches. Acid and Base Systems. Flow of acid and base was regulated by specially constructed solenoid hose clamps held in a closed position by a spring and opened when the solenoid was energized. Flow of neutralizing agents was restricted by capillary tubes to prevent overshooting the control band by too rapid entry into the vessel. The capillaries were enclosed in glass chambers which enabled the additions to be viewed as they were being made. Vessel and acid and base reservoirs were connected with rubber tubing. The reservoirs were maintained at vessel pressure via an equalizer line. They consisted of 1000-ml. graduated c) linders with standard-taper ground-joint tops, having their bottom ends drawn down to a diamrter small enough to attach rubber tubing Table I.
GROUND Q L A S S
jOlNT
Specially constructed salt bridge
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Process Baker’s yeast Cyanocobalamin 2-Ketogluconic acid Novobiocin Penicillin Prednisolone
Sterilization Methods
pH Electrodes. I n two designs (76, 79) suitable for most fermentations, the pH electrodes were mounted in the fermentor and sterilized by steam along with the fermentation vessel and medium. Experience with similar designs in these laboratories has demonstrated repeated short lives and failures of the eIectrodes, especially the glass electrode, after being subjected to steam sterilization. The inability of glass electrodes to withstand steam sterilization is substantiated, also, by the refusal of manufacturers to guarantee performance afrer exposure to such conditions. Because of the inherent difficulties encountered in steam sterilization, it was decided to sterilize the electrodes clremically and place them in the fermentor aseptically. Although ultraviolet irradiation had been claimed as an effective sterilizing agent for electrodes ( 7 4 , its poor penetrability discouraged its use. Because ethylene oxide involved special equipment for gas handling, it was not used. It has been reported as satisfactory for electrode sterilization ( 5 ) . Alter several chemical liquid disinfectants had been tested, a 0.5% solution of benzalkonium chloride (a high molecular alkyl dimethylbenzyl ammonium chloride, sold under the trade name of Roccal, Winthrop-Stearns, Tnc., New York 38, N. Y . ) was chosen as the sterilizing agent. The electrodes were exposed to a sterilized solution for 24 hours, wiped dry with a sterile cloth, and seated in place: the glass electrode in the adapter and the calomel electrode in the salt bridge, the latter having been sterilized in place in the fermentor. Salt solution for replenishment of the bridge during fermentation was steamsterilized separately. Fermentation Vessel, Medium,
and Acid and Base Systems The vessel containing the medium was steam-sterilized in an autoclave at 120’ C. for the required time. The empty acid and base systems were attached to and sterilized with the vessel. The salt bridge was sterilized in place in the fermentor. Immediately after sterilization was complete and the vessel had cooled, sterile paraffin wax was poured around the adapter joint to make a leakproof seal with the salt
Typical Processes Using pH Apparatus Type of Principal Medium Organism Yeast Bacterium Bacterium Actinomycete Mold Bacterium
Constituents Molasses Molasses Sugar, corn steep liquor Sugar, distillers’ solubles Sugar, corn steep liquor Lactalbumin digest
bridge. Acid and base were steamsterilized separately and added to their respective reservoirs aseptically.
ACID AND CAUSTIC RESERVOIRS
EQUALIZER LINE
Results and Discussion
*
1
Pure-culture fermentations have been carried out equally well a t numerous p H levels between 4.50 and 8.50 for processes ranging from very low viscosity bacterial fermentations to high consistency mold fermentations. Table I lists the variety of fermentations in which the pH apparatus was employed successfully. The novobiocin and penicillin fermentations were very viscous, yielding broth rigidities of close to 200 centipoises, yet their control was as good as the less viscous bacterial and yeast fermentations. Various types of -suspended solids were also present in the media listed, but they did not present a problem. A recorder chart from part of a run illustrating the performance obtained is shown in Figure 1. T h e frequency of acid and base additions can be noted by the saw teeth in the p H curve. Over the 24-hour period shown, the batch was held at p H 6.50 k 0.10 units. T h e p H potential of this fermentation was such that, during the first 4-hour period starting at 8:45 A.M., the p H was driving downward. For the next 8 hours, the p H drove upward, but much more slowly. About 8 : 3 0 P.M., the p H began driving downward again, becoming rapid within 3 hours and then slowing down. Fifteen additions of 2 N sulfuric acid totaling 132 ml. and 77 additions of 2 N sodium hydroxide totaling 507 ml. were made. The frequency and amplitude characteristics which comprise the saw-tooth shape of the controlled p H curve are affected by the concentration of acid and base used, the rate of flow of these neutralizing agents through the capillaries and into the fermentor, the setting of the Mercoid switches, and the p H potential of the fermentation. The first three factors are easily controlled. Figure 2 illustrates the €requency and amplitude characteristics obtained using varying degrees of control. ‘4 shows a portion of a run in which the control point was set a t p H 6.55 with a control band of 1 0 . 0 5 unit allowed. Thus, with a basic p H potential in the fermentation, control was obtained on the high side of the control point in a band only 0.05 pH unit wide. A wider band and the one more often employed is shown in B, where the control point was set a t p H 7.50 with a 1 0 . 1 0 pH unit band. This curve shows part of a fermentation in which p H potential changed direction twice within 2 hours. Control was always within the 0.10 p H unit band on either side of the control point. In C, close control was not critical. This fermentation had an
BOX
0 U BROWN RECORDER-CONTROLLER
BECKYAN pH INDICATOR
G
pH indicator and recorder controller assembled with fermentor and acid and base systems D. Salt bridge A. Solenoid hose cocks Flow chambers C. Calomel reference electrode
E.
extreme p H potential in the acid direction only. A control point of p H 7.00 with a band of 0.30 pH unit was used. A batch controlled a t p H 7.50, in which acid additions built up to as fast as one addition per minute, is shown in D. These additions were made within an 0.08 p H unit control band. The fermentation itself often presents another factor more difficult to controlfoaming conditions. Generally, two types of foaming conditions are encountered in aerobic fermentations. One type is chaiacterized by a rather stable and slow-rising head foam above the main fermentation broth; in the second the entire broth volume is flooded with air. Both conditions present problems, illustrated by the abnormal p H control shown in the two parts of Figure 3. In A, a head of foam during several portions shown on chart caused a lag in the response of the controller in stopping the addition of acid, as the acid had to drain down through the foam before it could change p H ; the limits of the control band were overshot in several instances. In each instance, the control became normal again after addition of antifoaming agent suppressed the foam layer. .4n attempt was made to compensate for the lag by narrowing the. control band, but large overshots still occurred. The second type of foaming condition caused the erratic behavior shown in B. Apparently, air pockets momentarily surrounding the electrodes caused the indicator to fluctuate widely. Here too, however, additions of antifoaming agent eliminated the adverse condition and control returned to normal.
E.
Glass electrode
F. Temperature compensator Ground wire
G.
After initial standardization of the p H indicator, very little drift occurred. The indicator was checked once or twice daily against a p H reading of a sample withdrawn from the fermentor measured on another p H meter. This was often unnecessary. Disagreements between meters seldom exceeded 0.05 and never exceeded 0.10 p H unit. pH Control in Production Vessels. I t would be desirable to have a glass electrode capable of withstanding steam sterilization before direct insertion of electrodes into production fermentors is attempted. The chemical method of electrode sterilization and aseptic insertion, although successful on a bench scale, is too hazardous for larger batches LEVER ARM JUMBO
HOSECOCK
34
r I
I S P R I N G LOAD LEAD TO
MERCOID SWITCH Specially constructed solenoid hose clamp VOL. 49, NO. 8
AUGUST 1957
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Optimum pH Range for Several Industrial Fermentations ReferProcess
pH Range
ences
Baker’s yeast 2,J-Butanediol Dextran sucrase Gluconic acid Itaconic acid Lactic acid Penicillin Torula yeast
4.5 7.0-7.6 6.5-7.0,6.75 6.0-7.0 1.8-1.9 5.4,5.7
(6) (19) (7, 12) (3) (16) (8, 1 1 )
7.0,7.4 5.0
(18)
( 4 , 10)
Brown, W. E., Peterson, W. H., Ibid., 42, 1769 (1950). Callow, D. S., Pirt, S. J., J . Gen. Microbiol. 14, 661 (1956).
Figure 1.
Typical DH recorder chart
possibly worth thousands of dollars. p H control could be applied routinely to batches where it has proved advantageous by inserting the electrodes in a small stream flowing continuously from the fermentor on either a recirculating or thieving basis. These methods, however. have disadvantages over p H measurements made directly in the fermentor. They offer additional operational problems. In the case of a recirculating stream a pump is necessary. For a thieving stream, continuous sterilization of the discharge is required. Where the medium contains a large amount of suspended solids, solids deposition in the external leg also becomes a problem. From a purely aseptic standpoint, additional process piping is undesirable. These methods offer analytical problems, when the pH of the fermentation is changing rapidly. During the time between actual p H measurement of a stream and its leaving the fermentor, large changes in p H can occur. Figure ZC, shows the pH changing 0.4 unit in less than 10 minutes in one fermentation, The oscillations which could result from p H control via an external leg might be too great for the range of
Figure 2.
Campbell, L. S., Can. Chern. Processing 37 (October 1953). Dworschack, R. G., Lapoda. A. .\.. Jackson, k. W., Afipl.-Mic;obiol. 2;
190 (1954). (8) Finn, R.K., Halvorson, H. 0..Piret, E. L., IND. ENG. CHEM.42, 1857 (1950). ( 9 ) Finn, R. K., Wilson, R. E.! J . Agr. Food Chem. 2, 66 (1 954). (10) Hosler, P., Johnson, M. J., IND. ENG.CHEM.45, 871 (1953). (11) Kempe, L. L., Halvorson, H. O., Piret, E. L., Zbid., 42, 1852 (1950). (12) Lakata, G. D., Afifil. Microbiol. 2,
control desired. Certain lag-compensating features could be built into the control system, but this introduces a problem not encountered with direct insertion of the electrodes in the vessel. Despite successful p H control in benchscale vessels, routine application in larger vessels undoubtedly will await the development of steam-sterilizable glass electrodes.
2 (1954). - ,. \ -
(13) Longsworth, L. G., MacInnes, D. A., J . Bacteriol. 29, 595 (1935). (14) Neish, A. C., Ledingham, G. A., Can. J . Research 27B, 694 (1949). (15) Nelson, G. E.. Traufler, D. €I., Kelley, S. E., Lockwood, L. B., IND. ENG. CHEM.44, 1166 (1952). (16) Nelson, H. A, Maxon, W. D., Elferdink, T. H., Zbid., 48, 2183 (1956). (17) Prescott, S. C., Dunn. C . G., “Indus-
Acknowledgment The authors wish to acknowledge gratefully the helpful advice and assistance of N. R. Trenner in the design and construction of the pH-indicating apparatus.
trial Microbiology,” McGraw-Hill, New York, 1949. (18) Reiwr, C. O., J . Agr. Food Chem. 2, 70 (1954). (19) Wheat, J. A., Can. J , Technol. 31, 7 3 (1953).
Literature Cited ( 1 ) Anderson, R. F., Tornqvist, M.,
Peterson, W. H.? J . Agr. Food Chem. 4, 556 (1956). ( 2 ) Bartholomew, W. H., Karow, E. O., Sfat, M. R., IKD.ENG. CHEM.42, 1827 (1950): ( 3 ) Blom, R. H., Pfeifer, V. F., hloyer, A. J.. Traufler. D. H.. Conwav.
H. F:, Crocker, C. K:, Farisoi; R. E., Hannibal, D. V., Zbid., 44, 435 (1952).
RECEIVED for review October 24, 1956 ACCEPTED March 26, 1957 Presented in part before Division of Agricultural and Food Chemistry, Symposium on Fermentation Process and Equipment Design, 130th Meeting? ACS, Atlantic City, N. J., September 1956.
Portions of pH recorder charts illustrating control characteristics A.
Control a t p H 6.55 i 0.05, narrow
bond
6. Control a t pH 7.50 & 0.10, usual
Figure 3. Portions of pH recorder charts illustrating effect of foam-
band
Control a t pH 7.00 band D. Control at pH 7.50 addition C.
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INDUSTRIAL A N D ENGINEERING CHEMISTRY
f 0.30,
wide
i 0.08, r a p i d
A. B.
Effect of head f o a m Effect of flooding-type
