Development of a Typical Aerobic Fermentation 0. G. WEGRICH
AND
R. A. SHURTER, JR.
Commercial Solvents Corp., Terre Haute, Ind.
D
URING the past decade, microbiological screening programs have been organized throughout the country to search for new antibiotics, vitamins, and growth promoting substances. Basic techniques and philosophies have been developed which are used to eliminate the known or undesirable products as soon as practical. From such screening programs, a few new products filter through to the pilot plant. The function of the fermentation pilot plant is t o scale up the fermentation from laboratory data and to produce larger amounts of the product for evaluation. As with laboratory screening programs, basic methods and philosophies have been developed for use a t the pilot plant level. In addition to the development of new fermentation processes, it is the function of the pilot plant to develop and improve processes already in production. A prime example of such a process
Figure 1.
improvement can be found in the history of the penicillin fermentation. In the Spring of 1944 a penicillin deep culture fermentation plant was put into operation based on broth titers of 50 units per ml. Penicillin was selling for $7.50 per 100,000 units of product. Today penicillin titers of about 2000 units per ml. of broth have been reported (5) using Penicillium chrysogenum, Wisconsin Q-176, and penicillin is selling for about 1.8 cents per 100,000 units (4). It is reasonable to assume t h a t titers in the range of 4000 to 5000 units per ml. have been obtained. The low price of penicillin today is the direct result of higher broth potencies, better recovery techniques, improved material handling methods, and plant expansion throughout the industry, The pilot plants within the fermentation industry have played an important role in the development of this remarkable achievement.
Laboratory Fermentors in Operating Position
1153
INDUSTRIAL AND ENGINEERING CHEMISTRY
1154
All Phases of Product Development Evolve through Pilot Plant When a fermentation process is introduced into the pilot plant it is first studied in small laboratory fermentors of about 5-gallon capacities. From data obtained in these small units the process is scaled up to 100-gallon fermentors. In these units, development of the fermentation process continues, and broth is produced for expanded recovery studies and product evaluation. Nest, the fermentation is moved into 2000-gallon units R-here scale-up data
Figure 2.
Vol. 45, No. 5
However, it is not always possible to obtain adequate laborntory information prior to the introduction of a process into the pilot plant. In many cases, management recommends that pilot plant development proceed before 1aho1,atoryinvestigations have been completed. Laboratory Fermentor Design. Initial investigitt ions in our pilot plant organization are effected in small aerated 12-liter laboratory fermentors such as those shown in Figures 1, 2, and 3. Figure 1 illustrates the ferment,ors in operating position in the bath. The bath is divided into t7.i.o sections one of which is shown. Temperatures on each side of the bath ma>- be maintained independently. Thus it is possible to run six fermentntions a t one temperature on one side and six at anot,her temperature on the other side of the bath. It.em 1 shows the tempemture recorder-controller. From this instrument, leads run to heaters located in the bath. Water is circulated throughout the bath constantly, and a thermostatic arrangement 'nieeds cold water into the circulation line if the temperature hccomes Too high. When the temperature drops, the heaters automatically bring t'he temperature up to the required point. Item 2 is a tachometer n-hich indicates the speed a t which a group of three fermentors is operated. Each group of three can be maint'ained a t independent speeds. The drives for the fermentors are located underneath the unit, A '/*-hp. motor furnislzes the power which is trarisniitt,ed through a silent chain drire, through a Vicker's hydraulic drive, and then through a gear system. Finally, through flexible shni'ts (item 4) the power is transmitted to the shaft of the ferment.or unit.
Twelve-Liter Fermentation Unit
are evaluated and verified. Large quantities of product ale produced and packaged for evaluation. Equipment and personnel are available in the pilot plant to carry on all phases of process development from fermentation through recovery, including the bioengineering and final product packaging studies. The methods and philosophies used in any one organization are by necessity governed by the equipment and technical personnel available. The object of this paper is to describe the methods and equipment that have been used by one organization to develop and scale up a n aerobic fermentation process. Prior to the introduction of a neiT- fermentation process into the pilot plant there are certain basic data that should be obtained from the laboratory. These data should include techniques of culture maintenance and inoculum development methods, recommended medium and suggested medium variations, and suggested aeration and agitation conditions. In the laboratory, the fermentation work is done in flasks using either rotary or reciprocal shakers. Some indication of the degree of agitation and aeration required can be determined by comparing results obtained on slow reciprocal shakers with those obtained on high speed rotary shakers. The biochemical picture of the fermentation should be described and supported with adequate data to illustrate potency curves, p H drift, carbohydrate utilization, and cell growth rate. Techniques for assay of these variables should be available and standardized, with some information on the accuracy and precision of the anal\-tical methods.
Figure 3.
Laboratory Fermentor H e a d
Item 3 shows a rotameter JT hich is used for rneasuiing air late% These rotameters allow measurements of flow up to 4.0 feet per minute average superficial air velocities a t 15 pounds per square inch gage. Item 5 is a header system from a batch stei~hzer located in another part of the building. The medium is sterilized in this vessel, blown through a sterile header, and then through a flexible line (item 6) into the inoculum port, When not in use, this entire system is kept undei qteam pressure, and excess steam is bled through a trap a t the end ot thP hath. Item 7 show:
May 1953
INDUSTRIAL AND ENGINEERING CHEMISTRY
TIME, HOURS
Figure 4. Biochemical Fermentation Picture, Laboratory Fermentor per Standard Operating Procedure P. chryrogenum, Wisconsin Q-I 16
reservoirs which are used for slow feeding of precursor or nutrient during fermentation. Figure 2 is a photograph of a 12-liter fermentation vessel. This vessel is a 5-gallon borosilicate glass jar. Usually, these fermentors are charged with 12 liters of medium. The vessel is 9.5 inches in diameter and is filled (12 liters) to a depth of about 10.5 inches. Item 1 shows the baffle arrangement. These baffles are mounted on a coiled strip of stainless steel and are the tank diameter. Item 2 shows two flat, six-bladed radial flow turbines (9). However, one turbine is frequently used in the work. Variously sized turbines from 3 to 6 inches in diameter permit investigation of this variable. Item 3 is a probe for automatic antifoam detection. Item 4 shows a ring sparger which delivers air downward to the bottom of the tank. Item 5 shows a stainless steel sample outlet which extends to the bottom of the fermentor. Figure 3 shows a close-up of a fermentor head, Item 1 is a n air filter which is used t o remove organisms from the air. It is packed with glass wool. Air leaving the filter flows through a line (Item 2) down into the sparger ring. Item 3 shows a well in which thermometers or thermocouples may be placed t o indicate the temperature of the fermenting broth. Item 4 shows a sight glass for observing the addition of antifoam, precursor, or nutrient. Item 5 is a solenoid valve which can be actuated manually or in conjunction with the automatic detecting device. Item 6 is a n antifoam reservoir. Item 7 is a combination inoculating port and safety valve. A stainless steel ball seated in a hemispherical smoothly ground cup moves up when the pressure in the fermentor exceeds 3 pounds. Excess air is vented through a fluted arrangement around the edge of the cap. Item 8 is a sample line. Item 9 is a vent line. Item 10 is a n electrode for the automatic foam detection system. Item 11 shows the agitator shaft. Use of Laboratory Fermentors. The function of t h e small equipment is t o produce batches of fermented broth of approximately 12 liters in size. This broth is then turned over t o t h e laboratory recovery group for the isolation of material which will be used for the continuation of product and biological phases of evaluation. During the fermentation, samples are removed a t regular intervals. These samples are assayed for potency of product, pH, carbohydrate content, and cell growth. The exact technique used for assaying these samples will vary with the product under investigation. From these fermentations, preliminary d a t a are obtained on the process under conditions of aeration and agitation. Larger quantities of broth are produced which in turn will yield
1155
more product for evaluation. Up to this point, very little experimentation will be done if yields have been comparable t o those obtained in shake flasks. Just enough experimental work is done t o develop and produce broth of reasonable potency. During the period of broth production a standard operating procedure (SOP) is established for the fermentation. A biochemical picture, based on the average of several runs made per the SOP for the process in the 12-liter fermentors is plotted. This plot serves a s a base line for evaluation of future experiments. Figure 4 shows a plot of the SOP biochemical picture for a penicillin process using Penicillium chrysogenum, Wisconsin Q-176. As product evaluation proceeds and the data indicate t h a t fermentation development should proceed, a n experimental plan is outlined which is designed to obtain additional d a t a for the development of the fermentation. Some of the variables which are usually investigated in the 12-liter fermentors are new cultures, agitation and aeration variations, medium variations, optimum p H level for the production of the product, and media sterilization techniques. Other variables and techniques can be evaluated as development of the process proceeds or a s data from the laboratory dictate. In general, the fermentation is evaluated in two phases which are the lag phase and the slope or rate of production (Figure 5).
As the lag phase is decreased and the rate of production is increased the factors responsible are incorporated into the SOP for that fermentation. Again a biochemical picture is made based on the average of several runs and a new SOP fermentation is established. As limiting factors are removed, the development process proceeds, producing higher potency broths for the isolation of product. If the product evaluation continues t o look promising the fermentation is moved into larger equipment. Product Evaluation Program Controls Extent of Work in Small Equipment As in the laboratory, it is not always possible t o complete the fermentation investigations in the 12-liter fermentors prior t o introducing the process into larger equipment. The extent of the program in the small equipment is controlled by the progress of the product evaluation program. If the product is “hot,” research management needs more of the material immediately, and it is necessary to move into larger equipment with data available from laboratory and small fermentor work. The fermentation is standardized in 100-gallon fermentors and
Vol. 45, No. 5
INDUSTRIAL AND ENGINEERING CHEMISTRY
1156
a n SOP biochemical picture is established as was done in the 12liter fermentors. The fermentation is held per the SOP to provide broth as uniform as possible for the recovery group. This is the most important job of the fermentation section at this time. Material is then supplied for a n accelerated product evaluation program. Fermentation variables investigated in the 100-gallon fermentors are usually factors that have been found to be limiting in the 12-liter fermentors and need to be verified in larger equipment. When the development of the particular process to be described here was investigated, properly designed equipment intermediate between the 12-liter and 2000-gallon fermentation vessels was not available. As a result, the investigation of the process proceeded directly from the 12-liter scale to the 2000-gallon level. There are a t least three disadvantages in proceeding in such a manner. First, extremely large quantities of material are not usually needed a t this stage of development for either recovery investigations or for clinical testing. Raw material costs are therefore unnecessarily high per fermentation, as the same data oftentimes might be obtained on a smaller scale. Secondly, in our own organization we are not yet confident that a scale up from about 3 gallons to 1600 gallons is justified. It is hoped that future xork will allow rapid scale up of this magnitude. Thirdly, most pilot plants have some definite limits to the number of 2000-gallon vessels that can be installed. Approximately twenty 100-gallon vessels can be installed in the space required by four 2000-gallon fermentors. The use of smaller vessels wherever possible frees
the larger equipment for final fermentation investigations and the production of large quantities of material for large scale recovery investigations and clinical work. There are circumstances, of course, which invalidate one or all of the above disadvantages. One such circumstance, the lack of proper intermediate equipment, has been mentioned. Because the investigations conducted in vessels of the 100-gallon size are similar in scope and methods employed, only those experiments conducted on the 2000-gallon scale will be described. The principles, the variables investigated, and the methods of measurement are equally applicable to either scale.
Intermediate Size Equipment Provides Basic Data for Final Scale Up Figure 6 is a schematic diagram of t h r 2000-gallon fermentation system. The fermentor proper is a nominally 2000-gallon vessel, Batch sizes of 1600 gallons are usually used. The inside diameter of the tank is 5.0 feet and the operating depth when 1600 gallons are charged is 11.85 feet. The vessel contains an agitation system which can be changed hy means of a variable speed drive to deliver up to 15 hp. The agitator proper is composed of three flat six-bladed radial flowturbines (9). The diameter of each turbine is 28 inches. The bottom impeller is located 21 inches from the bottom of the vessel, and the distance betm-een succeeding impellers IS 42 inches. The blade length of each impeller is 7 inches, and the blade width is 5.5 inches. The vessel contains four baffles, each 4
AIR PRESSURE REGULATOR
&1
AIR HEADER 5 dASH HEADER- BATCH 81 CONTINUOUS STERILIZERS
G WATTMETER NUOUS STERILIZER
BACK PRESSURE CONTROL VALVE
I I
AIR RATE b PRESSURE REGULATOR
I
MICROMAX IRIFICE
E 3
AUTQMATI( AIR RATE VALVE
F 4
i
I
!I
I
.
AIRYLTER
I
I
Figure 6.
f
DROP L I N E
Schematic Diagram of 2000-Gallon Fermentor
May 1953
INDUSTRIAL AND ENGINEERING CHEMISTRY
inches wide, which are located 1.5 inches from the wall of the tank. Located in another part of the building is a recording wattmeter which continuously records the total horsepower input to the motor. There are several lines leading into the top of the fermentor. One line leads from a batch sterilizer where a 2000-gallon batch may be sterilized and passed through a cooler into the fermentor. Tying into this line is another line which leads from a continuous sterilizer. When the continuous sterilizer is used, the mash is slurried in the batch sterilizer, is passed through the continuous sterilizer and cooler, and then into the fermentor. The top of the vessel also contains openings through which the inoculum is blown. In addition to the openings described, there are still further openings through which antifoam, precursor, and nutrients may be fed. When slow feeding of nutrients is desired, they may be fed either from the large continuous sterilizer or from a reservoir through the small continuous sterilizer located on the top of the vessel. Antifoam and precursor may also be fed through the other small sterilizer, sterilized continuously, and passed into the vessel. Rotameters located immediately upstream of the continuous sterilizer indicate the flow rate. The 2000-gallon vessels do not contain automatic temperature controlling equipment. However, the temperature is recorded continuously on a Micromax. I n addition, another manual thermometer well is provided near the bottom of the vessel. Air used during the fermentation enters the building from a plant source and is filtered through large carbon beds located in a central area of the plant. The air from these primary filters passes through a pressure regulating valve which throttles it to 24 pounds per square inch gage and into a n air header serving the fermentors. From the header, the air for each unit passes through a metering orifice and air operated flow control valve, through a secondary air filter, and into the fermentor through a sparger. The air flow rate is measured in terms of the drop in pressure across the calibrated metering orifice and is indicated on a recording chart a t the top of the fermentor in cubic feet per minute a t 14.7 pounds per square inch absolute and 70" F. The air leaving the fermentor passes through a n automatic back-pressure control valve in the vent line and thence down and out of the building. The back-pressure control valve may be set t o maintain the fermentor pressure a t any desired value by loading it with the proper combination of weights. The fermentor back pressure is transmitted to a recording instrument where it is continuously recorded. Air rates may be changed from 0 t o 150 standard cubic feet per minute, and fermentor back pressures may be varied from 0 to about 20 pounds per square inch gage.
Power Requirements Are Calculated and Equipment Specifications Checked Figure 7 is a log-log plot of horsepower versus agitator speed with superficial air velocities a s the parameters. These data were obtained when the 2000-gallon unit was calibrated. Calibration of a unit with a water charge and a t different air flows serves two purposes. First, equipment specifications can be checked in this manner and the over-all range of the equipment can be determined. Secondly, the proper baffling arrangement can be checked by looking at the slopes of these lines. I n the turbulent range, curves of these sort, when plotted, should have a slope of 3.0. It can be seen from these curves t h a t the slopes are not exactly 3.0, but they approximate this figure. Deviations from a slope of 3.0 probably indicate that our measurements still leave something t o be desired. I n addition, it'is possible to gain a n indication of the speeds a t which certain experiments are to be effected under varying conditions of air flow. The approximate speeds for the investigational conditions can then be set in advance without recourse to making trial runs each time to determine the average horsepower under
1157
which operations are effected. There are slight differences when penicillin or other fermentations are carried out versus the effects of a pure water charge. However, the differences are not great, and the relative differences in horsepower remain about the same. It might be interesting to go through the exact steps t h a t are taken in making such a calibration or in determining horsepower for a n actual fermentation. Besides the actual motor (which in this case is a 15-hp., variable-speed motor), a wattmeter and certain basic data are necessary before the information can become available. Actually the wattmeter is probably not the best instrument for use in determining horsepower. Torsion dynamometers or strain gages can probably be used with more precision and more accuracy. A wattmeter, we feel, is cheaper and seems to give results that are sufficiently precise for our purposes.
The operations required during the calibration are a s follow: 1. With just enough water in the fermentor t o cover the bottom step bearing, if one is used, the speed should be varied through approximately e ual stages from low speed to full speed. Wattmeter readings be taken at each of the stages and from these readings a no-load power curve for the system may be drawn. 2. After the no-load power curve has been drawn, the vessel should be filled with water to approximately the level which will be used during the fermentation. The motor speed is again changed in approximately equal increments and power readings are again taken at each of the stages. Care must be exercised during this work because if a motor is purchased which can deliver 15 hp. during aerated conditions, powers far in excess of the rated motor capacity will be developed with a water charge if no air is used. Therefore, this power curve cannot be investigated to much more than 10 or 20% over rated load for the motor and must be extrapolated the remainder of the distance. 3. After the foregoing data have been gathered, the process is repeated, using air. However, a t each stage air rates should be varied and after steady conditions are achieved, wattmeter readings should be taken and recorded with the appropriate air rate or superficial velocity. These investigations of air rate may be repeated at different fermentor head pressures.
.%auld
The data obtained may be treated as follows: 1. The average power drawn by the agitator motor during the run a t each stage must be determined from the wattmeter record chart. 2. Using a motor efficiency curve obtained from the manu-
1158
INDUSTRIAL AND ENGINEERING CHEMISTRY
facturer of the motor, the shaft or brake power delivered by the motor can be calculated. 3 . From the no-load curve for the agitator in question, the power drawn by the motor while turning the agitator with no beer in the fermentor may be obtained. 4. Again using the efficiency curve, the brake poaer delivered by the motor under no-load conditions is calculated. 5. The no-load brake horsepower is then subtracted from the brake horsepower at each stage, and the difference is the horsepower actually expended in agitating the liquid. 6 . These points may be plotted then on logarithmic paper, and the straight lines which result should have a slope which approximates 3.0 if turbulent and fully bafffed conditions exist. T h i s in essence is a description of the calibration. During the experimentation a slightly different method is used. T h e n the experimental design is formulated it uill be determined t h a t several different horsepowers and air rates will be investigated. From the previously plotted curves, the spproximate speeds a t different air rates and head pressures, which must be set on the motor t o obtain horsepowers in the region of those desired, can be determined. The experiment is then effected using those speeds, and for all experiments, say, a t 0.5 hp., the same speed under the same conditions of aeration and agitation will then be used. The actual horsepower input to the fermentor may be slightly different from t h e 0.5 hp. which was desired, but if increments of, say, 0.2 hp. are desired, these increments will be effected even though the absolute level may deviate slightly from the 0.5 desired For example, using such curves it may be found t h a t the horsepowers instead of being 0.25, 0.50, and 0.75 run something like 0.22, 0.53, and 0.72.
Figure 8. Section of Wattmeter Chart Showing Power Consumption Fluctuations during Foaming Tendencies
The wattmeter records continuously during the feimentation. I n addition t o the power information a s obtained from the wattmeter readings, foaming characteristics may be determined quite readily from visual observation of the chart. When foaming occurs, power consumption decreases. As soon as the necessary amount of antifoam is added, power consumption increases sharply and then gradually decreases a s foaming again has a tendency t o occur. Figure 8 is a photograph showing such power consumption fluctuations with foaming tendencies. Since growth is taking place during some of the stages of the fermentation and since viscosity and density characteristics are rhanging with the ability of the broth t o retain air, it is necessary to actually average the horsepower consumption over a run. I n many cases, it is found t h a t during 40 t o GO hours of the fermentation a relatively smooth curve will result especially if antifoam is added continuously or in small increments over short intervals of time. There are several ways of averaging the wattmeter record. The average operation b y whatever niethod is used,
I
I 0
I
I 20
40
Vol. 45, No. 5
I
I
I
100
120
I
I 60 80 T I M E , HOURS
(
Figure 9. Biochemical Fermentation Picture 2000-gallon Fermentor per Standard Operating Procedure
P. chryregenum,
Wisconsin 0 - 1 763 0.5 hp./lOO gallons
hoxever, consists essentially of evaluating the total kilowatt hours of electricity consumed by the motor and dividing this quantity by the running time in hours. Usually either graphical integration or numerical integration is used in the absence of a planimeter which is a n expensive instrument. I n the graphical integration method the power curve is divided into segments of equal length, such a s 1 or 2 hours, and the average ordinate for each segment is determined by eye. The average power readings for the individual segments are then added and divided by the number of segments considered t o obtain the average power for the run. I n cases where little or no foaming is encountered, this method is probably a s fast and accurate a s any other. Another method used is numerical integration. This method permits the evaluation of the area under a data curve from a series of numerical values read from the curve a t regular intervals thus avoiding the tedium and uncertainty of visual averaging. A number of formulas are available for numerical integration. The one t h a t best combines accuracy with simplicity, however, is known as Simpson’s one third rule (8). If Simpson’s rule is used over a 120-hour fermentation, it is recommended t h a t a t least 50 values be used for accuracy. This number of values was determined by analyzing wattmeter data from many runs over a period of time in the pilot plant. The average horsepower obtained by the foregoing methods thus represents the total power input including motor and no-load losses. The data are then treated in accordance with methods described previously t o obtain the actual horsepower input to the liquid for the run.
Superficial Air Velocity Formula Translates Air Rates from One Vessel to Another Agitation and horsepower studies are important because not only are nutrient and precursor gradients between liquid and mycelia reduced t o a minimum by proper agitation, but air is dispersed, dissolved, and delivered t o the mycelia through the action of the mechanical agitators. Several good papers (1,9,5, 7 , 10) are available in which the correlation between oxygen uptake rates or diffusion rates with such factors &s horsepower requirements, sparger design, and agitator design are discussed. Diffusion rates must also be correlated with fermentor potencies t o be of value, and the experimental techniques of measurement which seem most successful are rather specialized. On the other hand, it has been possible in several instances in our pilot plant to correlate fcmientor potencies with superficial air velocities and to
INDUSTRIAL A N D E N G I N E E R I N G C H E M I S T R Y
May 1953
translate such requirements to fermentation effected in variously sized vessels when the superficial air velocity is defined in a particular manner. The particular superficial air velocity formula which is used in this organization for scaling up rates of air flow from one vessel to another is:
qT v, = 0.0686 pzD2
14.58 VspzD2 =
T
where Ti, = average superficial linear air velocity (SAV), feet/ minute a t p z q = volumetric rate of air flow, standard cubic feet/ minute (14.7 Ib./sq. inch absolute and 0' C.) p l = pressure at exit of fermentor, Ib./sq. inch absolute (14.7 Ib./sq. inch plus gage pressure, Ib./sq. inch gage) p~ = effective pressure, Ib./sq. inch gage = pl plus (0.217) (feet) of operating depth above sparger outlet D = diameter of fermentor, feet T = absolute temperature of fermentation, K.
0 z
< *:/
n W
,//
400
40
;'
-----0.749
G R S E P O W E R / i 0 0 GALLONS
1159
Experiments in 2000-Gallon Equipment Establish Standard Operating Procedure Using the methods described for the measurement of power input by the agitator and for the calculation of air rate, fermentations were conducted in the 2000-gallon fermentors t o establish an SOP fermentation process for the production of penicillin. The conditions under which these fermentations were made \\-ere selected on the basis of information available. The culture used was Penicillin chrysogenum, Wisconsin Q-176. The inoculum consisted of vegetative mycelium equal to 8% of the operating volume of the fermentor. The medium consisted of corn steep solids, 3.5700; lactose, 3.5700; calcium carbonate, 1yo, monopotassium phosphate, 0.4%; corn oil, 0.25%; p H after sterilization, 5.1 to 5.3. A quantity of phenylacetic acid equal to 0.02.!17~ of the broth weight was added every 6 hours a s sodium phenylacetate starting a t 18 hours. The medium was batch sterilized at 121' C. a t 15 pounds pressure for 30 minutes. An average superficial air velocity (SAV) of 2.1 feet per minute at 27.2 pounds per square inch absolute effective pressure was delivered through a 2inch open pipe. The agitator speed was 92 r.p.m. which was calculated to deliver 0.5 hp. per 100 gallons of medium. An incubation temperature of 24" C. was maintained for the first 36 hours, then increased t o 26.5" C. for the remainder of the fermentation. The p H was controlled by the addition of 6% Alkaterge C in lard oil added at a rate to maintain the p H of the fermentation between 7 and 7.5 and t o control foaming. At the end of each run, the average horsepower input by the agitator t o the liquid was calculated from wattmeter records. The average input per 100 gallons of broth was based on the final volume of broth in the fermentor. The average horsepower input obtained based on wattmeter records was 0.497 hp. per 100 gallons of broth a s compared to the precalculated input of 0.5 hp. per 100 gallons based on calibration curves.
0 664
-.-- 00 439272
0
Air rates have been reported and used on a volume of air/ volume of beer basis. It can readily be seen t h a t the actual flow conditions existing in two fermentors of equal capacity and using the same volume/volume of air will be quite different if the units are of widely differing shapes. The scale-up procedures presently used in this organization are briefly as follow: 1. Superficial air velocities are calculated, using the appropriate formula shown, a t an effective pressure, p ~ . 2. Back pressures for the larger fermentor are chosen to equal the back pressures used in the standard fermentation. This choice is made in the absence of data t o indicate what pressures should be chosen. By choosing the back pressures t o be equal, instead of, say, the average pressures, a safety factor is introduced, since (all other quantities being equal such as bubble size, agglomeration tendencies, bubble rise, holdup, etc.) at all points in the large fermentor below a point equal to the depth of the original fermentor, the partial pressure of oxygen in the air will be greater. 3. Using the same average superficial velocities as were used in the small fermentor, the air rate necessary for duplication of the average velocity is then calculated. It can be seen t h a t the velocity at the bottom of the larger fermentor will be slightly less than in the small fermentor, and at the top i t will be slightly greater. If velocities were chosen t o be equal at the bottom of each fermentor, velocities should be greater through most of the large fermentor. If velocities at the tops of the fermentors were chosen to be equal, velocity through most of the large fermentor would be less.
T I M E , HOURS
Figure 11. Biochemical Fermentation Picture, 2000-gallon Fermentor per Standard Operating Procedure
P. chrvsosenum, Wisconsin
Q-176; 0.7 hp./l00 gallons
Penicillin titers were determined by biological assay, carbohydrate by the anthrone method and calculated a s lactose. T h e p H was followed with a Beckman G potentiometer, and t h e mycelial dry weight was determined by filtering 100 grams of whole beer and drying the mycelial mat to a constant weight. The data obtained from nine fermentations are plotted in Figure 9. A penicillin potency of about 1600 units per ml. of broth was obtained. Using this plot as a base line or control, a n exDeriment was de-
INDUSTRIAL AND ENGINEERING CHEMISTRY
1160
Vol. 45, No, 5
Comparison of Geometric Ratios for Different Sizes of Equipment . .
Table 1.
T = tank diameter D = impeller diameter H = liquid depth C = height of impeller off bottom Ratio 5 Gallon 1.9
T/D E/H
Einn $-: J / T
1.25 7.89
E L
= =
distance between impellers blade length
W = blade width J = baffle width 2000 Gallon
24,000 Gallon
2.13 0.30 0.5 1.27 6.71
2.73 0.26 0.5 1.23 7.62
One turbine in 5-gallon unit.
3 0
20
40
80
BO
100
120
TIME, H O U R S Figure 13.
Potency Curves for Standard Operating Procedure
Fermentation in 2000-gallon and 24,000-gallon fermenton
0
20
40
60
80
I00
120
TIME, HOURS Figure 12. Biochemical Fermentation Picture, 24,000-gallon Fermentor per Standard Operating Procedure
P. ehryrogenum,
Wisconsin Q-176; 0.7 hp./100 gallons
signed t o investigate the effect of horsepower input by the agitator to the liquid, All operating conditions for the fermentation were held constant except the speed of the agitator shaft. From the horsepoiTer calibration curves, agitator speeds were selected t h a t should give power inputs in the range of 0.325, 0.50, 0.65, and 0.75 hp. per 100 gallons of broth. In Figure 10, the penicillin titers obtained are plotted against time; these increased a s the power input increased. The actual range of power input obtained by wattmeter readings was 0.322, 0.407, 0.654, and 0.749 hp. per 100 gallons of broth. A t 0.749 hp. per 100 gallons oi broth, titers of 1975 units per ml. were obtained. With this information a t hand, it was evident that our standard operating fermentation was being limited by improper agitation. A new standard operating procedure for the fermentation was then established increasing the horsepower input by the agitator shaft from approximately 0.5 to 0.7 hp. per 100 gallons of broth. I n Figure 11,the new standard operating procedure biochemical fermentation picture is plotted. The rate of lactose utilization increased and more penicillin per gram of lactose charge was produced.
Production Rates in 24,000-Gallon Equipment Confirm Accuracy of Scale-up Methods The next step in our development program B-as to scale up the fermentation to 24,000-gallon production equipment. Aeration was scaled up by use of the formula in Figure 8. As the large equipment was not geometrically similar to the 2000-gallon units, the agitation scale up was not based on equality of Reynold’s numbers (9) but on equality of horsepower per 100 gallons of broth using agitating equipment of the same type but not geometrically similar. 4 comparison of a few of the geometric ratios is presented in TabIe I.
Medium ingredients, sterilization methods, inoculum development, pH control, temperature, etc., were held constant as per the final SOP fermentation (Figure 11)in the 2000-gallon fermentors. The air rate was held a t an average superficial air velocitv of 2.1 feet per minute. The air rate used was 430 standard cubic feet per minute (TO” F., 14.7 pounds per square inch absolute) at a head pressure of 10 pounds per square inch gage. If this air rate had been scaled up on the basis of volume of air per volume of medium, the air rate selected for the 24,000-gallon fermentor would have been 1125 cubic feet per minute. Figure 12 s h o w the SOP biochemical picture obtained when the data taken from the first nine runs made in the 24,000-gallon fermentors are plotted. The average penicillin titer for the nine runs was about 2000 units per ml. after 120 hours incubation. Over 9570 of the total penicillin produced was benzylpenicillin (penicillin G) ( 6 ) . It is evident from the data presented in Figure 13 that the empirical methods described can be used for the scale up of pilot plant data to production size equipment even though geometric and kinematic similitude of the two fermentors is not exact. ThcA lag phase of the potrncy curve is a little shorter in the 24,000gallon units, However, the rate of penicillin production is about the same in both fermentors.
Acknowledgment The authors are indebted to Mr. Paul Greenman for his assistance in the preparation of photographs and graphs.
Literature Cited (1) Bartholomew, W.H., Karow, E. O., and Sfat, A f . R., IND. ENG. CHEM.,42, 1827 (1950). (2) Bartholomew, W.H., Karow. E.O., Sfat, M. R., a n d Wilhelm, R. H., Ibid., 42, 1810 (1950). (3) Brown, W. E., and Peterson, W. H., Ibid.,42, 1769 (1950). (4) Chern. Eng. X e z o s , 31, NO. 4, 383 (1953). (5) Cooper, C. &I., Fernstrom, G. A , , and Niller, S. A,, IND.ENG. CHEM.,36, 504 (1944). (6) Craig, J. T., “C14 Isotope Dilution Analysis,” unpublished. (7) Hixson. A. W., and Gaden, E. L.. Jr., IKD.ENG. CHEM.,42, 1792 (1950). (8) Iielson, A. L., Folley, K. W., a n d Borgmau, W-.M., “Calculus,” p. 266, Boston, D . C. Health and Co., 1942. ( 9 ) Rushton, J. H., IND.ENG.CHEW,44, 2931 (1952). (IO) Wise, W. S., J . Gen. M i c r o b i d . , 5 , 167-77 (1951). .‘iCCEPTED March 2, 1953. Presented before the Symposium of Microbiology, Section P, A . A . A S. Meeting, December 27. 1952.
RECEIVED for review February 11, 1953.
