September 1950
INDUSTRIAL AND ENGINEERING CHEMISTRY
of 3% distillers’ dried solubles with 2% wheat bran in the propagation medium also shows a beneficial effect. Under the most favorable conditions thus far, the fermentation of corn mash converted with submerged fungal culture can be completed in 42 hours instead of the usual 60 hours. The secondary fermentation was found t o follow the course of a firseorder reaction. There is a better correlation between the velocity constant, K,and the maltase activity of the fungal culture than with a-amylase or limit dextrinase activity. LITERATURE CITED
Adama, S. L., Balankunr, B., Andreasen, A. A., and Stark, W. H., IND.ENQ.CHEM.,39,1615 (1947). Andreasen, A. A., Am. Brewer, 82, No. 4,30 (1949). . Back, T. M., Stark, W. H., and Scalf, R. E., Anal. Chsm., 20, 56 (1948). (4) Back, T. M., Stark, W. H., and Vernon, C. C., IND. ENQ.CHBM., 40.80 (1948). ( 5 ) Corman, J., and Langlykke, A. F., Catsal Chem., 25, 190 (1948). (6) Foth, G., “Handbuch der Spiritus-Fabrikation,” Berlin, Paul Parey, 1929. (7) H o p k i ~R. , H., Advances in Enwmol., 6, 389 (1946). (8) Hopkina, R. H., Dolby, D. E., and Stopher, E. G., Wallsratain Labe. Cotnmuns., 5, 125 (1942).
1789
Leibowits, J., 2.phyeiol. Chem., 149, 184 (1925). Leibowits, J., and Mechlinski, P., Ibid., 154, 64 (1926). Le Menae, E. H., Corman, J., Van Lanen, J. M., and Lsnglykke, A. F., J . Bad.,54,149 (1947). Le Mense, E. H., Sohna, V. E., Corman, J., Blom, R. H., Van Lanen, J. M., and Langlykke, A. F., IND.ENQ.CHEM., 41, 100 (1949).
Lippa, J. D., Roy, D. K., Andreasen, A. A., and Kolachov, P., Abstracts of Papers, 114th Annual Meeting, AMERICAN CHEMICAL SOCIETY, p. 14A, 1948. Meyer, K. H., Advances in Colloid Soi., 1, 143 (1942). Myrblick, K., A d v a m Curbohydrate Chem., 3,251 (1948). Sandatedt, R. M., Kneen, E.,and Blish, M. J., Cereal Chem., 16,712 (1939).
Schwimmer, S., J . BioZ. Chem., 161,219 (1945). Somogyi, M., Ibid., 160,61 (1945). Stark, I. E., and Somogyi, M., Ibid., 142, 579 (1942). Stark, W. H..Adams. 8. L., Scalf. R. E.. and Kolachov. P.. IND. ENQ.CEEM.,ANAL.ED.,15, &3 (1943). Thorne, C. B., Emeraon, R. L., Olson, W. J., and Peterson, W. H., IND.ENG.CHEM., 37, 1142 (1945). (22) Tsuchiya, H. M., Corman, J., and Koepsell, H. J., Abstraota of Papers, 49th General Meeting, 80oiety of American Bacteriologists, 1949. (23) Vola, G. W., and Caldwell, M.L.,J.BioE. Chem., 171, 667 (1947). REaEZVED
February 23, 1950.
Sterile Air for Industrial Fermentations W. H. STARK Vickers-Vulcan Procam Engineering Company, Ltd., Montreal, Canada C. M. POHLER The Vulcan Copper & Supply Company, Cincinnati, Ohio
Air sterilization is a major problem in many industrial fermentations. When reciprocating compressors are installed and operated as described, sterile air is delivered by the compressor, and the need for the customary carbon or glass wool filters is eliminated. The development and use of this method is discussed as well as the economic factors to be considered in selecting air compreesion and sterilization systems over a wide capacity range.
T
HE
*
increasing w e of aerobic fermentation procesw on a commercial scale for the production of antibiotics, vitamins, enzymes, and other commodities has necessitated the develop ment of systems which will deliver sterile air at a high rate. The initial investment and the operating costs are substantial and warrant careful study. Furthermore, a poorly designed or improperly operated system may result in financial loeses of considerable magnitude. The criteria of the ideal sterile air system may be stated as follows: 1. Complete elimination of all viable microorganisms 2. A high degree of reliability 3. Ease and simplicity of operation 4. Minimum capital and operating costs Filtration through cotton, glasa wool, or carbon, and various scrubbing systems have been used. Glam wool and carbon filters are in extensive use and are perhaps the most popular methods for the production of sterile air in industry. Terjeaen and Cherry (3)reported reaently on the results of a series of careful investigations on the filtration efficiency of glam wool and slag wool. Aside from this report little published information exists, probably because of the rapid expansion of the fermentation industry. It is probable that each organization that has been faced with the problem has worked out its own solution on an empirical basis. This paper describes heat-of-compreasion systems for air
sterilization and presents a brief discussion of some of the economic factors to be considered in the selection of an air sterilization system. The experimental work was done a t Azucarera Cooperati valefayette, Arroyo, Puerto Rico, and at Joseph E. Seagram & Sons, Inc., Louisville, Ky. AIR STERILIZATION WITH HEAT OF COMPRESSION
There have been no previous reports in the literature on the use of heat of compression to sterilize air. The first known commerical installation is one that was made by Langlykke ( I ) at the Lafayette butyl alcohol plant in Puerto Rico. This unit was installed to provide sterile air for fermentor, seed tank, and line cooling. It is illustrated in Figure 1. The installation is straightforward and consists of a water-cooled reciprocating compressor designed to operate at 100 pounds per square inch gage. The water-cooled unit was an old compressor and was replaced after a short period of time with an air-cooled unit. The latter was selected in the interest of obtaining higher discharge temperatures than were possible with the water-cooled unit. The conventional air inlet filter has been replaced with a Winch pipe, approximately 55 feet high, which is packed with chain. A tee with a safety valve and a main line cutoff valve waa installed in the air discharge line as close as practicable to the cylinder head. The valves, discharge line, and the first receiver in the system were insulated to maintain high air temperatures for aa long a time M posaible. This system was operated as follows:
The unit was sterilized with steam at 100 pounds per square inch age for 30 minutes or steam at 15 pounds per square inch age for 2 hours on the downstream side of the main cutoff valve. %he cornpressor waa started and was run for a proximately 30 minutes, with the air discharged through the reIef valve. The heat so generated was relied on to sterilize the compressor and the short pipe aonnection between the cylinder head and the msin valve. As won as the steam was turned off, the main
INDUSTRIAL AND ENGINEERING CHEMISTRY
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n I1
I
COMPRESSOR
RECEIVER
Figure 1. Air Sterilization by Compression-Original Installation
valve was opened and compressed air was admitted to the receiver. Every 24 hours 5 ]gallons of lubricating oil containing 5 % phenol was poured down the air inlet at a time when the compressor was not operating. The oil was drained from the bottom of the inlet leg and collected for re-use. Since compressor operation was intermittent, this presented no operating problem, and the chain filled leg also served to draw inlet air from the higher levels, thus reducing the load. As long as the pressure in the receivers remained above 40 pounds per square inch gage the compressor might be shut down and restarted; if provision was made for 30 minutes' operation through the relief valve to resterilize that part of the system before again admitting compressed air to the receivers. When the demand for sterile air was great, for instance wh'en cooling the fermentors, the pressure in the receivers occasionally dropped to as low as 35 pounds per square inch gage. This system could be operated for 20 to 30 days without resterilization, and frequent checks of the condensate that accumulated in the receivers showed no evidence of microbial contamination. There was no evidence of contamination in the butyl alcohol plant which could be attributed to the air supply, with a single exception when the compressor was overlubricated and the valves became badly fouled. The system was installed in 1942 and was known to be operating in 1948. Although it is recognized that the requirements with respect t o air sterility are more stringent with aerobic fermentation processes than where sterile air is used only for cooling fermentors and lines, as in a butyl alcohol plant, modifications of this system have been used successfully in aerobic fermentations. =AIR
INLET
-
STERILE AIR
COMPRESSOR
RECEIVER
Figure 2. Air Sterilization by Compression-Modified Installation
One of the authors had personal experience with the described unit in Puerto Rico; this led to a decision to modify the system for pilot plant and semicommercial work with aerobic fermentations a t Seagrams. The modified installation is shown in Figure 2. The principal difference is the elimination of the oil-phenol chain-packed scrubber on the upstream side of the compressor
Vol. 42, No. 9
and the absence of insulation on the receiving tanks. The former eliminates the danger of entraining quantities of disinfectant which might reach inhibitory concentrations due to absorption in the fermenting medium. The ordinary air filter for compressor protection was the only filter used on inlet air. Two installations of this type have been used, one with a 100-cubic foot-perminute, 100-pound-per-square inch-gage oil-lubricated watercooled compressor, and the second with a 200-cubic foobper-minute oil-lubricated water-cooled 100-pound-per-square inch-gage compressor. Both systems worked well, and each was used over reasonably long periods of time. Air to the compressor was room air a t approximately 60" to 70" F. The first installation was used for pilot plant studies on the production of 2,Sbutylene glycol with 8000-gallon and 1000-gallon fermentors. The air flows ranged from approximately 0.03 to 0.1 volumes of air per volume of liquid per minute. Later the same unit was used to produce an Aspergillus oryzae supplement for the continuous alcoholic fermentation of acid hydrolyzed mash aa reported by Ruf et al. (2). The mold supplement was grown in a 300-gallon fermentation with an aeration rate of 0.25 volume of air per volume of liquid per minute. A large number of ordinary aeroscope tests were run on the air supply during the course of both of the development programs with no evidence of air contamination, despite the fact that the inlet air was from the basement of the pilot plant building and must have had a reasonably high load of contaminants.
1
TURBO-COMPRESJOR e 3 0 LB./SQ.lNCH 5,0PO~---41T0 F.
- -.- . . I -30,000----39PF, 1O4OO0---4OF m-,__ m - - - . ~ a eF. P
Ir-'
REDUCEiO%FOR UNIT5 BELOW 50 HP. U5CASGlVLN FORUNITS 50-25OHP. INCREASE SXFOR UNITS 250-500HR INCREASLIO~FORUNITS 500-125OHP lNLtT AIR TEMPERATURE- 70'F.
I I
400
Y
350 ul
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200 . .
LB/SQ.
Figure 3.
INCH
Discharge Air Temperatures for Reciprocating Compressors
Mold culture so produced in a semicontinuous or intermittentbatch manner was used as a supplement (7.5 to 11.0% by volume) in a continuous fermentation process with runs varying from 4 days to 9 days in length. The fact that these runs were conducted without contamination is good evidence that sterile air was delivered by the compressor. Here again the pressure in the system was not allowed to fall below about 40 pounds per square inch gage at any time and usually was carried a t 100 pounds per square inch gage with excess air exhausted through the relief valve. The second installation, which has been mentioned, was used in semicommercial fermentation runs on microbial amylase production with Aspergillus niger. Fermentations of 6000 gallons were aerated at a rate of 0.2 volume of air per volume of liquid per minute. Approximately 25 fermentations of this siae were run in this system. Aeroscope and fermentation results again confirmed the efficacy of air sterilization by compression under the described conditions. There is no doubt that sterile air for commercial aerobic
September 1950
INDUSTRIAL AND ENGINEERING CHEMISTRY Table I.
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Effect of Inlet Air Temperature on Discharge Air Temperatures Discharge Air Temperature, O F. Inlet air, O°F. Inlet air, 70* F.
Disoharge Pressure Lb./Sq. Inch Gage' 80 40 60 60
80
100
of the air may also be a factor. Consequently, turbocompressors which may have air discharge temperatures as high as or higher than reciprocating compressors, will not necessarily produce sterile air. It would be of value to conduct a systematic study of air sterilization by heat of compression under standard conditions, varying the type compremor, the discharge pressure, and inlet air temperature, with a system of air contamination and air sampling similar to the one described by Terjesen and Cherry (9).
Figure 4. Power Requirements for Single-Stage Compressor at Several Discharge PraS8UrO8
fermentation may be produced by compression alone under proper conditions without resorting to filtration. This has certain advantages: first, the simplicity of the system lends itself to ease of operation and will therefore tend to reduce the probability of operational failure; secondly the cost of filter systems is eliminated; and thirdly, over certain capacity ranges t h i system may be the most economical. These advantages might be increased by further work since no exact study of this method has been reported, nor have the authors had rtn opportunity to conduct a development program designed to yield the necessary data for adequate evaluation and process design. It is known that sterile air is produced in an oil-lubricated water-cooled compressor with an inlet air temperature of 60' to 70' F. and a discharge pressure of 100 pounds per square inch gage. There is some evidence that lower discharge pressures and consequently lower air discharge temperatures may be effective since units have been operated on occasion at aa low a pressure as 40 pounds per square inch gage. The design of these compressors is such that cylinder pressure and temperature are governed by the back pressure of the system. The effect of air inlet temperatures cannot be ignored, aa shown in Table I. If the inlet air temperature is 0" F., there is a substantial drop in discharge air temperatures as compared with the discharge temperatures obtained with 70" F. inlet air. It may be assumed that if the back pressure is maintained at 90 to 100 pounds per square inch, the heabof-compression system will produce sterile air when the inlet air temperatures are as low as 0' F. This is based on the observation that with 70' F. inlet temperatures] a discharge pressure of 40 to 60 pounds per square inch has been found adequate to sterilize air. Since the contaat time at the high temperature ie extremely short, there is some difficulty in reconciling this method of air sterilization with conventional laboratory practice and theory on dry sterilization. The presence of oil vapor and oil mist may greatly accelerate the sterilization rate, and the moisture oontent
A graph of air discharge temperatures is presented in Figure 3. These are subject to some variation dependent on the temperature of the cooling water and other factors. However, the data are representative of normal average operating conditions. The discharge temperature falls off rapidly with a decrease in pressure, and for a given pressure the temperature increases appreciably with capacity. Turbocompressor discharge temperatures are also presented. These exceed the 370" F. temperature obtained at 100 pounds per square inch gage with a single-stage compressor but may or may not result in sterile air. Twostage units discharge air at relatively low temperatures, and i t is most unlikely that the exit air is sterile. It is difficult to make a general economic evaluation of the h e a t of-compression method of air sterilization as compared with filtration systems such as activated carbon and glass wool. The initial and operating costs of the compressors are major factors in any type system, and an economic comparison involves a comparison of compremor costa for any given capacity, to which the cost of filters must be added if they are required. The selection of the system with the maximum over-all economy will depend on several factors such as the volume of air required, steam or electric costs, and the degree of variation in air demand. An air system with low pressure drop filters does not require compression beyond 30 pounds per square inch gage. Con13.
f2 11
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\
I
I
I
I
I
I
I
S 6
7 6
5 4
3
" 00
5
10
15
20
25
30
THOUSANDS OF CU. FT/MIN?ACTUAL
Figure 5. Approximate Cost and Horse ower Requirement8 for Turbocompre88or8, 30 Founds per Square Inch
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INDUSTRIAL A N D ENGINEERING CHEMISTRY
sequently, power for compression to higher pressures for the purpose of sterilization must be offset by savine in investment, hbor, and materials and a reduction in lost production, if any. Figure 4 presents the approximate brake horsepower per 100 cubic feet per minute a t several pressures for single-stage units. Air at 30 pounds per square inch gage requires one half the power required at 100 pounds per square inch gage. Where power costs are low, heat of compression may prove to be the most economical method unless sufficient air is required to justify the use of turbocompressors and filters. As shown in Figure 5 these units require approximately 10% more power than singlestage reciprocating compressors at 30 pounds per square inch gage. However, the installed cost a t 10,OOO cubic feet per minute actual capacity is 20 to 25% less than reciprocating units and roughly 50% less a t 30,000 cubic feet per minute. Turbocompressors are not suitable where the total demand i s less than 5000 cubic feet per minute, and the maximum savings in investment are not realized unless 25,000 to 30,000 cubic feet per minute of air are required. Furthermore, thase units have leas flexibility than reciprocating compressors. Based on present information, it is believed that reciprocating compressors delivering air at 100 pounds per square inch gage
Vol. 42, No. 9
without filters will be the most economical for air rates up to about 2000 and possibly to 5000 cubic feet per minute. Singlestage units at 30 pounds per square inch gage equipped with suitable filters may be more economical for the range from 5,000 to 20,000cubic feet per minute and turbocompreasors with filters above this range. These are generalizations, and it must be emphasized again that several factors must be considered in the determination of the most economical unit for a given set of conditions. A special study is required for any specific installation ACKNOWLEDGMENT
The authors wish to acknowledge the assistance of the IngersollRand Company, which provided many of the data on compressors. LITERATURE CITED
(1) Langlykke, A. F., personal communication. (2) Ruf, E. W., Stark, W. H., Smith, L. A., and Allen, E. E., IND. ENO.CHEM.,40, 1154-8 (1948). (3) Terjesen, S. J., and Cherry, G. B., presented before the Northwestern Branch, British Institution of Chemical Engineere (Oct. 11, 1947). RECEIVED May 3, 1950.
Oxygen Transfer in Submerged Fermentation ARTHUR W.
HIXSON AND ELMER L. CADEN, JR, Columbia Unioersity, New York 27, N . Ye
T
HE various microbioWhen the transfer of oxygen supplied by aeration in are of practical significance. logical processes collecsubmerged fermentation is treated as a series of rate They are first static surprocesses, quantitative expressions (oxygen transfer face growth in flat, shallow tively called i n d u s t r i a l equations) for each step can be developed. The physical fermentations comprise one pans with high surface to of the oldest branches of absorption of oxygen is shown to be a function only of volume ratio, and secondly, the chemical industry. Rethe design and operating characteristicsof the equipment submerged in tanks cent developments have and the physical properties of the medium. The experfemploying forced aeration caused greatincreases in the mental determination of oxygen absorption coefficients with or without mechanin a small laboratory fermentor, designed for this study, value of the materials proical agitation. Forced is described. A voltammetric method for determination duced but, despite these adaeration techniques have of the instantaneous concentration of dissolved oxygen, v a n c e s , fermentation rebeen extensively studied using the dropping mercury electrode, is described. mains essentially an art. Variations in the calculated absorption coefficients over in connection with yeast the course of a single fermentation are ascribed to changes propagation and a and Lee (I1) have discussed some of the reain the absorbing phase. The absorption coefficient is review Of the equipment correlated with aeration rates for different systems of and Practices used h a m~ for this have agitation and air dispersal giving relationships whi& aid been Presented bY d e drawn attention to the need in predicting the most suitable aeration systems for Becze and Liebmann (3). for fundamental studies of various phases of the gendifferent processes. These authors have era1 fermentation process pointed out the concentration on proper technology from a combined biologicalrather than on fundamental studies of the biological requireengineering viewpoint. It is toward this end that the present etudy is directed. ments for oxygen and the efficiency of methods for supplying it. One phase of fermentation technology well suited to treatSubmerged fermentation methods are now almost universal in the development of new antibiotics and in practically all other ment by “bioengineering” methods is the problem of oxygen fermentations of industrial significance. The provision of an upp ply in aerobic processes. Methods for the design of fermentation equipment and for the evaluation of its performame oxygen supply adequate to meet the metabolic requirements of will be greatly improved by increased knowledge of the factors the organism employed is absolutely essential to the successful use of these techniques. There is, however, very little quantiaffecting oxygen transfer in those systems requiring some degree tative data on this problem in the literature, and that which h of aeration. Aeration, taken in the general sense to mean the provision of available is restricted to the system from which i t was obtained. an adequate oxygen supply, is required in some degree for all The general procedure has been to relate the yields of product, aerobic processes. Many methods for providing sufficient or sometimes metabolic rates, to the superficial aeration rateoxygen for growing cultures have been proposed but only two volumes of air per volume of culture in unit time-with no
