ENGINEERING A D V A N C E S IN FERMENTATION PRACTICE tions, as it is easier to control and uses the raw materials more efficiently. Of major importance to the operation of continuous cultures is the influence of foreign organisms. These may be external contaminants or mutants. A fast-growing foreigner will quickly displace the original population. This problem is serious but not beyond solution. The case may arise where multistage continuous operation for cell propagation is advantageous. The optimum number and relative size of stages must be chosen by economic considerations. Frequently an organism will grow a t maximum rate until the substrate is nearly exhausted. I n this case it is hard to imagine an advantage for more than one stage. I t may sometimes be desirable to recycle a fraction of the organism being produced, especially when the substrate concentration available is low-for example, in sulfite waste liquor. Recycling permits a higher population of cells and thus higher productivity. A two-phase system, where the organism is held in the tank by a supporting matrix of twigs, shavings, glass wool, etc., is similar in theoretical relationships to the recycle case.
Product Formation While product formation and growth may be similarly affected by environ-
mental conditions, especially in growthassociated systems, it is more likely that conditions optimal for one will be far from optimal for thd other. T h e conditions in a single-stage continuous fermentation for product formation can only be a compromise. I t is, therefore, to be assumed that at least two stages should be employed and that conditions in the first stage should be chosen for most rapid growth, in the second stage for most efficient product formation. The procedures for design of such processes from batch data have been worked out only for the growth-associated case. Calculations similar to those used for cell production may be applicable, but experimental verification is wanting. Many people are interested in making continuous fermentation work. Work it does for the growth of nonfilamentous organisms. The advantages of productivity, not to mention uniformity, ease of control, and so forth, are so great that batch procedures need hardly be considered. The difficulties from contamination and mutation may have effect, but it seems possible to minimize these. T h e growth of filamentous organisms is also most efficiently accomplished (theoretically) in continuous culture; less so than for the yeast and bacteria, perhaps because of the longer generation times that are usually encountered. An additional trouble here is that the
filamentous material is prone to plug up the works. Most of us have an eye to using continuous methods for product formation. The difficulties here multiply. We are probably faced with multistage operation and more equipment problems. Furthermore, there is less to be gained. The product formation sequence of reactions cannot be speeded up as much as cell production. Growth is an autocatalytic process and continuous culture maintains the catalytic agent at its maximum level at all times. I n non-growth-associated production formation, however, it makes little basic difference to the rate whether the cells were grown continuously or batchwise. I n other words, we must not expect large improvements in productivity. We may gain in ease of control, uniformity, and labor costs, but we must face the problems of handling filamentous organisms and the hazards of contamination and mutation. These hazards may be especially difficult with the slowgrowing organism frequently used in this type of fermentation. T h e future of this aspect of continuous fermentation, while not rosy, will surely be interesting. We need more thoroughly considered theory and especially more supporting data.
W. D. MAXON The Upjohn Co., 301 Henrietta St., Kalamazoo 49, Mich.
Control Application in Fermentation Processes
IN
THE development of current fermentation processes, various types of automatic instrumentation have become standard in industry. These include temperature, air flow, and pressure, antifoam, and p H control as well as gas stream monitoring devices. Because most industrial fermentations, as practiced today, are batch systems, control of a function such as p H may be difficult, since the amount of acid or base required to maintain a constant p H can increase exponentially with time. However, when continuous processes come into common usage, this part of the control system will be simplified, because the amount of acid or base required per unit time will be constant. O n the other hand, continuous processes will require instrument systems that can operate continuously and without failure for thousands of hours, as well as aseptic equipment construction, a problem unique in the biological field. Laboratory systems have maintained “pure” strains in continuous culture for over 100 days (3).
This problem of asepsis prevented the use of automatic p H control in industrial scale fermentations until recently, because no p H electrodes were capable of withstanding repeated steam sterilization. Because of this inherent difficulty several authors described flow-type control systems (2), but with new electrodes now available the system of choice would be an immersion system. Within the past several years, two companies have been able to supply steam-sterilizable electrodes, and p H control is now feasible industrially. Automatic antifoam control is now commonplace in industry ; Bungay, Simons, and Hosler discuss the field adequately (7). A number of the antifoam control systems described in the literature did not use a low voltage high level probe, and a definite electrical shock hazard to personnel would exist in its use. Other more complex control in fermentations has not been studied outside of the laboratory as yet, mainly because of the inadequacies of proposed systems.
Dissolved oxygen in a broth may be related to oxidation-reduction potentials (7) or to exhaust gas composition (6), but there is often no simple relationship to dissolved oxygen. Automatic nutrient control, using such measurements as refractive index, has not proved practical in batch systems, but may be useful in continuous systems. Little utilization of programmed control in fermentation has been reported. There is no reason to expect that a constant p H or a constant dissolved oxygen concentration in a bath process will yield optimum process conditions. Some definite variation with time might result in better yields. Obtaining experimental data on such systems is not simple, but some of the newer statistical optimization techniques should be applicable to this problem. I n addition, when multiple stage continuous fermentations become practical, undoubtedly optimum results will require different controlled conditions in each stage ( 5 ) . Another field in the study of fermentations is opening u p through the applicaVOL. 52, NO. 1
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JANUARY 1960
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tions of techniques derived in control engineering for the study of the dynamics of processes. Some recent studies, using a continuous fermentation, where a sinusoidal forcing input function was utilized, have shown that this technique can yield valuable data about transient responses of a fermentation system (4). I t may be possible to utilize even more refined methods of control engineering which do not require a forcing function input to a system, but rely on random fluctuations of the system for analysis. In fermentations, one is dealing with a unique chemical problem. The study of the controllability, dynamics, and opti-
mization of systems involving living organisms is a fascinating one. There is an urgent need for specialized measuring devices applicable to these complex systems. I n the coming years, great advances can be made in the control of fermentation processes by the application of some of the methods outlined here.
( 3 ) Ellsworth, R., Meakin, L. R. P., Chem. and Znd. (London) 1954, p. 926. (4) Fuld, G. J., Mateles, R. I., Kusmierek,
B., Symposium on Continuous Fermentation, SOC.Chem. Ind., London, March 1960. (5) Gaden, E. L., Jr., J . Biochem. Microbiol. Technol. Eng. 1, No. 4 (1959). (6) Shu, P., IND. ENG. CHEM.48, 2204 (1956). (7) Squires, R. W., Hosler, P., Ibid., 50, 1263 (1958).
literature Cited (1) Bungay, H. R., Simons, C. F., Hosler, P., J . Biochem. Microbiol. Technol. Eng., in press.
(2) Dennison, F. W., Jr., West, I. C.: Peterson, M. H., Sylvester, J. C . , IND. END.CHEM.50, 1260 (1958).
GEORGE J. FULD Department of Food Technology, Massachusetts Institute of Technology, Cambridge 39, Mass.
Sterilization of Media for Biochemical Processes P U R E culture fermentations are presumed to require sterile media, yet attainment of true sterility is neither practical nor desirable (7). Although a number of alternatives have been proposed for sterilizing biochemical systems, industrial operations still rely on heat processing. Nevertheless, recent developments have resulted in the use of ionizing radiations for bone sterilization in surgery ( 8 ) )for specialized pharmaceutical applications (4), and potentially in many other situations (72). Agents such as ethylene oxide (74), 8-propiolactone ( 5 ) ,and high frequency sound waves (7 3 ) are of only minor industrial interest a t present. Hence the improvement of heat processing and the possible development of radiation sterilization techniques hold the most promise for improvement of industrial sterilization practice in the immediate future. Perhaps the most notable advance in heat processing during the past decade has been the development of continuous, high temperature processing. This was adapted for fermentations by Pfeifer and Vojonovich in 1952 (769, although it had been well developed for foods a t least a decade earlier (7). The technique should receive increasing application in new fermentation installations, because it produces commercially sterile products with less damage to the nutrients ( 7 , 76) and its use can increase the capacity of present units by shortening the down time between fermentations. Both Qlo values and activation energies are used to characterize temperature effects on spore killing rates. However, these expressions are not synonymous, because the former vary with temperature while the latter are relatively constant (2). Nevertheless, both concepts are used industrially. Almost a decade ago Gillespy (70) pointed out that these two bases for calculation produce similar processing schedules a t 250’ F., but a t
66
“flash” sterilization temperatures considerable differences develop. These differences may be as much as 35-fold at 310’ F., the Z value method indicating the longest time. When experimental data for destruction of Clostridzum botulinum spores in phosphate buffer (9) are plotted both as a function of the temperature and as the reciprocal of the absolute temperature, the precision of the data is not adequate for assuming a straight line in either case; neither is it sufficient for the extrapolations that are standard practice for flash sterilization calculations today (3,6, 7, 76). Studies of the use of ionizing radiations for sterilizing fermentation media have been reported only for the lactic acid (7 I), tissue culture (75), and alcohol (77) systems. An extensive literature exists on the use of these rays for sterilization of food (72), biologicals (8), and drugs (4). Basically, two processes are involved, radiopasteurization and radiosterilization. T h e former may become useful in dominant culture, the latter in pure culture fermentations. Heat sterilization is still the method of choice wherever it can be used. Continuous, high-temperature heat sterilization has been successfully adapted to fermentations from previous use in food processing. T h e contradiction between Z value and activation energy calculations has not been resolved; in fact, it has not even been adequately recognized. Hence, improved flash processing schedules should be possible when data become available from spore destruction studies a t higher temperatures. Such studies should also justify one or the other of the calculation methods, if either is valid at high temperatures. Radiation sterilization may become useful in the beverage alcohol and perhaps other fermentations. However, its adaptation to industrial use is contingent
INDUSTRIAL AND ENGINEERING CHEMISTRY
upon development of better and cheaper irradiating equipment. literature Cited (1) Ball, C. O., Olson, F. C. W., “Sterilization in Food Technology,” McGraw-Hill, New York, 1957. (2) Buchanan, R. E., Fulmer, E. I., “Physiology and Biochemistry of Bacteria. Effects of Environment on Bacteria,” Williams & Wilkins, Baltimore, Md., 1930. (3) Charm, S . E., Food Technol. 12, 4-8 (1958). (4) Controulis, J., Lawrence, C. A., Brownell, L. E., J . A m . Pharm. Assoc., Sci. E d . 63, 65-9 (1956). ( 5 ) . Curran, H. R., Evans, F. R., J . Znfectious Diseases 99, 212-18 (1956). (6) Deindoerfer, F. H., Humphrey, A. E., Appl. Microbiol. 7, 256-64 (1959). (7) Zbid., pp. 264-70. (8) DeVries, P. H., Kempe, L. L., Brinker, W. O., Univ. Mich. M e d . Bull. 21, 29-33 (1955). (9) Esty, J. F., Mayer, K. F., J . Infectious Diseases 31, 650-63 (1922). (10) Gillespy, T. G., “Heat Resistance of the Spordiof Thermophylic Bacteria. 111. Thermophilic Anaerobes,” .4nn. Rept. Fruit & Veg. Research Sta., Campden, Endand. 1948. (11) Eillie;, R. A., Kempe, L. L., J . Agr. Food Chem. 5, 706-8 (1957). (12) Hannan, R. S., “Scientific and Technological Problems Involved in Using Ionizing Radiations for the Preservation of Food,” Special Rept. 61, Department of Scientific and Industrial Research, H. M. Stationery Office, London, 1955. (13) Homre, D., J . Bacteriol. 57, 279-95 \
’
(1 , -949’1. .~,(14) Judge, L. F., Pelczar, M. J., Jr., A,b,b1. Microbiol. 3, 292-5 (1955).
5]-Merchant, D. J., Stewart, R. D., Kempe, L. L., Graikoski, J. T., Proc. 506. Exptl. Biol. @ M e d . 86, 128-31 (1954). G ) Pfeifer, V. F., Vojonovich, C., IND. END.CHEM.44, 1940-6 (1952). 7) Stratton, J. R., Coulter, J. F., Day, W. H., Boruff, C. S., J . Agr. Food Chem. 4, 260-2 (1956). LLOYD L. KEMPE
University of Mich.
Michigan,
Ann Arbor,
