a classification scheme for apparent or over-all “type” reactions, which can be described easily by simple volumetric observations made during the course of most fermentations. Figure 2 shows an example of a simultaneous reaction which obviously involves an “overflow” or “shunt” metabolism ( 4 ) . Figure 3 is one example of the stepwise reactions unique to enzymatic processes. Similar reactions occur in a number of fermentation processes. Mostly, however, the processes involve a combination of several over-all type reactions. Kinetic information, coupled with biochemical evidence, provides a sound basis for studying reaction mechanisms in a fermentation process. Such studies can eventually lead to improvements in batch
fermentations through a program of control that optimizes the rate-determining steps occurring during a fermentation, once they are established, and can also lead to the successful design and operation of multistage continuous systems. Analytical support for such studies is now available in the form of commercial automatically operated analytical instruments. Electronic analog computers permit rapid process simulation and can aid greatly in the interpretation of kinetic results previously difficult to analyze. By a judicious combination of the engineer’s interpretation of reaction kinetics, the biochemist’s understanding of reaction chemistry, and the microbiologist’s ability to maintain active cells, which provide the catalysts for the numerous reactions occurring, fermentation technology can realize a substantial advance in process understanding and control over the next decade. literature Cited
!ICE
Figure 2. Simultaneous conversion of sugar into cell protein and cell fat during Rhodoforula glutinis growth (3)
(1) Calam, C. T., Driver, N., Bowers, R. H., J . Appl. Chem. 1,209 (1951). (2) Deindoerfer, F. H., Humphrey, A. E., IND.ENG.CHEM.51, 809 (1959). (3) Enebo, L., Anderson, L. G., Lundin, H., Arch. Biochem. 11,383 (1946). (4) Fosfer, J. W., “Chemical Activities of Fungi,” Academic Press, New York, 1949. (5) Gaden, E. L., Jr., Chem. and Ind. (London) 1955, p. 154. (6) Herbert, D., Elsworth, R., Telling, R. C.. J. Gen. Microbiol. 14, 601 (1956). (7) Luedeking, R., Piret, E. L., Division of Agricultural and Food Chemistry, 134th Meeting, ACS, Chicago, Ill., September 1958. (8) Maxon, W. D., Appl. Microbiol. 3, 110 (1955). (9) Monod, J., Ann. Rev. Microbiol.3, 371 (1949).
i
n u
Phase of Glucose Utilization
L
Figure 3. Diphasic utilization of energy sources during the growth of Escherichia coli ( 9 ) (10) Monod,
J., “Recherches sur la croissance des cultures bacteriennes,” Herman & Co., Paris, 1942. (11) Moser, H., “Dynamics of Bacterial Populations Maintained in the Chemostat,” Carnegie Inst. of Wash., Publ. 614 (1958). (12) Novick, A,, Szilard, L., Proc. Natl. Acad. Sci. U. 5‘. 36, 708 (1950). (13) Piret, E. L., Luedeking, R., Division of Agricultural and Food Chemistry, 128th Meeting, ACS, Minneapolis, Minn., September 1955. (14) Pirt, S. J., J . Gen. Microbiol. 16, 59 (1957). (15) Spicer, C. C., Biometrics 11, 225 (1955). (16) Teissier, G., Rev. xi.,Extrait du No. 3208, 209 (1942).
FRED
H. DEINDOERFER
School of Chemical Engineering, University of Pennsylvania, Philadelphia 4, Pa.
Continuous Fermentation
THE
major emphasis in this discussion of continuous fermentation is on the single-stage homogeneous fermentation or stirred-tank reactor, where feed and product streams are continuous and equal and the fermentor contents, cell3 and liquid, are kept homogeneous by agitation. The modifications of this basic system-multistage, recycle, two-phase, etc.-are considered to a lesser extent. The table a t right gives some material balance relationships for the basic system. The complete balance given in Equations l and 2 reduces to Equation 3 when steady state is assumed. Then a t steady state each synthetic or degradative rate is simply equal to the appropriate concentration term multiplied by the dilution rate as shown in Equations 4,5, and 6 (X“ = substrate concentration, X p = production concentration). To relate the concentration of one material (such as the substrate) to that of another (such as the cells) the yield
64
The batch growth curve can be used directly, appropriate equations can be developed, or a graphical analysis can be used.
Material Balance Relationships
IUPJT t GROWTH
(-341 = OUTPUT t ACCJMULAT
S O E C I F ’ C GROWTH
RATE CONSTON? l k l =
Mechanism for Culture Control
Oh (1 )
$ = DILUTIOW R A T E 13)
- - - - - - - - - - - - -RATE
OF
CE! L SYUTHESIS = X3
(I!
F 4 T E OF S U E S T R A T E U T , L I Z A T ’ O N = [ X i - X ‘ ! R A T E OF F P 3 D U C T F O R M A T I O N = -
-
f ELD C 3 \ S T A N T
-
IY1 =
Xi-X
(6
kPD
_
4
D
1
(6 ) _
-
_
(7
constant, J‘, is employed as in Equation 7. T o design a continuous fermentor for cell production it is necessary to have kinetic data, to evaluate k. This can be done to some extent with batch fermentation data.
INDUSTRIAL AND ENGINEERING CHEMISTRY
There are two mechanisms for control of continuous cultures. One is exemplified by the Turbidostat of Bryson; the other, by the Bactogen of Monod and the Chemostat of Novick and Szilard. I n the Turbidostat the cell population is held constant by a device which measures the culture turbidity and regulates the feed accordingly. In the Chemostat the feed and withdrawal rate are held constant at a value less than the maximum growth rate. Under these conditions the growth rate is regulated by a limiting nutrient concentration. This can be shown to constitute a self-regulating and stable steady state. The Chemostat principle is more widely applied to continuous fermenta-
_
_
_
_
_
_
I
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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