1792
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 characteristics of 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. in connection with yeast Variations in the calculated absorption coefficients over 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 ment by “bioengineering” methods is the problem of oxygen in the development of new antibiotics and in practically all other 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
September 1950
INDUSTRIAL AND ENGINEERING CHEMISTRY
attempt to determine and separate the rate processes involved (19, 13). Over-all data of this type. are of limited value b e c a w they fail to differentiate between the purely physical process of oxygen absorption and the biological factors involved in ita utilization. Basically the problem includes two phases which may be summarized as follows: 1. Determination of the oxygen requirements of an biological system such that oxygen supply shall not be a limiting &tor in the rate of metabolism 2. Development of some quantitative system for expressing the relative and absolute ability of the equipment and aeration system to supply these requirements
A study of the whole problem of agitation, aeration, and oxygen transfer in antibiotic fermentations by Bartholomew et al. (1) is presented concurrently in this journal. The reader may consult these papers for a somewhat different approach and for extended studies in different scale units. DEVELOPMENT OF OXYGEN TRANSFER EQUATIONS
Fundamentally, the problem is to transport oxygen from the air to the respiratory enzyme system of the cells a t a rate sufficiently high that metabolic activity will not be limited by the availability of oxygen. The situation can be clarified if the overall oxygen transfer mechanism is separated into a series of individual rate processes as follows: 1. Diffusional transfer (absorption) from the gas to the liquid medium 2. Diffusional transfer through the liquid to the cell 3. Chemical reaction with oxidizable substrates through the oxygen-carrying enzymes of the cells respiratory system
Each individual process must have a rate a t least equal to the over-all rate-i.e., the over-all rate of transfer cannot exceed the lowest individual rate. Before proceeding, it is necessary to point out some important assumptions which have been made. First, it is implied that maximum respiration is desirable. There are case8 where this is not necessarily true, of which antibiotic production is a notable example. Gottlieb and Anderson (7)have shown that maximum streptomycin formation is not associated with maximum synthetic metabolism in submerged cultures of Streptomyces g~iseus. Maximum antibiotic production occurred during a period of low oxygen consumption in the later stages of the fermentation and was not detected a t all in the early period when respiratory activity was a t its highest level. For the purposes of this discussion, however, it will be assumed that the organisms are to be supplied with as much oxygen as they can use a t any time in the culture cycle. The second assumption is that only oxygen dissolved in the suspending medium is available to the cells for respiration. Arguments can certainly be offered (supporting evidence for which may be gained from mineral flotation studies) for direct transfer of oxygen from gas bubbles to cells in contact. Bartholomew et d.(1)have discussed the importance of this phenomenon in fermentations where extensive mycelial networks are formed capable of entrapping large numbers of bubbles. Since measurement of the amount of oxygen transferred to the cells by this mechanism is not practicable a t this time only dissolved gae will be considered in the following discussion. Lastly, it is implied that respiratory metabolism will constitute the only oxygen demand by the system. It is possible to postulate other than respiratory requirements-for example, direct, nonenzymatic oxidation of some substrate componentsbut for this discussion any such oxygen uptake that may exist will be considered negligible in comparison with respiratory uptake. With these conditions established it is possible to disc- the component rate processes in more detail.
1793
The optimum conditions for oxygen transfer exist when the over-all rate of transfer, ro, from the air to the substrate is determined only by the rate of formation and degradation of the ensyme-oxygen complexes by means of which oxygen is t r a m ferred to oxidizable metabolitea. I n other words, the rate of uptake is controlled by the final, or chemical, rate in the sequence. Under these conditions the mass transfer rates from the gas to the liquid and through the liquid to the cell must be equal to or greater t h n the chemical rate, ro,and ro
-
r.
(1)
Because of limitations in the measuring techniques now available and incomplete knowledge of the actual mechanism of respire tion, it 13 not possible to separate the last two mass transfer rates in pravtice. Experimental methods for determining oxygen uptake rates do not eliminate the factor of oxygen diffusion from the liquid to the cell. Values of oxygen uptake rate obtained are, therrforr, observed over-all rates for the oomposite process of diffusion and chemical reaction. Regardless of which of these two fartors actually controls the rate, the measured value of the uptake prorrss, r,, does indicate the effective rate a t which oxygen is removed from solution by the cellular respiratory processes. It must, however, be remembered that since it is an empirical rate it will bo valid only under the conditions of the experimental method uwd for its determination. The rate of oxygen uptake, rr, may then be partially resolved aa follows rr kr(Cm) (2) where k, rate of oxygen uptake per unit weight of tissue and C, = weight of tissue per unit volume of medium. Concurrently with this respiratory uptake from solution, oxygen is being added to the liquid by diffusion from the stream of bubbles supplied by forced aeration. The rate, Td, of maw transfer across the gas-liquid interface is given by the masa transfer coefficient, ko for the gas phase and kr, for the liquid, multiplied by the area of transfer, A , and the concentration gradient available as a driving force, or =I
= ko(A)(pa
- pi) = k ~ ( A ) ( c-,
(3) For the gas the driving force is equal to the difference between the partial pressure of oxygen in the bulk of the gas stream, p ~ , and a t the interface, pi. For the liquid it is equal to the difference between the concentration of dissolved oxygen a t the interface, cd, and in the bulk of the liquid, CL, all expressed in suitable units. For a sparingly soluble gas like oxygen one may neglect the gas film term and consider the entire resistance tn transfer to lie in the liquid phase. Equation 3 may be reduced to: Td
kLA(ci
-
CL)
(4) Since oxygen follows Henry’s law very closely, equilibrfum conditions a t the interface will be represented by the expression Td
3 .
CL)
cc = H(pc) or with negligible gas film resistance, pc = pa,
(5)
The value of oxygen concentration a t the interfaoe, CJ,is then very nearly equal to its solubility, or equilibrium concentration, a t the partial pressure of oxygen in the entering air stream and the temperature of the fermentation. Since, in practice, oxygen is supplied by forcing air through the liquid in bubbles, the area for mass transfer ia the effective interfacial area of the bubbles. This is extremely difficult to measure accurately and, therefore, must be combined with the liquid film coefficient, kL, to give a modified mass transfer co. efficient, kd, where
kd = kLA
(7)
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
17S4
ABSORPTION EFFICIENCY
Substituting in Equation 4 rd
=
kd(ci
- CL)
(8)
If oxygen is not accumulating at any point in the system, steadystate conditions exist and one may write:
r,
=
r, =
rd
(9)
That is, the rate of oxygen absorption from the gas is just equal to the rate of removal by the organism. The expressions for the individual rates, rr and r d , may then be substituted and equated, r, k,(Cn) = k d ( C i - C L ) (10) For the more general case where the concentration of dissolved oxygen is varying in time (unsteady state), the rate of change of the oxygen concentration in the liquid, dcL/dt, must be taken into account. For these conditions r, # r d , and one may write:
Again substituting the expressions for the individual rates, r, and rr kd(C{
- CL)
Vof. 42, No. 9
= k,(Crn)
- dcL -&
If, for the system undet study, the magnitude of the term dcL/dt is negligible in com arison with the other terms in the expression, it is possible to isregard the rate of change of dissolved oxygen concentration and use the simple form (Equation 10). With values for kr, Cm, ci, and CL obtained experimentally the absorption coefficient, kd, for any arrangement of physical variables may be calculated from Equations 10 or 12. Qualitatively the rate of absorption of oxygen, T d , is known to be a function of the area of contact, time of contact, agitation intensity (turbulence), and concentration gradient. The absorption coefficient, kd, will be affected only by the first three of these, contact time, area, and agitation intensity, and therefore will be dependent only on the design and operation of the equipment and on the physical properties of the absorbing phase. The coefficient may be expected to change with the following operating variables:
Since the maximum possible economy of air utilization is desired some expression of the efficiency of oxygen absorption is desirable. The absorption efficiency, E, will be equal to the oxygen absorbed divided by the oxygen supplied in the air stream per unit time. The weight rate of absorption is given by T d and the weight rate of oxygen supply, G, may be calculated from the aeration rate in volumes of air per unit time and the operating volume of the fermentor. The absorption efficiency, E, is then given by
E
= ;(100)%
(13)
OXYGEN UPTAKE RATES
The determination of k,, the oxygen consumption rate per unit weight of tissue, is important because it indicates the oxygen demand of the biological system a t any point in the fermentation cycle. The rate of respiration is normally determined by observing the decrease in oxygen partial pressure in the gas phase in contact with a cell suspension in any suitable manometric apparatus. For cases involving only oxygen-carbon dioxide exchange the direct method of Warburg, described by Umhreit et al. (16),may be used. The voltammetric method for determination of dissolved oxygen concentration offers, in many cases, a more convenient and rapid method for the measurement of k,. It has the additional advantage that the actual rate of decrease of dissolved oxygen concentration rather than the oxygen partial pressure in the gas is observed. It is necessary only to equilibrate the suspension with air, stop the flow of air, and then observe the decrease of CL with time t. Figure 1 is a plot of this type for a suspension of yeast in phosphate buffer with glucose as a substrate. The slope of the curve, -dcL/dt, is proportional to the respiration rate and may be converted to any set of units by introducing the proper proportionality factor involving the dry weight of tissue present.
1. Aeration rate or superficial air velocity, Vs 2. Bubble size or type of air dispersal device
3. Agitator speed and design Physical properties of the liquid phase (particularly viscosity, density, and surface tension) 4.
In nonbiological gas absorption systems changes in the absorbing phase are normally negligible and the initial choice of the absorbing fluid will determine the physical properties which may be considered to be constant throughout. I n fermentation systems, where liquids containing complex substrate materials are employed, this is an important consideration. The surface activity of the liquid will not only affect the initial bubble ske but, even more important, the ability of the liquid to sustain small bubbles once formed and minimize coalescence. Surface activity will vary greatly over the course of a fermentation both because of the chemical changes in the medium brought about by the metabolic activities of the organism and because of the deliberate addition of surface active agents for foam control. It is possible for very small amounts of added surface active agents to alter the foaming properties of the liquid enough to change the effective bubble size many times. This means that the transfer area, and with it the observed values of k d , will also be altered accordingly. In so far as oxygen transfer is concerned, k d values obtained with one biological system will apply equally well to any other system in the same equipment provided that changes in the physical properties of the absorbing fluid are accounted for, The absorption coefficient is concerned only with the transfer of oxygen from the gas to the liquid phase.
Time -minutes Figure 1. Dissolved Oxygen Concentration vs. Time for Bakers' Yeast in pH 6.8 Phosphate Buffer a t 30" C.
The respiration rate, kr, is not constant throughout the fermentation cycle and may exhibit wide relative variations. Gottlieb and Anderson (7) followed the variation in uptake rate with culture age in submerged cultures of Streptomyces griseua. A similar effect, but of much less magnitude, was obsdrved for cultures of baker's yeast grown in aerated mediums in the present work (Figure 2). Data for this plot were obtained by volt ammetric measurement of kr and have been given elsewhere ( 6 ) .
INDUSTRIAL A N D ENGINEERING CHEMISTRY
Soptombar 1950
1795 \
complex fermentations where the relationship between respiratory activity and productivity is obscure, the proper level of respiratory activity must be evaluated by experiment. While this is not the most desirable method it nevertheless enables us to obtain values of k, through the course of the fermentation which can then be used in the oxygen transfer equations previously developed.
t
EXPERIMENTAL METHODS
Run.27
Legend: 0
Runom
+ Aun.280nd 28a
1
O*t
Time -hours I
0
0
I
Figure 2.
I 2
I
3
I 4
I
L
I 6
I 7
Respiration Rate VI. Culture Age for Bakers' Yeast in Aerated Malt-Base Medium Compodte curve
This variation can be ascribed, more or less, to several factors like depletion of carbohydrate substrate, decreased activity of older tissue in organisms forming mycelial networks, and to an actud physiological aging process. Another factor which may affect the value of k, in any system is the dissolved oxygen concentration, CL, in the medium, The literature contains somewhat divergent opinions but it is generally assumed that cellular respiration rates are independent of oxygen concentration down to very low values of CL (8). Below these oriticd values of CL marked dependence of the respiration rate on oxygen concentration is observed. Values of the critical concentration reported by different workers, in many cases for the same tissue, have differed considerably. The techniques usually employed involve the equilibration of cell suspensions with gas mixtures containing various amounts of oxygen. Critical oxygen levels &re then expressed as partial pressures of oxygen in the gas rather than as aotual dissolved oxygen concentrations. The critical concentration for any cell suspension can often be determined most readily from a plot of CL versus time as in Figure 1. With data like these, obtained voltammetrically, it is possible to plot kr, expremed in any suitable units, against CL to determine the critical concentration (Figure 3). Actually the second plot is not needed, for as long as the rL versus t curve remains linear, the slope, and therefore kr, must remain constant. Below the critical CL value, k, (or the slope) falls off rapidly. This discussion of oxygen uptake rates, krl may be concluded by briefly summarizing the main points: Basically the oxygen uptake (respiration) rate, kr, will be determined by the choice of organism and the physical and chemical roperties of the environment. Since these are, however selected From biological considerations that are fundamental to the entire fermentation process, they cannot rightly be considered aa operatin variables so far as oxygen transfer is concerned. $c, will var , in greater or less degree, over the fermentation cycle, depening on physiological a ng, etc. Icr will be a function of the availsle oxygen concentration below a so-called critical value. Above the critical level the uptake rate will be constant. The absolute value of the critical concentration is generally rather low but will vary considerably with the organism employed and the growth and fermentation conditions. As a corollary to that just mentioned, one ma note that if the available oxy en concentration in the liquid is &owed to fall below the critic3 level, res iration, and with i t growth and metabolic activities associated wit! respiratory activity, will be retarded,
It is necessary, therefore, to determine k,, the oxygen uptake rate, empirically over the course of the fermentation cycle. These determinations must be made under the environmental conditions best suited to the process in question. I n some casesfor instance, the oxidation of glucose to gluconic acids-the maintenance of maximum kr a t all times is probably desirable. On the other hand, for antibiotic production and many other more
Biological System. In order to obtain data for use in oxygen transfer equations the simplest biological system that will permit the necessary measurements waa selected. The propagation of baker's yeast in aerated medium, besides being one of the most completely studied of microbiological systems, offers several important advantages in the present application. Among them are simplicity of operation and analysis, short fermentation times, good reproducibility of results, and, with reasonable cam, freedom from contamination. A supply of fresh press yeast was provided regularly by the Fleischmann Laboratories from their stock propagators to give an organism of constant characteristics throughout the experiments.
0
t
0
I
2
3
L49*10-'
Figure 3. Respiration Rate us. Oxygen Concentration for Bakers' Yeart at 30' C.
The yeast was grown in a medium of diluted Fleischmann Diamalt Syrup (nondiastatic) supplemented by the addition of ammonium phosphate and adjusted to pH 4.5 with sulfuric acid. Straight-set fermentations, in which a11 the nutrients are added a t the start, wem used in all experiments. All experimenta were run a t 30' C. Feimentor. A laboratory fermentor of stainless steel and glass with a working capacity of 1.5 liters was constructed for this study. Means were provided for varying the agitation intensity and the air dispersal mechanism. The essential parts are: 1. A glass jar (4.5 X 8 inches) with a %liter total capacity. 2. Stainless steel base plate with threaded tie rods for clamp
ing the cover down. 3. A stainless steel cover plate fitted with an agitator shaft and stuffig box with soft carbon bearings, a sparger tube for the introduction of air, a eamplmg tube d r & F g from the approximate midlevel of the charge, vent and additron tubes. Ports were included for glass and calomel electrodes to enable the continuous measurement and control of pH. The complete unit, assembled with electrodes in place, is shown in Figure 4. A great variety of sparging devices have been reported, particularly in the patent literature. To illustrate the effect of dispersal conditions on the absorption coefficient, kr, two extreme types were employed. One (singlebubble type) releasea air from a single hole, 0.060 inch in diameter, directly under the rotating agitator. The second sparger is a disk of fritted stainless
1796
INDUSTRIAL AND ENGINEERING CHEMISTRY
Figure 4. Laboratory Fermentor Assembled for Use
steel, 2.5 inches in diameter, with average pore openings of 65 microns, to provide a cloud of fine bubbles. It may be seen beside the assembled fermentor in Figure 4 and can be threaded on to the outside of the sparger tube over the single hole. A vaned-disk impeller, serving as a combination agitator and air disperser, was used only with the single-bubble agitator sparger. Measurement of Oxygen Concentration. Chemical methods for the determination of dissolved oxygen concentrations are not applicable to this study for several reasons. Perhaps the most important is the need for rapid determination of the instantaneous oxygen concentration. Manipulative difficulties in chemical methods may result in contamination of unsaturated samples and, secondly, the complex substances present in the medium being analyzed may interfere with the analytical reactions, These difficulties are avoided by the use of the v o l t ammetric (polarographic) method for the determination of dissolved oxygen using the dropping mercury electrode. The use of the dropping mercury electrode for this purpose is discussed in detail by Kolthoff and Lingane (8). When oxygen is reduced a t the dropping mercury cathode a current-voltage curve showing two waves is obtained, but for quantitative determinations i t is not necessary to record the entire curve. A single potential method like that described by Lewis and McKenzie (Q),and earlier by Baumberger (2) and Winzler (16),is well suited to rapid determination of dissolved oxygen concentration and has been adapted to the conditions of the present study. A single potential, chosen perferably to lie on the relatively flat portion of the wave, is applied to the sample contained in the polarographic cell and the galvanometer deflection is noted. The sample is then freed of oxygen by bubbling with tank nitrogen. A second, “residual current,” galvanometer deflection is then noted and the difference between the two readings, D, is considered to be the deflection due only to the oxygen present. The applied potential used in this work was -0.5 volt against the standard calomel electrode. For repeated quantitative determinations it is unnecessary to calibrate the galvanometer in terms of microamperes of diffusion current and the initial instrument calibration can be made to relate directly galvanometer deflection and oxygen concentration.
Vol. 42, No. 9
Calibration of the dropping mercury electrode is normally accomplished by observing the corrected deflection (sample reading minus residual reading) a t an appropriate single potential for solutions containing known concentrations of oxygen. A plot of corrected galvanometer deflection, D,versus concentration should give a straight line. The calibration methods used previously all involve the possibility of serious error (2, 9, 16), and so a modified technique was used here. To calibrate the instrument a series of solutions of sodium chloride were prepared ranging inc oncentration from 4 to 0.1 M and the solubility curve of oxygen in these solutions a t 25’ C. was plotted from the literature (IO). Each solution was then saturated, a t 25’ C., with air and nitrogen and the corrected deflection, D, a t -0.5 volt applied potential was observed. A plot of dissolved oxygen concentration, taken from the solubility curve, versus corrected deflection gives a straight line as expected. The use of residual deflection readings from the sample substantially freed of oxygen by sweeping with nitrogen permits the use of the voltammetric method in the most complex fermentation mediums. I n a simple supporting electrolyte like 0.1 M sodium chloride solution, the residual current is small and usually negligible. For complex fermentation broths this is seldom true, A few sample determinations of dissolved oxygen in various s u p porting liquids are given in Table I to illustrate this. A large concentration of electroreducible substances which give appreciable diffusion currents a t the potential being used will distort the reading considerably, as in the case of the riboflavin broth, unless a residual current correction is made. The instrument used in this work was a manually operated polarograph (Fisher Elecdropode). The recommendations of Kolthoff and Lingane (8) for the assembly and maintenance of the dropping mercury electrode were followed. A dropping time of about 6 seconds per drop was maintained. An H-type cell with permanent external anode, similar to the one described by Kolthoff and Lingane (8) but modified by adding a draining stopcock to the sample chamber to allow for rapid emptying and rinsing, was constructed. A bubbling tube was included in the sampling chamber for saturating the sample with oxygen (air) or tank nitrogen as required. All applied potentials are referred directly to the standard calomel electrode in this type of cell. Table I.
Determination of Dissolved Oxygen in Various Supporting Liquids Galvanometer Deflection after Saturation with Corrected Air Nitrogen Deflection, D
Sample NaCl solution 84.0 Diamalt medium and yeast cella from laboratory fermentation 76.8 Riboflavin broth from riboflavin fermentation 86.0
0.1 M
4.5
79.5
7.0
69.8
38.0
48.0 ~
Methods for obbaining representative samples present the greatest problem in the voltammetric determination of dissolved oxygen, Since the samples removed for analysis will usually be unsaturated, handling may result in the addition of oxygen from the atmosphere. Any sampling techniques which cause splashing or foaming of the sample are therefore useless. The second problem is that of loss of oxygen due to continued respiratory activity after the sample is removed from the fermentor. Therefore, it is necessary to end respiration (“kill” the sample) instantly on removal. Many methods have been devised but it is usually very difficult to find techniques for killing the sample rapidly which do not cause contamination with air. The method which is outlined below was actually used only at the end of this work but is by far the most satisfactory developed so far:
1. A reased lCbml. hypodermic syrin e equipped with a large neehe (13 gage) of suitable length is &ecked for air-tightr
September 1950
1797
INDUSTRIAL AND ENGINEERING CHEMISTRY
nesa and 1 ml. of a saturated aqueous solution of phenol, previously saturated with air, is drawn in. 2. While the sample is bein taken nitrogen is passed through the em ty Sam le chamber of &e polarographc cell. 3. %e n e e i e is immersed in the fermentation broth and held so as to avoid sucking in large bubbles. Medium is then sucked in to.the 10-ml. mark and the syringe is shaken to ensure thorough muung. 4. The nitrogen flow is stopped and the sample slow1 injected into the cell sample chamber. If care is taken an$ the needle tip is a t the bottom of th? cell, there is little agitation of the sample while it is being placed m the cell. The use of the syringe technique keeps the sample isolated from the moment it is taken until it is placed in the polarographic cell and practically eliminates any contact with the atmosphere. Th edeflection is read immediately. Then the sample is saturated with air and the deflection, CI, is read, Nitrogen is bubbled through the sample and the residual reading obtained is used to correct both of the previous readings. In this work it was possible to detect differences in oxygen concentration of as little aa 0.05 milligram per liter (parta per million). While the sensitivity remains unchanged the precision or reproducibility of the voltammetric determination falls off as the oxygen content decreases, Actually the loss of precision lies mostly in the sampling method. Since the lower range of oxygen concentration is also the range of maximum cell ooncentration and, therefore, high total oxygen uptake, small differences in the time required to sample will result in different oxygen incrementa consumed by the organism before killing. With less satisfactory sampling techniques than the one deBcribed above (used through most of this work) the precision of measurement was estimated to be of the order of 6 to 10% in the lower concentration, CL, range and 2 to 5% a t higher disnolved oxygen levels. The syringe sampling method greatly improves the precision in all concentration ranges. No provision for temperature control has been noted. The decrease in oxygen solubility with temperature is fortunately balanced exactly by the increase in its diffmivity, over the range from about 10' to 35' C. (8,9). The diffusion current, therefore, fs independent of temperature in this range. The use of the dropping mercury electrode to determine the oxygen uptake rate, k,, of a cell suspension ww discussed earlier. The cell suspension is saturated with oxygen by bubbling rapidly with air in the polarographic cell. The air stream is stopped, a few seconds are allowed for turbulence to subside, and the stop watch is started. A curve of deflection (or CL)versus time is then plotted and k, is taken from the slope of the curve. Ex erimental Procedure. The fermentor and attached cotton air fifter were sterilized prior to use and sterile medium waa prepared separately in 1.5liter batches. Just before use the medwm waa filtered to remove matter precipitated during sterilization since this interfered with dry tissue weight determinations. The medium was then added to the fermentor and allowed to come to operating temperature by immersing the whole assembly in a conetsnt tem rature bath while air waa bubbled throu h to saturate the l i q u i r i t h ox gen. The air flow w w then a&usted to the value desired and {grams of fresh press yeast were addeddurried up in a small amount of medium. After allowing for thorou h mixing, the first tissue sam le waa withdrawn from the samde line and this time waa consifered to be the start of the fermentstion. Each run waa then continued for 8 hours. Dissolved oxygen (aand CL) determinationswere made every 30 minutes in the manner described above. Eve hour a 10-mI. sample waa withdrawn and centrifuged. The c z s were washed, recentrifuged, and dried to constant weight a t 65" C. Sugar analyses, when desired, were made on the clear broth from the centrifugingoperation, just noted, by the method of Stiles, Peterson, and Fred (14). Calomel and glaaa electrodes were installed a t the beginning of the fermentation, in the porta provided, and the pH waa continuously observed and maintained in the range 4.3 to 4.7 by the addition of aqueous ammonia. To minimize the loss in volume caused by the withdrawal of samples, 10 ml. of sterile, fresh medium were added after each samplin Any effects from the addition of this small volume to the 1.5 tters in the fermentm are negligible and this procedure
d0
1
3
2
I
Figure 5.
4
5
6
':. 7
b0
Fermentation time- hours Complete C w e a of Experimental Determinations for Run 21 Single-bubble rparger Air rate, 0.1s CU. ft./min. Agltatton, 300 r.p.m.
eliminates the otherwise serious cumulative depletion which would have resulted. Sterile methods were em loyed in the o n 'nal set up only. No attempt waa made to awere to sterde tecfniques in samplin shce the exercise of reasonable cleanlinw waa ade.quata under conditions of low pH and rapid propagataon whch prevailed.
th
RESULTS AND DISCUSSION
Presentation of Data. During the c o m e of each fermentation determinations of dry tissue weight Cm, instantaneous dissolved oxygen concentration, CL, and the saturation concentration or solubility, GI, were made in the manner described previously. Since the method of calculation is the same in all cams, the complete data from one typical fermentation (run 21) will be used to illustrate the application of oxygen transfer equations (Figure 5). AI1 other experiments were similarly handled. Determinations of sugar present, aa maltose, were made to check the ooulge of the fermentation. The sugar utilization curve, though included, is not neceesary for oxygen transfer calculations. 1. The growth curve (dry tissue weight) is typical. The maximum growth rate coincides with the maximum rate of sugar utihation and growth begins to fall off at the end of the fermentstion period due to the complete utilization of available carbohydrate. 2. The equilibrium concentration (solubility) of oxygen in the medium ia constant for the first 4 to 5 hours after which it bes to rise. This is the result of reduction in the concentration of iaaolved sugar. The solubility of oxygen is dependent on the concentration of solutes but is relatively unaffected by the amounta of suspended matter (cells, etc.).
r
Table 11. Time,
Calculation of h from Run 21 (Figure 5) and Values for R, from Table 111
-
k, Cm k,, ci CLO ci CL 6.30 X 10-8 3.57 X 10-8 2.73 X 10-8 0.0775 0.46 13.1 6.30 X io-' 3,is x io-' a.14 x 1 0 - 8 0.0850 6.29 X 10-3 2.70 X 10-8 3.59 X 10-8 0.0870 0'66 0 54 14 15'76 6.30 X 10-8 2.18 X 10-8 4.12 X 10-1 0.0850 0:sO 16:s 6.30 X 10-8 1.86 X 10-8 4.45 X 10-8 0.0830 0.89 16.6 0 ' s 6.30 X 10-8 1.50 X 10-8 4.80 X 1 0.0820 0.99 16.9 6.30 X 10-8 1.08 X 10-8 6.22 X 10-8 0.OS04 1.11 17.1 3.00 6.30 X 10-8 0.72 X 10-8 6.58 X 10-1 0.0792 1.26 17.7 V ~ U - of c L are correoted for the rasidual oxygen oonoentration of 0.60x 10-8prrm perliter (Figure5).
Hr.
0.50 1 .oo 1.50 2.00 2.25 2.60 2.75
INDUSTRIAL A N D ENGINEERING CHEMISTRY
1798 I
I
‘I
I
I
1
1
I
/-
d
Data from runs: 15
I
0
23
24
I
I
1
I
I
I
2
3
4
5
-
1
6
Time hours
Figure 6. Calculated Values of Absorption Coefficient us. Fermentation Time
3. The instantaneous concentration of dissolved oxygen CL, decreases as growth progresses until a steady level is reached. The point where the curve levels out represents the practical zero concentration, for below this the oxygen uptake rate, r?, exceeds the rate of supply by aeration and no true CL value can be measured. This apparent “residual” oxygen concentration is really due to the presence of oxy en dissolved in the 1ml. of phenol used to kill the sample. The scfubility of oxygen in saturated aqueous phenol solution a t 25’ C. was determined to be 4.8 x 10-8 grams er liter and since a 1 to 10 dilution of phenol in sample is made t\e residual value should be about 0.48 X 10-8 gram per liter. It was actually found to vary from 0.40 to 0.50 x IO-* gram per liter due to manipulative errors. To correct for oxygen in the phenol used each value of CL taken from Figure 5 is corrected for the residual value determined from the same curve. Calculation of Absorption Coefficients. For the general case Equation 12 may be solved for kd. As noted previously the term &/dt may be neglected if i&s magnitude is insignificant in comparison with the magnitude of the uptake rate, k,Cm. In these experiments this simplification was possible. Absorption coefficients were calculated both by Equation 12 and by the simplified form, Equation 10, for sample points on all runs and the values compared. In all cases they agreed closely. The maximum deviation, in runs a t very low air rates which constitute extreme cases, was about 5 to 10% but the bulk of the points calculated agreed within less than 1%. As these errors are well within the precision of the measurement of oxygen concentration itself, the simplified form has been used for all calculations of kd, Table 11 summarizes the calculations for run 21. AI1 values used are taken from the appropriate curves (Figure 5) and, therefore, may differ slightly from the actual experimental values. The instantaneous concentration of dissolved oxygen, CL, is taken from the curve in Figure 5 and corrected for the residual dissolved oxygen discussed previously. The values of CL recorded in Table I1 are corrected as noted. I n order to satisfy the conditions of the original development, the oxygen uptake rate, k,, should be determined a t frequent intervals throughout the course of each run and a curve of k, versus time should be included with those shown in Figure 5 . Unfortunately manpower limitations made it impossible to make determinations of oxygen uptake rates and oxygen concentrations both on each fermentation. Consequently data from a group of runs in which k, only was measured were plotted separately as a Cwve of k, versus time (Figure 2 ) and values from this composite curve were used for subsequent calculations of ka for all other runs. Table I11 gives the values of k, at different t i e s in the fermentation cycles taken from this plot. The use of values from a composite curve like this introduces a considerable and
Vol. 42, No. 9
variable error which, however, can be avoided if facilities are available for making all the necessary measurements in each fermentation. Discussion of Experimental k d Values. It is evident from the data of Table 11, and from calculations for all the other experiments, that the absorption coefficient is not constant over the fermentation cycle. When propagation rates are highest, a twofold increase in k d may be observed in the course of a single fermentation. The chief factor responsible for vanation in k d &s the fermentation proceeds is the inclusion of the area term, A . The addition of foam control agents and the metabolic activities of microorganisms cause changes in the physical properties of the medium and consequently the average bubble size and the area of transfer. It is possible to observe visually these changes in the character of air dispersion. At the start of the fermentation a hard foam of large bubbles builds up and i s difficult to control, requiring the addition of large amounts of antifoam. This type of foam, characteristic of the early stages of many fermentations, is associated with poor air dispersion, large bubbles, and much coalescence in the body of the medium. As the fermentation proceeds the foam changes gradually to an easily controlled froth of fine bubbles. The character of air dispersion in the body of the medium changes accordingly and the tendency to coalesce is greatly reduced. As a result, the area of transfer will increase as the fermentation proceeds depending on the rate of metabolio activity and the quantities of antifoam added. Since the growth and metabolic activity of yeast is enhanced by increased aeration, the greatest incresses in area, and, therefore, the greatest increases in ka, may be expected where the highest aeration rates are used. The data indicate that this is the case. Variations in kd due to changes in the actual area of transfer as the fermentation proceeds are not in any sense errors. The absorption coefficient has increased, due to the inclusion of the area term, and the trend of the increase is in agreement uTith qualitative observations of air dispersion. A limitation on the amount of data which can be used for transfer calculations in each run exists. The concentration of dissolved oxygen must be above the critical level, for, once the value of CI. falls below the critical, conditions exist about which only limited knowledge is available, and these equations can no
ai
05
LO
Superficial oir ve1ocify.V~
Figwe 7. Effect of Aeration Rate on Absorption Coefficient No mrshrnical aritation
INDUSTRIAL AND ENGINEERING CHEMISTRY
September 1950
Ionger be applied. The critical level which was determined earlier (Figure 1)to be about 0.5 x 10-8 gram per liter of oxygen was used and, in general, calculations of k d were not made at concentrations below about 0.7 X 10-8 gram per liter. In order to correlate the absorption coefficient with operating variables it is desirable to have a single value of kd for any particular set of operating conditions. Since the main variations in k d result from real changes in the transfer area and not frOm experimental error it is not possible to consider m y one value of k d to be truly representative of the absorption system under study. Under such conditions the best approach is probably to use some mean value for each set of operating variables employed. In calculating ka values it was noted that there is a period in each run over which kd is fsirly constant, This can be seen best if the coefficients are plotted against the fermentation time (Figure 6). A reverse curve is obtained with k d nearly constant over a range on both sides of the point of zero slope. It might be argued that the value of k d a t this point is the best ingle value to use, but when it is comparedwith the arithmetic mean ofvalues calculated at hall-hour intervals the two are found to agree very closely. The arithmetic mean has been used in this dieowion for simplicity but values obt&mi by both methods are given in the tables which follow. The first (Table IV) contains the results of experimentg in which air agitation alone was used and the second (Table V) those in which mechanical agitation was employed. The superficial air velocity, Va, for each case is based on the cross-sectional area of the fermentor, 0.1045 square foot. Correlationof Absorption Coeftlcientawith OperatingVariables. Cooper, Fernstrom, and Miller ( 4 ) studied the absorption of oxygen in aqueous sodium sulfite solutions and found that the absorption coefficient, K,, expressed in pound moles of oxygen absorbed per (hour) (cubic foot) (atmosphere partial pressure difference), could be correlated with the superficial air velocity and the agitator power for one vessel and air dispersal system. The logarithm of the absorption coefficient was found to be a linear function of the logarithm of the superficial air velocity up to the “loading point” of the agitator. Below this point the relationship K. = kVa0.” was found to hold. A family of parallel lines of the same slope was obtained with agitator power as the parameter and the values of K. at each velocity increased with power. It is possible to correlate k d values for the fermentor in the same manner. The logarithm of the absorption coefficient is also a linear function of the logarithm of the superficial air velocity and the slope of the line changes with different dispersal conditions. Tho data for fermentations employing air agitation only (Table IV) are plotted in Figure 7 and it is clear that the relationships between k d and V8 for the two types of spargers employed, single and fine bubble, are markedly different. For the single-bubble sparger the relationship is kVa0.82
kd
1799
Table 111. Valuer of Rr for Calculation of Absorption Coefficients [Values taken from a general plot.ot kr against fermentation time for yeast grown under oonditione identical with those of typical runs. Data from Figure 2 and (6) 1 Fermentation Time, Hr. kr 0.26 0.50 0.76 1.oo 1.50 2.00 2.60 3.00
0.0700 0.0775 0,0820 0.0850 0,0870 0.0850 0.0820 0.0792
iX6
0.0748 0.0730 0.0710 0.0695 0.0678 0.0660 0 0645 0.0620
a
n .n77o
hn
4.60 6.00 6. 60 6.00 6.60 7.00 8.00
I
by both types increases but the single-bubble sparger introduces, relatively, much more turbulence in the medium. The great increase in turbulence with higher air rates using the singlebubble sparger can easily be noted by visual observation. As a result, k d for the single-bubble sparger increases much more rapidly with increased air rate than is the case for the fine-bubble type. The data for fermentations in which the singlebubble sparger was used with mechanical agitation (Table V) are plotted in Figure 8. Two lines are drawn to indicate the influence of agitator speed. The data are not precise enough to allow better than an estimate of the relative positions and slope of the curves. The slope of the lines appears to be the same for different levels of agitation, and the relationship between kd and V8 approximatea that for nonbioiogicsl systems (8). It is approximately kd
= kV80*’8
The acceptance in the literature of the more or less universal desirability of fine-bubble aeration in aerobic fermentations may require some reconsideration in the light of the results presented here, While it appears that, under similar operating conditions, fine-bubble aeration will provide more oxygen a t low air rates, this advantage is rapidly lost with increased air velocity. Since
Table 1V. Table of Absorption Coefficients for Fermentations Using Air Agitation Only Aeration Rate, kd Run Sparper Type Cu.Ft./Min. Bt.y&in. =e Curve 10 11 18 14 16 16 17 20 22 23 24
Single bubble Single bubble
Fine bubble Fine bubble Fine bubble Fine bubble Single bubble Single bubble Fine bubble Bin& bubble Fine bubble
0.246 0.188 0.28 0.28 0.146 0.04 0.04 0.15 0.16 0.28 0.28
2.34 1.32 2.66 2.68 1.38 0.38 0.38 1.43 1.43 2.86 2.66
26.8 16.8 41.4 31.4 24.4 17.1 6.0 10.6 28.8 28.0 39.0
23.8 17.8 37.5 30.0 26.0 19.0 6.0 10.0 27.6 31.0 38.0
and for the finebubble sparger kd
3
kVaD.**
While the fine-bubble sparger gives much higher oxygen absorption coefficients at relatively low air rates, this advantage disappears 88 the air rate is increased. The reason for this lies in the nature of the coefficient, kr. Since it includes both the liquid-film transfer coefficient, B, and the transfer area, A, two effects are present. A t lower air rates both spargers givo limited turbulence in the medium but the he-bubble sparger, because of the greater transfer area provided, gives higher k,+ values. As the air rate is increased the t w f e r ama provided
Table V. Absorption Coefficients for Fermentations Using a SinglcBubble Sparger with Mechanical Agitation Run 1 2 3
5 8 7 8
9 18 19
21
Agitator 8 eed, Aeration Rate, R.P.d Cu. Ft./Min. 300 300 300 300
800 480 430 430 300 430 800
0.06 0.06 0.12 0.12 0.20 0.04 0.246 0.188 0.04 0.04 0.16
Va Ft./Min. 0.67 0.57 1.15 1.16 1.91 0.88 2.84 1.32 0.88 0.38 1.43
kd
Average
Curve
13.3 17.2 21.8 22.1 25.9 12.0 43.6 23.2 7.3 13.9 16.2
1710 18.5 20.0 30.0 11.5 38.0 22.0 7.6 14.0
17.0
INDUSTRIAL A N D ENGINEERING CHEMISTRY
Vol. 42, No. 9
fermentor operation, from the standpoint of oxygen transfer only, would be at a dissolved oxygen level just above the critical concentration for maximum respiratory activity. Operation a t this level would permit full respiratory activity with the maximum possible aeration efficiency. Even under the best conditions absorption efficiencies are very low and the great part of the oxygen supplied passes through and out the vent. This is characteristic of gas absorp tion in this type of equipment and is found to be true even in the relatively efficient case of Ytraight chemical oxidation (4). SUMMARY
A correlation of engineering variables in a submerged fermentation system has been made from theoretical considerations, and a satisfactory method for expressing the performance of aeration systems independent of the biological system employed has been developed and substantiated by experiment. In addition, the following points can be noted in summary:
Figure 8. Effect of Aeration Rate on Absorption Coefficient Single-bubble sparger
this effect is ascribed to the relatively low agitation intensity induced by fine-bubble aerators it is also necessary to consider other effects which may result from poor agitation. Since successful fermentations require not only sufficient oxygen for metabolic activity, but also intimate contact between organism and substrate, lower agihtion intensity may result in detrimental effects which more than offset the increased oxygen supply. This is particularly true in entire fermentations or periods of fermentations where the oxygen demands are low. The choice Of an aerating system should be made on the basis of the particular characteristics of each fermentation. For a process largely respiratory in character or where mediums of low viscosity and solids content are used, like yeast propagation, a finebubble aeration system without mechanical agitation may be the most desirable due to the economy gained by the elimination of power costs. For the production of some antibiotics and similar products where respiratory activity is low in the critical stages of the fermentation, and where high solids mediums are often used, a fermentor with mechanical agitation may be better. Absorption Efficiency. The absorption efficiency, E, has been defined previously m the weight of oxygen absorbed per weight supplied by aeration in unit time. Calculations of absorption efficiencies for run 21, which was used previously as an example, are given in Table VI. In this case, values of rd and G are expressed in pound-gallon units. The absorption efficiency incresses over the fermentation as a result of the increased concentration gradient with lowering of the dissolved oxygen concentration. The optimum point of
1. The propagation of bakers’ yeast in aerated medium has been used as an experimental tool for the study of oxygen transfer and offers many advantages for studies of the physical processes associated with biological systems for chemical production. 2. The voltammetric determination of dissolved oxygen haa been adapted to the measurement of instantaneous concentration values in fermentation systems. 3. For any one sparger type and degree of mechanical agitation the oxygen absorption coefficient for a fermentor was found to vary as a power of the aeration rate. The power changes for different physical arran ements. 4. An absorption egciency may be calculated from the data obtained and is found in all cases to be very low. NOMENCLATURE
A
=
ci
=
CL
=
E
= = =
H kd
ko =
k~ = k,
=
po = pi
transfer area (interfacial area of bubbles), sq. ft. or sq. cm. dissolved oxygen concentration at the as-liquid interface (saturation concentration), gramsjiter dissolved oxygen concentration in the fluid bulk, grams/ liter oxygen absorption efficiency Henry’s law constant oxygen absorption coefficient, grams 0 2 abso;bed/(hr.) (liter)(unit concentration difference) gas film mas8 transfer coefficient, grams o2transferred/ (hr.)(sq. cm.) (unit concentration difference) liquid-film mass transfer coefficient, grams O2transferred/ (hr.)(sq. om.) (unit concentration difference) specific oxygen uptake rate by organism, grams 0%consumed/(hr.) (gram of dry tissue) partial pressure of oxygen in the gas bulk, atm. or mm.
H$ or mm.H4
= partial pressure of oxygen at the gas-liquid interface, atm. . -
rC = rate of reaction (oxygen with oxidizable substrate) = rate of oxygen absorption, grams 0 2 absorbed/(hr:)(liter) Td r0 = over-all rate of transfer of oxygen from the gas to the substrate r, = total oxygen uptake rate by the organism from the liquid, grams O2consumed/(hr.) iter) = mass flow rate of oxygen to ermentor, Ib. 02/@r.)(gal.) = superficial air velocity, ft./min. = dry tissue weight, grams dry tissuefliter = time
c
LITERATURE CITED
Table VI.
Calculation of Absorption Efficiencies for Run 21 (Figure 5)
Singlebubble sparger; agitator speed, 300 r..p.m.: a, 0.47lb. Ov’(h~.)(gal.), supphed by aeration] Time, Hr. k, =I CL rda % 0.064 13.1 2.73 x 10-3 0.299 X 10-1 0.50 14.6 3.14 X 10-3 0.383 X 10-1 0.082 1.00 15.7 3.69 x 10-8 0.454X 10-8 0.097 1.50 16.5 4.12 x 10-1 0.688 X 10-1 0.126 2.00 16.9 0.144 4.80 X 10-8 0.678X 10-8 2.60 0,179 5.68 X 10-8 17.7 0.840 x 10-1 3.00 0 rd hers has been converted to pounds of O r sbaorbed/(hour)(gallon).
-
Bartholomew, W. H., Karow, E. O., Sfat, M. R., and Wilhelm, R. H., IND.ENQ.CHEM.,42, 1801 (1950). Baumberger, J. P., Cold Spring Harbor Symposia Quant. Biol., 7, 195 (1939). Beoze, G. de, and Liebmann, A. J., IND.ENQ.CHEW,36, 882 (1944). Cooper, C. M., Fernstrom, G. A., and Miller, S. A., IND.ENO. CHEX.,36,604 (1944). Gaden, E,L.,Jr.. thesis, Columbia University, N. Y., 1949. Goddard, D.G.,in “Physioal Chemistry of Cells and Tissues,” by R. HBber, section 6, Philadelphia, Blakiston Co., 1945. Gottlieb, D., and Anderson, H. W., Science, 107, 172 (1948). Kolthoff, I. M.,and Lingants, J. J., “Polarography,” New York, Interscienoe Publishers Inc., 1946.
September 1950 (9)
INDUSTRIAL AND ENGINEERING CHEMISTRY
Lewis, V. M., arid McKenzie, H. A,, ANAL. CHEM.,19, 043 (1947).
Seidell, A,, “Solubilities of Inorganic and Metal-organic Compounds,” 3rd ad., Vol. I, New York, D. Van Nostrand Co., Inc., 1940. (11) Silcox, H. E.,and Lee, 8. B., IND.ENG.Caem.,40,1602 (1948). (12) Stansley, P. G., Schlosser, M. E., Ananenko, N. H., and Cook, M. H., J. Bact., 55, 673 (1948). (13) Stefaniak, J. J., Gsiley, F. B., Brown, C. S., and Johnson, M. J., IND.ENO.CHEM.,38, 666 (1946). (14) Stiles, H. R., Peterson, W. H., and Fred, E.B., J. Bad., 12, 427 (10)
(1926).
1801
(16) Umbreit, W. W., Burris, R. H., and Stauffer, J. F., “Msnometrio Techniques and Related Methods for the Study of Tissue
Metabolism,” Minneapolis, Burgeas Publishing Go., 1946. R.J., J . Cellzrlar C m p . Phyewl., 17, 263 (1941).
(16). Winder,
RECEIVED February 2, 1960. Presented before the Division of Agriculture and Food Chemistry at the 118th Meeting of the A M E R r C r N CHEMICAL BOCIETY, Atlantid City, N. J. Abstracted from a dissertation submitted by Elmer L. Oaden, Jr., in partial fulfillment of the requirements for the degree of doctor of philosophy in the faculty of pure science, Columbia University, 1949. Major part of work waa completed during Gaden’s tenure a8 a Du Pont fellow in chemical engineering. Contribution No.4 from the Chemical Engineering Laboratories, Enmneering Center, Columbia University N. Y.
Oxygen Transfer and Agitation in Submerged Fermentations MASS TRANSFER OF OXYGEN IN SUBMERGED FERMENTATION OF Streptomyces griseus W. H. BARTHOLOMEW, E. 0. KAROW, AND M. R. SFAT Merck dt Co., Inc., Rahway, N. J., R. H. WILHELM, Princeton Uniusroity, Princeton, N. J.
A theory of oxygen absorption by suspended mycelia in aerated nutrient broth is proposed. Diffusion mechanism steps include an oxygen transfer resistance at bubbles and other air-liquid interfaces, a resistance through cell clumps and liquid films around individual cells, and a mechanism which involves direct contact between cells and air bubbles. Direct and indirect experimental evidence is presented In support of the mechanisms. Air supply and agitation rate affect oxygen transfer resistancesin various ways and determine whether the local oxygen concentrations at the cells lead to a state of oxygen saturation or deficiency.
0
XYGEN transfer and agitation are important in maintaining a desirable environment for mycelial growth and antibiotic synthesis in submerged cultures. The design of deep tank fermentors and the establishment of optimum fermentation conditions depend upon proper choice and control of these variables. The problem of supplying adequate oxygen to fermentors arises because of the limited solubility of this element in water. A program was started several years ago in this laboratory to explore, measure, and analyze fermentation variables such as aeration and agitation from a biological-engineering point of view. The work was undertaken with the object of acquiring increased understanding of mechanisms by attempting to distinguish between and study separately the physical and biological rate variables involved in oxygen transfer. A background was thereby to be provided for logical procedures of translation from one scale of fermentation to another. Strains of Penicillium chrysogenum and Streptomyces gTiseus were the organisms used, but the techniques that were developed and conclusions that have been reached may also be of interest in other fermentations. An initial step in the program was to develop a versatile, laboratory scale fermentor, which in multiple unite would provide a rapid and economic means of investigating fermentation variables. Such a bench scale type of fermentor was constructed with a capacity of 5 liters (2). Fermentations in the unit were
found to be consistent with pilot plant and factory fermentations. The concepte and techniques set forth in this and the succeeding paper (8) have been used with success in translation of results among the three scales of operation. The laboratory fermentor was found to reduce the extent of research and development effort necessary on the pilot plant scale. The present pap& is concerned with the transfer of oxygen from air to the suumerged mycelium. Component rate steps and their interrelations were studied in a quantitative manner. Experiments were limited to studies with Streptomyces griseus and with uninoculated mediums. A second paper (8)deals with the effect of aeration and agitation upon mycelial growth, sugar utilization, and biosynthetic formation of penicillin and streptomycin. The literature (I, 7, 11, 14, 16, 17,28) dealing with oxygen transfer and agitation in various fermentation systems is extensive, but the number of quantitative studies in the field is limited. The aeration of water alone by means of spargers in agitated vessels has been studied by Cooper, Fernstrom, and Miller (IO). Becze and Liebmann (8) reviewed the literature on aeration in compressed yeast manufacture. Studies in aeration and agitation in penicillin fermentation have recently been reported by Brown and Peterson (@, and in yeast fermentation by Olson and Johnson (26). THEORY OF OXYGEN ABSORPTION B Y SUSPENDED MYCELIA IN AERATED NUTRIENT BROTH
Oxygen for mycelial growth and biosynthesis in industrial fermentations frequently is supplied by diffusion from air bubbles suspended and rising through the broth. The air usually is introduced through spargers near the bottom of the fermentor. Mechanical agitation is supplied by rotating propellers, turbine impellers, and similar devices. There is present a three-phase system of liquid, gas, and suspended solids. As a suggested theory, oxygen transfer from gas to organism may be divided into a sequence of primary steps, which are illustrated in a schematic manner in Figure 1. Within a bubble or at
