2554
INDUSTRIAL AND ENGINEERING CHEMISTRY
GRIGNARD SYNTHESIS. Following the method used by Overberger and Saunders (12) in the preparation of 3-chlorophenylmagnesium bromide, 2,5-dichlorophenylmagnesium iodide was prepared using 68 grams (0.25 mole) of 2,5-dichloroiodobenxene and 6.1 grams (0.25 mole) of magnesium ribbon in anhydrous ether. This was treated with acetaldehyde in the usual manner t o produce upon purification 30 grams (62.5%) of 2,5-dichlorophenylmethylcarbinol. This reaction is of interest in view of the difficulty frequently encountered in the formation of Grignard reagents from dichloro compounds having a chlorine adjacent t o the active halogen. DEHYDRATION. The method used was the same as in Procedure 1 to given an 85% yield upon dehydration and an over-all yield of about 35y0. Procedure 3. The 2,5-dichlorostyrene was prepared in the manner used by Brooks (3). The over-all yield was 34 to 35%, somenvhat higher than that given in the literature for this procedure. This is accounted for by the higher yield in dehydrating the carbinol over alumina rather than with potassium acid sulfate. Procedure 4. The 2,5-dichlorocinnamic acid was prepared in 94y0 yield using the method of Walling and Wolfstirn (IC). Successive attempts to decarboxylate the acid did not produce sufficient material to afford purification and identification. Procedure 5. BROMINATION AND ESTERIFICATION. Bromine (96 grams; 0.60 mole) was added to 105 grams (0.60 mole) of 2,5-dichloroethylbenzene in carbon tetrachloride. The product was isolated but not purified. The mixture of CY- and 8-bromoethyl-2,5-dichlorobenzene was treated with fused potassium acetate in acetic anhydride by the method of Marvel et al. (10). This produced a 63% yield of 1-(2,5-dichlorobenzene)-ethylacetate, boiling at 124’ to 127’ (7 mm.) and having the following physical
Vol. 45, No. 11
constants: n g 1.5242; d;: 1.2353. Molecular refraction: theoretical 57.89; found 57.34. PYROLYSIS. The pyrolysis was performed by dropping the ester through a vertical column containing glass beads that was heated to 425’ to 450’. tert-Butyl catechol was used as an inhibitor. The yield was 31% of the theoretical and gave an overall yield for 2,5-dichloroiodobenzeiie of 19%. LITERATURE CITED
Arndt, F., Org. Sgntheses, 20,27 (1940). Basdekis, C. H. (to Monsanto Chemical Co.), U. S. Patent 2,485,524 (Oct. 18,1949). Brooks. L. A.. J. Am. Chem. SOC..66. 1295 (1944). Brooks; L. A:, and Nazzewski, Matihew (to SGague Electric Co.), U. S. Patent2,406,319(Aug. 27, 1946). Carbide & Carbon Chemicals Corp., Brit. Patent 1316,844(Jan. 27,1949). de Crauw T., Rec. trav. chim., 51, 757 (1931). Erickson. E. R.. and Michalek, J. C. (to Mathieson Alkali Works, Inc.), U. S. Patent 2,432,737 (Dec. 16, 1947). Kshatriya, K. C., Shodhan, N. S., and Nargund, K. S., J . Indian Chem. SOC.,24,373 (1947). Lund, H., Ber., 70, 1520 (1937). Marvel, D. S., Overberger, C. G., Allen, R. E., Johnston, 1%. W., Saunders, J. H., and Young, J. E., 6.Am. Chem. SOC., 68,863 (1946). Michalek, J. C. (to Nathieson Alkali Works), Brit. Patent 564,828 (Oot. 16, 1944). Overberger, C. G., Saunders, J. H., Allen, R. E., and Gander, R., Org. Syntheses, 28,28-31 (1946). Roberts, E., and Turner, E. E., J Chem. Soc., 1927, 1855. Walling, C., and Wolfstirn, L. B., J . Am. Chem. SOC.,69, 852-4 (1947). RECEIVED for review April 1 5 , 1953.
A C C E P T E D July 23, 1953. Presented at Southeastern Regional hfeeting, AMERICANCHENICAI, SocmrY, Auburn, Ala., October 25, 1952. Performed under Contract CST-1020, Ordnance Division, National Bureau of Standards, Washington, D. C.
Aeration Studies on Propa ation of Baker’s Yeast WILLIAM D. ilIAXON AND MARVIN J. JOHNSOS University of Wisconsin, Madison, Wis.
T
HE study of the transfer of oxygen from the air into liquid cultures of microorganisms has been greatly stimulated in recent years by the increased industrial importance of aerobic submerged culture fermentations such as are used in large-scale production of antibiotics. The influence of air throughput rate, agitator power and speed as well as fermentation vessel, agitator, sparger, and baffle design upon the absorption of oxygen has been investigated by many workera in several different manners. Thorough investigations of the oxygen uptake rates of tsodium sulfite solutions in fermentation-type vessels under various conditions were made by Cooper, Fernstrom, and Miller (3). Data of this sort have value for the empirical evaluation of the aeration effectiveness of fermentation equipment but are limited by the physical dissimilarities between the liquid phase in such an aqueous system and that in an actual fermentation. Karow el al. ( I f ) have described the use of such data for fermentor design. Bartholomew et al. ( 8 ) and Wise ($8) have made direct studies of aeration in penicillin and streptomycin fermentations using polarographic measurements of oxygen concentration in the medium. Similar experiments have been carried out by Hixson and Gaden (9) in the submerged propagation of bakers’ yeast. The system chosen for the present study was the aerobic
propagation of the baker’s yesst organism, Saccharomyces cerevisiae. It is ideal in the following ways: 1. The organism grows well on a conipletely synthetic medium with glucose as the sole source of carbon. 2. The over-all metabolism of the organism is simple. The only major products are yeast cells, ethanol, and carbon dioxide. 3. The process requires air. The absence of air causes an easily measured change in metabolism, the increased rate of formation of ethanol. 4. The organism is enzymatically stable and is relatively easy t o keep free of contaminating microorganisms, There are three general methods for the propagation of haker’s yeast. When the complete growth medium is initially charged (a batch fermentation), the sugar is partially glycolized to ethanol, and the remainder is oxidized to carbon dioxide. Recent studies of this type of yeast propagation have been made by Swanson and Clifton ( 1 7 ) . I n order to provide a more economical conversion of sugar to cellular material, it is common practice to limit the rate of supply of sugar to the point where the yeast is able to convert i t completely to carbon dioxide. The usual means to this end is the addition of glucose a t exponentially increasing rates to a growing yeast culture-a slow feed fermentation. This is the type of
fermentation used for the manufacture of baker’s yeast. White and Munns (91)have recently made a study of this kind of operation. The third type of aerobic yeast propagation is the continuous fermentation. Here, a steady state is maintained. Glucose and other nutrients are fed slowly to a high population of yeast cells; this population is held constant by removing yeast from the fermentor at the rate at which it is growing. Such a system is most commonly used in the manufacture of food yeast, employing the organism Torula utilis. Descriptions of several largescale installations have appeared in the literature (10,14, 18). Pilot operations have been described by Harris et al. (7, 8 ) and by Thompson et al. (19). Continuous propagation of 8. cerevisiae has been studied on a small scale by Harris et al. (7), by Unger et al. (% andI) by,Adams and Hungate (1). In the present study, use has been made of both the batch fermentation and the continuous fermentation. The purpose of this research was to define the characteristics of these systems thoroughly so that their advantages and limitations for the study of aeration in submerged fermentation should be well understood. THEORETICAL RELATIONS
OXYQENTRANSFER IN FERMENTATIONS. The development of theoretical equations for oxygen transfer in submerged fermentations has been presented by Hixson and Gaden (Q), by Bartholomew et al. (b), and by Wise (98). A brief review of these considerations is necessary in order to define terms. Oxygen, in transferring from the bulk of an air bubble to the enzyme surfaces of a microorganism in submerged culture, may be considered to pass through three hypothetical films or concentrations of resistance. Between the bulk of the air bubble and the gas-liquid interface is the gas-film resistance. Between this interface and the bulk of the liquid medium is the liquid-film resistance. The remaining resistances, between the bulk of liquid and the cellular enzymes, are grouped as the cell-film resistance. The concentrations of oxygen a t each significant point have been designated as follows: p = partial pressure of oxygen in bulk of gas (atmospheres) p , = partial pressure of oxygen a t gas-liquid interface p , = partial pressure of oxygen in equilibrium with its concentration in bulk of liquid p c = partial pressure of oxygen in equilibrium with its effective concentration to the organism
If the diffusion process is occurring in steady state, the three transfer coefficients may be defined in the following manner:
rd
= ka(p
- Pi)
= kt(pi
- P*)
ko(p*
- Pc)
(1)
= rate of oxygen transfer in a fermentation, gram moles per
liter of medium per hour k, = transfer coefficient, gas to interface, gram moles per liter per hour per atmosphere kl = transfer coefficient, interface to liquid kc = transfer coefficient, liquid to cell Regrouping this equation leads to another relation that is useful in discussing the presently involved fermentation system: Td
where Kt
Since the rate of oxygen absorption from air by copper-containing sulfitq solutions is independent of sulfite concentration over wide ranges, the absorption reaction must be gas-film controlled. Since the oxygen concentration in the liquid is essentially zero, the equation for the chemical system becomes:
(6)
rs = k,p
All these transfer coefficients are, of course, highly dependent on the characteristics of the gas, the liquid, the microorganisms, and the manner in which they contact each other. They are not necessarily constant, even with respect to oxygen partial pressure. They cahnot be evaluated directly, but must be calculated from measured values of transfer rates and oxygen partial pressures according to the above equations. MATERIALBALANCE IN FERMENTATIONS. The study of metabolic rates in fermentations is greatly facilitated b y consideration of material balance equations for such systems. When a fermentation is operated in such a manner that there are streams both entering a n d leaving the fermentation vessel continuously, one may write a balance on each contributing substance according to the following equation:
FX, = XV = F’X
+ VdX/dO
(7)
where
F X,
rate of feed stream entering, liters per hour concentration of substance in entering stream, moles per liter X = rate of formation of substance, moles per liter per hour V = volume of li uid in fermentor, liters F’ = rate of with2rawal stream, liters er hour X = concentration of substance in w d d r a w a l stream d X / & = rate of increase of concentration of substance in fermentor, moles per liter per hour = =
I n a continuous fermentation in a steady state operation, F is equal to F’ and dX/dO is zero. For a product such as yeast which is not in the feed stream the equation becomes:
X/8h
5
(8)
where h = the holdup time, V / F . For a nutrient such as glucose
-x
=
(X,
- X)/Oh
(9)
For a product in the air stream such as carbon dioxide x = Fa(X
- Xo)/V
(10)
where F. = rate of air stream, liters per hour.
where Td
2555
INDUSTRIAL AND ENGINEERING CHEMISTRY
November 1953
5
= Kt(p
- pc)
(2)
transfer coefficient, over-all.
1/Kt = l/kg
+ l/kc = l / k o
(3)
I n the chemical system, the oxidation of sodium sulfite, the following relation is useful: re
= Kd(p
- p*)
(4)
where v8 = rate of oxygen transfer in sulfite oxidation, and Kd = transfer coefficient, gas to liquid. 1/Kd
l/k,
+ l/kr
(5)
These are the equations that lead to the calculation of metabolic rates in a continuous fermentation. In a batch fermentation, as in a continuous one, the growth of yeast behaves as a first order reaction. Thus:
x = KX
(11)
where K is a proportionality constant. I n a batch system it may be shown that
K = o.693/e9 (where e, in hours, is the generation time or the length of time required for the population to double itself. I n the continuous system, Equation 8 indicates that K = l / h . I f yeast is to be grown at the same rate in both types of operation, i t is evident that Oh s=
8g/O.693
(12)
This relation is useful in correlating results from one type of fermentation to the other.
2556
I N D U S T R I A L A N D E N G I N E E R I N G C H EM I S T R Y
Vol. 45, No. 11
When a high population of yeast is present in the fermentor, it becomes necessary to feed antifoam oil slowly in order to maintain a constant small amount present in the fermentation a t all times. Lard oil containing 6% Alkaterge C (Commercial Solvents Corp., Terre Haute, Ind.) is held in a reservoir at a head of about 3 feet above the fermentor. This head is kept constant with respect to the fermentor by means of a pressure equalization line. I n the antifoam feed line there is a metering valve which delivers oil aseptically a t a rate of less than 1 ml. per hour. FERMESTATIOAMETHODS.Three different synthetic media, as detailed in Table I, were used in these experiments. Medium A was developed by Olson and Johnson ( I S ) for maximum yields of S. cereaisiae y-30 in aerobic batch fermentation. Medium B is identical except that it contains no asparagine. Medium Figure 1. Schematic Diagram of Fermentor and Auxiliary Equipment Used C fulfills the requirements for use in coni n Continuous Propagation of Baker's Yeast trolled p H continuous fermentation at the 10% glucose level. APPARATUS AND EXPERIMENTAL METHODS Operation of a batch fermentation involves simply inoculation of the medium, which was previously sterilized in the fermentor, The fermentor that was used for batch propagation of yeast has been previously described by Olson and Johnson (13). The and agitation and aeration at appropriate rates. Inoculum is provided from pure cultures of S. cerevisiae y-30 that have been baffled vessel has a total volume of 3.5 liters, an operating capactransferred to 25 ml. of medium in 500-ml. Erlenmeyer flasks ity of 1.5 liters. Constructed of stainless steel, i t is provided and incubated on a Gump rotary shaker a t 250 r.p.m. for 18 hours. with a propeller type agitator of variable speed. Air is introThe inoculum is small; 0.2% by volume or 0.3 mg. of yeast (dry duced through a ring sparger located beneath the agitator. The weight) per 100 ml. of medium. fermentor is immersed in a constant temperature bath a t 30" C. Operation of a continuous fermentation is preceded by a batch Adaptation of this fermentor to continuous operation involved fermentation to provide the desired population of cells in the the auxiliary equipment diagrammed in Figure 1. Feed is fermentor. Sterile feed of the appropriate medium is then begun, added by a rotary positive displacement pump that can be operand the n-ithdrawal device is put into operation. Continuous ated aseptically. The pump may be adjusted to deliver from fermentation of medium A or B is somewhat simpler than with 50 to 1500 ml. per hour, the exact rate being estimated from the medium C since adequate pH control can be effected by elevation rate at which an enclosed, sterile, graduated tube is emptied. of the p H of the feed, and the automatic system described above The air outlet of the fermentor is arranged differently for batch was not used. Furthermore, it was not necessary to provide foam and continuous operation. In the continuous fermentation the control in the 1yoglucose fermentations. exhaust tube is introduced into the fermentor through packing For the continuous fermentation of medium C i t is not possible glands BO that it may be raised or lowered. I t s lower end is to provide a sufficiently high yeast population by a preceding placed close to the liquid surface so that as feed is added to the vessel, fermented medium is carried out with the air stream a t the same rate and this contant level is maintained in the fermentor. The exact level may be determined a t any time by use of a TABLE I. SYNTHETIC MEDIAFOR USE IN CONTINUOUS probe that is mounted in the fermentor top. This probe is inPROPAGATION OF Xaccharomyces cerevisiae y-30 sulated from the fermentor body and may be moved up or down For batch fermentations Medium A : adjusted t o p H 5.0 with 85% HsPO4 through two packing glands. As it touches the liquid it closes For continuous fermentation feed Medium A: 1 ml. 60% KOH/liter added after sterilizing a circuit and this indicates the surface level. Medium €3: 2 ml. 50% KOH/liter added, after sterilizing As air and liquid leave the exit line, they pass into a glass Medium C: no p H adjustment, automatic p H control separator. Fermented medium collects in the bottom where it Medium A or B. Grams Medium C, Grams may be sampled; air passes out the top. A slight pressure head Glucose is maintained in the separator in order that the air may be anaNH4HaPOc K&POa lyzed for carbon dioxide. hlgSOi. 7H10 When i t is desired to control pH, a medium of low nitrogen Nas Citrate L-Asparagine content is employed (Table I, medium C). The greater part of Medium A or B, the nitrogen requirement is then met by slowly feeding concenMg. Medium C, Mg. trated aqueous ammonia, A second rotary feed pump is used 0.02 0.2 Biotin 0.50 5.0 C a Pantothenate for this purpose. This pump will deliver up to about 1 ml. per 10.00 100.0 maso-Inositol minute but doee not operate continuously. It is actuated b y 4.40 44.0 Thiamine, HCI 1.20 12.0 Pyridoxine. HC1 an electronic controller. This controller is operated by a p H 1.76 17.6 ZnSO4.7HsO 1.05 10.5 FeSO~(NHdk30a.6HzO meter, which in turn responds to the condition of a glass electrode 0.096 0.96 cusec. 5Hz0 mounted in the side of the air liquid separator, This electrode t o 1 liter to 1 liter Distilled water is placed in such a position that liquid leaving the fermentor a Medium B is identical with medium A except that it contains no asparagine. drops directly onto it, constantly bathing it. The p H variation allowed by this equipment is rf 0.1 unit.
November 1953 I
INDUSTRIAL AND ENGINEERING CHEMISTRY I
I
1
I
I l l
I
I
1 ' 1
I
1 1 1
800
2557
showed the distribution coefficient a t 30" C. to be 3.7 x 10-8 (concentration in gas/concentration in li uid). The ratio of acetaldehyde to e t h a n a concentration in the medium is quite constant. Therefore, in some cases acetaldehyde was not determined but was assumed to be present in a constant ratio to ethanol. CARBONDIOXIDE.The exit gas stream from the fermentor was analyzed for carbon dioxide by the method described by Maxon and Johnson ( I d ) . Correction of all analyses for the evaporation of water and for the removal of samples was made when necessary. RESULTS AND DISCUSSION
I
I
I
I I l l
I
I
I
I
1
1 1 1
EFFECTIVEAERATION. The oxygen uptake rates of sulfite solutions (r.) in this fermentor have been determined for a variety of air throughput rates and agitator speeds. The values obtained are given in Figure 2. Logarithmic coordinates are used to demonstrate the expected exponential relation of r. and air rate. The slope of the lines is 0.4. Four rates of agitation permit the provision of effective aeration from 10 to 700 millimoles of oxygen per liter per hour by this fermentor. Variation of the operating liquid volume from 1500 ml. causes a percentage change in re as indicated in Figure 3. 180 160
Effective aeration measured by sulfite oxidation method of cooper, Fernstrom, and Miller (3)
batch fermentation alone. In this case the batch period must be followed by a period of continuous operation a t low feed rate continued until maximum population is reached. Use of automatic pH control and slow addition of antifoam is necessary in these 10% glucobe continuous fermentations. All fermentations were carried out under aseptic conditions in order to maintain a pure culture. A sensitive test for the presence of contamination was used, a modification of the method of Green and Gray (6). Suspected samples were inoculated into a rich medium containing 5 y per ml. of the antifungal antibiotic, actidione. This medium completely inhibits the growth of S. cerevisiae but most contaminating organisms will grow rapidly and are thus readily detected although present in the tested culture in only small numbers.
140
iao 100 80 80 1300
1500
1700
LIQUID VOLUME
lP00
- ML.
2100
Figure 3. Effect of Liquid Volume on Effective Aeration in Fermentor
ANALYTICAL METHODS
)r
EFFECTIVEAERATION( r e ) . The rate of oxygen transfer to aqueous solutions of sodium sulfite was determined according to Cooper, Fernstrom, and Miller (3). YEASTDRYWEIGHT. Samples were centrifuged, washed twice with distilled water, dried in an oven a t 110"C. for 12 to 24 hours, and weighed. Indirect measurement was often made by determining turbidity' in an Evelyn colorimeter with a 600-mp filter. Such determinations were calibrated frequently by direct weighing. GLUCOSE. Glucose was determined by the method of Shaffer and Somogyi (16). ETHAKOL.Five ml. from a 7-ml. sample containing 5 to 10 mg. of ethanol were distilled into 5 ml. of 0.2 N otassium dichromate in 5 N sulfuric acid. The mixture was tighify stoppered, steamed for 30 minutes, then cooled, and titrated with standard sodium thiosulfate after the addition of excess potassium iodide. The evaporation of ethanol from the fermentor was calculated from a determined value of the air to water distribution coefficient for ethanol a t 30" C . of 0.304 X 10-3 (concentration in gas/ concentration in liquid). This figure was found by aerating dilute aqueous solutions of ethanol in the fermentor and measuring the decrease in concentration. Friedemann and Graeser (6) determine lactic ACETALDEHYDE. acid by oxidizing it to acetaldehyde and collecting the acetaldehyde in a sodium bisulfite solution. The amount of bound sodium bisulfite is then determinediodometrically. Their method, excluding the oxidation step, was used here. Determinations of acetaldehyde in the fermentor gases were made by passing them through an absorption train containing sodium bisulfite solution. Determinations in liquid were made by distilling a suitable sample into sodium bisulfite solution. Simultaneous determinations
TIME
- HOUFS
Figure 4. Chemical Changes Occurring during a Typical Batch Fermentation by Baker's Yeast
Figure 4 is an illustration of the chemiBATCH FERMENTATION. cal changes occurring during the batch fermentation of medium A by S. cerevisiae y-30. It is evident that two phases of growth occur. I n the first phase glucose is used, yeast grows exponentially with a 1.7-hour generation time to a yield of 14 grams (dry weight) per 100 grams of glucose, and ethanol is formed; 67% of the glucose is glycolyzed to ethanol in this period. When the glucose initially present is completely used, the yeast, after a short lag, begins growth a t a lower exponential rate (generation time, 8.5 hours), using the ethanol formed in the first phase. The yield in the second phase is 60 grams per 100 grams of ethanol and the over-all yield is 33 grams per 100 grams of glucose. If the effective aeration provided to the fermentor was increased by increasing the air rate, a marked decrease in final yield resulted. Olson and Johnson ( I S ) iirst reported this effect.
INDUSTRIAL A N D E N G I N E E R I N G CHEMISTRY
2558
Increased agitation rates, however, did not appreciably affect the final yield. Closer study of batch fermentation conducted at an agitation rate of 100 r.p.m. and 5000 cc. per minute of air throughput led to a clarification of these observations.
I 0.04
0
c
e
--
4
ACETALDEHYDE/ETHANOL CARBON DIOXIDE EVOLUTION
8
12
16
TIME
-
20
24
28
32
HOUFS
Figure 5 . Comparison of Carbon Dioxide Evolution and Acetaldehyde to Ethanol Ratio in Medium during Batch Fermentation
A comparison of the carbon recoveries for the low air rate fermentation (93.1%) and the high air rate fermentation (67.4%) indicated that the loss of carbon could well account for the decrease in yield from 33 to 23%. Calculation of the loss of ethanol according to the air-water distribution coefficient increased the carbon recovery for the low air rate to 96.S% and for the high air rate to 87.3%. Acetaldehyde was present in exit gases in significant amount; Figure 5 shows the ratio of acetaldehyde to ethanol in the medium to approximate 0.03 throughout most of the fermentation. The higher volatility (more than ten times that of the ethanol) of acetaldehyde causes a significant loss of carbon. Recalculation of recoveries using this correction results in 9S.OoJ, for the low air rate and 94.3% for the high air rate. Search for further carbon losses is probably not warranted. Table I1 illustrates the major differences between the high and low air rate fermentations. If the amount of yeast formed in the second phase is corrected according to the loss of ethanol and acetaldehyde, the total yeast formed is the same in both cases. Also, the amount of yeast formed in the first phase and the amount of ethanol and acetaldehyde formed in the first phase after correction for evaporation are not decreased b y increased
OF HIGHAIR THROUGHPUT RATESON TABLE 11. EFFECT
METABOLISM O F YEAST I N BATCHFERMENTATION Medium A
Agitator speed: 1100 r.p.m. Low air rate: 400 ml /min * rt = 120 millimoles Oz/(liter)(hour) High air rate: 5000 ml./mih.; T S = 330 milhmoles Oa/(liter)(hour) Low Air Rate, High Air Rate, Grams/Liter Grams/Liter Yeast formed (first phasela 1.31 1.52 Yeast formed (second phase) b 1.93 0.80 Ethanol and acetaldehyde used (second phase)
3.10
1.36
Ethanol and acetaldehyde evaporated (over-all)
0.35
2.10
Ethanol and aoetaldehyde used (second phase) 3.45 3.46 Corrected for evaporation Yeast formed (second phase) 2.15 2.03 Correoted for evaporation Y e a h f o r m e d (total) 3.46 3.55 Correoted for evaporation a First 16 hours a t low air rate, first 18 hours a t high air rate. 1) Sixteen t o 32 hours at low air rate. 18 t o 28 hours a t high air rate.
Vol. 45, No. 11
effective aeration. Therefore the pattern of fermentation illustrated in Figure 4 is not in any way the result of a limited supply of oxygen. The fact that a high proportion of the glucose used in the first phase is glycolized must be due to some other limitation to oxidation. The figure for the ratio of acetaldehyde to ethanol as shown in Figure 5 is apparently of some significance. The variation in this ratio throughout a batch fermentation closely follows the changes in metabolic activity as measured by the rate of carbon dioxide output. Further investigation of this observation would seem warranted. Figure 5 also illustrates the interesting pattern in the carbon dioxide evolution during a batch fermentation. CONTINUOUS FERMENTATION. Further investigation Of the effects of aeration and other environmental factors on the metabolic behavior of growing yeast is difficult in the batch fermontation. Rapid changes in yeast population, pH, nutrient, and product concentrations mask the effect of independent variables such as agitation and aeration. The continuous fermentation at steady state eliminates the time variable and is, therefore, better suited t o these studies. The presence of asparagine in medium A as a secondary source of carbon is an undesirable complication to the study of metabolism. Although it is necessary for optimum growth rate in a batch fermentation, such apparently is not true in a continuous fermentation. Here, media without asparagine are satisfactory. A summary of the conditions employed for a typical continuous fermentation appears in Table 111. The determinations that were made after the achievement of steady state as well as the results calculated from them are also listed. The effective aeration was found from Figures 2 and 3 according to the measured agitator speed, air rate, and liquid volume. The pH was controlled at 4.65. There appears to be little p H effect in these fermentations. No differences were detected between control a t pH4.5 andpH5.3.
TABLE111. CONDITIONSAND RESULTS IN REPRESEXTATWE CONTINUOUS FERMENTATION
Medium C Air rate: 6100 ml./min. Agitator speed: 1100 r.p.m. Feed rate: 311 ml./hour Effective aeration (rs): 390 millimoles Oz/(liter) (hour) PH: 4.65 Liquid volume: 1440 ml. Holdup time: 4.63 hours Carbon hletabolism Rate Oxygen Demand, Concentration, hfillimoies' ~illirnoies Compound Grams/Liter C/(L.) (Hr.) 02/(L.) (Hr.) Glucose 103,9 - 746 In out 0.6 Yeast (dry weight) 43.5 35s -2s. 1 Ethanol 1.2 11.3 -5.6 Evaporated ... 4.0 , -2.0 Acetaldehyde 0.06 0.6 -0.2 Evaporated ... 2.5 -0.6 349 $349 Carbon dioxide 3.42 (net %) Results Carbon recovery: 728/746 X 100% =. 9,7.3% Calcd. oxygen uptake rate ( r d ) : 310 millimoles Oz/(liter) (hour) Yield: 43.5/103.4 X 100% = 42.1% Glucose glycolyzed: utilization rate: 3.7%2.86 millimoles/(liter) (gram yeast) Yeast production capacity: 9.4 gram/(liter) (hour)
Determinations were made of glucose in the feed and in the effluent liquid, of yeast and ethanol in the effluent liquid, and of carbon dioxide in the exit air. In all experiments less than 1% of the glucose fed remained in the effluent liquid. Acetaldehyde occurred in a ratio to ethanol of 0.05 in continuous fermentation. Use of the relations illustrated by Equations 8, 9, and 10 and conversion to the basis of carbon content permitted calculretion of carbon metabolism rates for each compound. The evapore tion rates of ethanol and acetaldehyde were calculated from the distribution coefficients. The oxygen demand, the amount of oxygen necessary to convert
INDUSTRIAL AND ENGINEERING CHEMISTRY
November 1953
TABLE IV.
2559
COMPARISON OF EFFECTIVE AERATION WITH OXYGEN UPTAKNRATEOF YEASTIN CONTINUOUS FERMENTATION UNDER LIMITEDAERATION
Aeration Conditions Agitator speed, Air rate, Liquid volume, r.p.m. ml./min. ml.
Effective Aeration (Ts) I
Millimoles Oi/ (L.)(Hr.)
01 Uptake,
Yeast Concn Grams/L&r
Glucose GlYCob’Zed‘~ % With excess 01 Observed
(7d)
Millimdles On/ (L.)(Hr.)
Ta/Vd
15.0 16.2 18.5
0.79 0.89 1.02
Medium A
a
500 500 500
150 250 480
2100 2100 2100
750 750 1100
2700 5800 1820 6100 6 100 5800
1700 1460 1600 1460 1440 1480
0
11.8 14.4 18.8 137 218 214 380 385 369
0 0 Medium C 17.0 5.0 9.5 40 0.5 0.5
21.6 16 3 6.4
3.7 4.6 5.4
62.9 46.2 41.9 15.8 3.7 2.7
19.0 29.1 24.1 38.3 43.5 41.6
1100 1100 1100 Average Increase in glycolysis over that expected with excess On (see Figure 5) indicates that aeration is limiting.
139 189 219 296 3 13 342
0.99 1.14 0.98 1.28 1 23 1.05 1.04
x
glucose stoichiometrically to each product, was determined. The following equation demonstrates the reaction involved for ethanol : CeHizOe - 302 .--)3CzHs0 3H20
-
Thus, -0.5 mole of oxygen per mole of ethanol carbon formed is used to convert glucose to ethanol. The data of Sperber (26) were used to determine an empirical formula for yeast and, thereby, the oxygen demand of this product. An algebraic summation of the demands of each product, expressed as millimoles of oxygen per liter per hour, results in the calculated oxygen uptake rate ( T d ) as given in Tables 111and Iv. 70
-
BO
-
MEDIUM B (PH 5.0
50
1
X
M E D N M C (pH 4.6
- 5.a) 4.7)
EXCESS AERATION
PERCENT YIELD
are present in greater abundance, operate t o supply additional energy for growth. The same phenomenon was noted in the first phase of the batch fermentation, where glucose was also supplied in excess. Similar results were obtained in the continuous fermentation of medium A. Here the additional carbon source (asparagine) increased the maximum yield from 46 to 57 grams of dry yeast per 100 grams of glucose and the additional load on the oxidative enzymes decreased the critical feed rate from 3.5 to 2.7 millimoles of glucose per hour per gram of yeast (holdup time, 3.6 hours). As the effective aeration supplied to a continuous fermentation becomes limiting the changes in metabolic pattern illustrated by Figure 7 occur. Since the feed rate of 1.4 millimoles of glucose per gram of yeast per hour is below the critical, effective aeration supplied in excess allows a high yield of yeast and little glycolysis. Decreasing r. below about 30 millimoles of oxygen per liter per hour limits the oxygen available to the oxidative enzymes of the cells, and glycolysis occurs to provide the required energy for growth. A sharp decrease in yield accompanies this change in metabolism.
- 25 @-E .NT
20
10
YIELD
4
GLUCOSE GLYCOLYZED GLUCOSE FEED RATE = 1.7
h
-
(LITER)(HR) MEDNM A
a
MILLIMOLES GLUCOSE PER HOUR PER GRAM OF YEAST (DRY WEIGHT)
Figure 6. Effect of Glucose Feed R a t e on Metabolism of Baker’s Yeast under Excess Aeration
.
8
4.8-5.0
20
P E!
r, is 240 millimoles of oxygen/(liter)(hour) for medium
B experiments; r, is approximately 10 X rd. r, ie not constant in medium C experiments, but is kept greater than 1.4 X rd.
Figure 6 shows the effect of varying the rate of glucose feed on the metabolic pattern in a continuous fermentation. Excess aeration was provided. Similar results were obtained with medium B (a low yeast population) and with medium C (a high yeast population). I n both cases there is a critical feed rate a t which the maximum yield of yeast is obtained. Below this value of 3.5 millimoles of glucose fed and utilized per hour per gram of yeast (holdup time, 3 hours) a higher proportion of carbon dioxide is evolved, and the yield decreases. More sugar is used for maintenance and less for growth. At feed rates above the critical a portion of the glucose is glycolyzed to ethanol. The operation of this lower yielding metabolic process causes the observed decrease in over-all yield. Apparently, the oxidative enzymes of the yeast have been saturated and the glycolytic enzymes, which
0
15W
PERCENT GLUCOSE
0
so
BO
E F F E C T N E AERATION
-
X120 150 mM. 02/(LITER)(RR) 80
Figure 7. Effect of Effective Aeration on Metabolism of Baker’s Yeast Fed Glucose a t Less than Critical Rate Effective aeration measured by sulfite oxidation method of Cooper, Fernstrom, and Miller (3)
The occurrence of fermentative metabolism in yeast fed a t less than the critical rate is due to limiting oxygen. At feed rates greater than the critical limiting oxygen can be recognized by an increase in the amount of glycolysis over that occurring with excess air. I n both cases it is probably true that the oxygen pressure a t the enzyme surfaces, peris negligible with respect to the
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
2560
oxygen pressure in the gas, p . With this assumption incorporated into Equation 2 the transfer of oxygen in an oxygen limited fermentation may be represented as rd
=
Ktp
Further combination with Equation 3 gives rJrd = 1 -t k , / h
+ k,/k
(15)
Equation 15 in the light of the facts of Table IV indicated that, in this fermentation, kl and IC, must be very large with respect to k,. Therefore, in this particular biological system, the transfer of oxygen is controlled by the gas film resistance. The combined resistances of the liquid film and the cell film average only 4y0 of the gas film resistance. The oband a t the highest are served deviation in rs/rd from the average is probably due to the accumulation of experimental errors. YEASTPRODUCTION CAPACITY.Since the fermentor described in these experiments is capable of providing extremely high effective aeration in comparison to commonly used fermentation equipment it is of interest t o compare the productive capacity of this unit with that obtained by other workers. The significant figure in this respect is the rate of production of yeast (grams dry weight) per unit time (hour) per unit of liquid volume (liter). Table V lists the conditions and results of the continuous fermentation that gave the highest yeast production capacity in this aeries of experiments--13.2 grams per (liter) (hour). Harris et al. ( 7, 8) achieved the following production capacities for the continuous fermentation of wood hydrolysates in a 34liter Waldhoff-type fermentor: T. utilis, 9.1 gram/(liter)(hour); and S. cerevisiae, 5.5 grams/( liter)(hour). These are the highest values that could be found in the literature for continuous yeast propagation.
TABLE V. CONTINUOUS FERMENTATION OF HIGHYEAST PRODUCTION
becomes limiting. A n equation relating yeast production capacity to this primary fermentor design characteristic, effective aeration, would therefore be useful.
(13)
With this in mind it is of interest to compare r d for a number of limited aeration fermentations with effective aeration ( T , ) determined chemically under identical conditions of agitator speed, air rate, and liquid volume. Table IV shows the result of such a comparison. The ratio of rs to r d is close to 1.0. This is true throughout a range of yeast concentrations from 4 to 40 grams per liter and is nearly independent of aeration conditions. A combination of Equations 6 and 13 for r, and r d , respectively, gives rs/rd = IC,/Kt (14)
CaPACITY
Conditions (Medium C) Air rate: 6100 ml./min. Effective aeration ( r a ) : 580 millimoles Oz/(liter) (hour) Liquid volume: 1480 ml. Holdup time: 3.14 hours Agitator speed: 1600 r.p.m. Feed rate: 464 ml./hour p H : 4.6 Results Yield: 39.9% Glucose used: 99.5% Glucose glycolyzed: 2.5% Yeast population: 41.5 gramsfliter Calcd. oxygen uptake rate: 410 millimoles Oz/(liter) (hour) Yeast production capacity: 13.2 grams/(liter)(hour)
Feustel and Humfeld (4)describe experiments in a small fermentor, in which T. utilis and S. cerevisiae were grown batchwise. Calculations show that this fermentor could supply the following yeast production capacities if operated continuously: T.utilis, 15.5 grams/(liter)(hour); and 8. cerevisiae, 9.3 grams/(liter) (hour). The primary limitation on production capacity is aeration. The maximum population of cells that will reproduce in good yield if red a t the optimum rate is reached when oxygen transfer
Vol. 45, No. 11
rd
= (3330,’~
- 41.3)C
(16)
where y = yield, grams of yeast (dry weight)/100 grams of glucose, and C = yeast production capacity, grams of yeast/(liter) (hour ). This equation depends upon several assumptions for validity; 1. Glucose is the sole carbon source, and it is completely used. 2. Carbon dioxide and r a s t are the only products. 3. The composition o f t e yeast is, according to Sperber (16): 45.9% carbon, 31.4% oxygen, 7.3% nitrogen, 6.7% hydrogen, ~ 3 . 7 7ash. ~ 4. Oxygen transfer is gas-film limited.
These assumptions are approximately true for the fermentation system described here if the feed rate is held below critical. The validity of the assumptions must be determined for other systems before the equation can be accurately applied to them. The equation would have value, where it is valid, in predicting the effective aeration (as determined by sulfite oxidation) required for a desired yeast production capacity. It could be used also in estimating the maximum capacity of an existing fermentor. For example, the fermentor used here has a maximum effective aeration of about GOO millimoles of oxygen per liter per hour. With an expected yield of 45%, Equation 16 predicts a maximum capacity of 18.3 grams per (liter)(hour). I n actual installations it is necessary to consider as a basis for production capacity not the quiet liquid volume but the m x e economically significant operating volume. This volume is that of the aerated liquid plus the accompanying quantity of foam. I n considering a practical aeration system, therefore, one must not only maximize the effective aeration, but also minimize the ratio of operating volume to quiet liquid volume. LITERATURE CITED
(1) Adams, S. L., and Hungate, R. E., IND. ENG.CHEX., 42, 1815 (1950). (2) Bartholomew, W. H., Karow, E. O., Sfat, M. R., and Wilhelm, R. H., Ibid., 42, 1801 (1950). (3) Cooper, C. M., Fernstrom, G. A., and RIiller, 8. A., Ibid., 36, 504 (1944). Feustel, I. C., and Humfeld, H., J . Bacteriol., 52, 229 (1946). Friedemann, T. E., and Graeser, J. B., J . Biol. Chem., 100, 291 (1933). Green, S. R., and Gray, P. P., Arch. Biochem. Biophvs., 32, 59 (1951).
Harris, E. E., Hannan, M. L., and Marquardt, R. R., IXD. ENC. CREM.,4 0 , 2068 (1948).
Harris, E. E., Saeman, J. h l . , Marquardt, R. R., Hannan, M. L., and Rogers, S. C., I b i d . , 40, 1220 (1948). Hixson. A. W.. and Gaden. E. L.. Jr.. Ibid.. 42. 1792 (1950). Inskeep, G. C., Wiley, A . J., Holdberry, J. k.,and Hughes, L. P., Ibid., 43, 1702 (1951). Karow, E. O., Sfat, M. R., and Bartholomew, W. H., Abst. 121st Meeting, AMERICAN CHEMICAL SOCIETY, 12A (1952). RIaxon, W. D., and Johnson, M.*J.,Anal. Chern., 24,1541 (1952). Olson, B. H., and Johnson, M. J., J . Bacteriol., 57, 235 (1949). Saeman, J. F., Locke, E. G., and Dickerman, G. K., Paper Trade J . , 123, hTo. 12, 38 (1946). Shaffer, P. A., and Somogyi, M . , J . Biol. Chem., 100, 695 (1933). Sperber, E., Arkiv. Rerni. Mineral., Geol., 21A, No. 3 (1945). Swanson, W. H., and Clifton, C. E., J . Bacteriol., 56, 115 (1948). Thaysen, A. C., Food, 14, 116 (1945). Thompson, J. H., Neal, G. H., and VanArsdel, W. B., Abst. 118th Meeting, AMERICAK CHEMICAL SOCIETY, 23A (1950). Unger, E. D., Stark, W. H., Scalf, R. E., and Kolachov, P. J., IND. ENG.CHEM.,34, 1402 (1942). White, J., and Munns, D. J., Wallerstein Labs. Comrns., 14, 199 (1951).
Wise, W. S., J. Gen. Microbiot., 5 , 167 (1951). RECCIVED for review February 28, 1953. ACCEPTEDAugust 17, 1953. Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported i n part by a grant from Red Star Yeast and Products Go., Milwaukee. Wis.
