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Fermentation of
Molasses
RAPID CONTINUOUS
FERMENTATION PROCESS
H. R. BILFORD, R. E. SCALF,
W. H. STARK, AND PAUL J. ICOLACHOV
Joseph E. Seagram & Sons, Inc., Louisville, Ky.
Fermentation Conditions an outlet for the gas which was passed to scrubbers to
remove the alcohol vapors. A third tube was employed
Introductory work involved the determination of experi- to remove samples for analysis during the run and to
mental conditions necessary for the rapid fermentation of add ammonia for pH adjustment. The feed medium en-
pure glucose solutions fortified with yeast extract and am- tered the fermenter through a storage flask and connect-
monium phosphate. No attempt was made at first to ing tube. The completely fermented material is withdrawn
conduct fermentations on a continuous basis. It was by a draw-off tube.
desired only to obtain conditions that would completely During the initial phase of operation the sugar was com-
ferment the sugar present in the shortest possible time. The pletely fermented in 4 to 10 hours, depending on the initial
experimental conditions obtained and applied to the con- cell count and other conditions. Continuous operation was
tinuous fermentation system appear in Table I. started by feeding fresh (uninoculated) fermentation medium
of high sugar concentration a t a constant rate from the
storage flask into the fermenter, and the completely fer-
TABLE I. EXPERIMENTAL CONDITIONS FOR RAPIDCONTINUOUS mented material was withdrawn a t the same rate. The rate
FERMBNTATION of feed and draw-off was dependent on the material being
Equipment Single fermenter plus storage tank fermented, initial cell count, and other conditions. The
Feed Reducing sugar concentration, 10-16 grams/lOO entire system was kept free from contamination. It is
cc. depending on material; pH, 4.6-6.0 important for successful commercial operation that a pure
Fermentation Reducing sugar concentration 0.1-1.5 grams/100 culture system be constantly maintained.
cc. depending on material; pH, 4.6-6.0 ad-
justed during operation with NHtOH; tem-
perature, 32.2' C.; yeast count, 150-350 Fermentation of Glucose-Yeast Water
million cells/cc. ; agitation, mechanical or Cot
(1.8-4.8 liters/min. per liter of medium) Initial work on this system involved the continuous fer-
Supplements Depending on fermentation medium used
Cycle time 4-10 hours, depending on above conditions mentation of a medium composed of 10-12 per cent glucose
dissolved in 10 per cent yeast water containing 0.1 per cent
(NH4)2HP04. The results of an experiment in which the
rate for the system under certain experimental conditions was
Under these conditions it was possible to ferment con- determined are presented in Table IIA. The initial yeast
tinuously and completely solutions containing 12 grams cell count in the fermenter was approximately 150 million
sugar per 100 cc. in a 4-10 hour cycle. Only a single vessel per cc. The strain of yeast was Seagram No. 1, a variety of
was required. The reducing sugar concentration of the feed Saccharomyces cerevisiae. To prepare the yeast inoculum,
medium ranged from 10 to 15 grams per 100 cc., and the pH large flasks of medium were inoculated and aerated for 18
of the medium was adjusted to 4.5-5.0. Reducing sugar was hours at 30" C. Upon the completion of the aeration period
determined by the method of Stiles, Peterson, and Fred (3). the cell concentration was determined, the count usually
I n the fermentation vessel during continuous operation, the varying from 150 to 200 cells X 106 per cc. The necessary
reducing sugar concentration varied from 0.1
to 1.5 grams per 100 cc., depending on the
material being fermented. The pH of the fer-
STORACIL FLASK Con
menting material was kept a t 4.5-5.0 by SUI.AR S O L U T I O N
adjustment with ammonia every half hour (APPROX. 12.0G/IOOCCJ
CONVENTIONAL
ALCOHOL REQUIRE
PLANTS
FERMESTATION A GREAT
MANYFERMENTATION
VESSELS
amount was centrifuged and cells resuspended in 3000 cc. of system could be put into continuous operation. This is
fermentation medium. The inoculated medium was then about half the time necessary with a cell count of 150 million
placed,in the fermentation vessel, and agitation with 4.8 liters per cc. After 2.5 hours about 98.5 per cent of the sugar was
of carbon dioxide per minute per liter was begun. The tempera- fermented. The rate for the first 3 hours was 20 per cent per
ture of fermentation was held a t 32.2" C. Sugar and Balling hour. This rate was satisfactory; the sugar concentration
determinations were made every hour, and the pH was
determined every half hour. The pH was maintained a t 5.0
during the run by adjustment with 3 N ammonium hydroxide.
Under the experimental conditions employed, a period of TABLE OF GLUCOSE-
11. RAPIDCONTINUOUS FERMENTATION
YEASTWATERMEDIUM
5 hours was needed to ferment 98.8 per cent of the sugar in a
3 N Reducing Yeast Throughput,
solution containing 10 grams of sugar per 100 cc. At this NHIOH, Sugar, G./ Milliln Total
point the yeast count had increased slightly from 133 to 182 Hours pH Cc. Balling 100 Cc. Cells/Cc. %ol./Hr.
million cells per cc., and the stationary phase was finished. A . Initial Yeast Count, Approx. 150,000,000 Cells/Cc.
Continuous operation was then begun a t this point; that is, 0 5.03 10.2 10.28 133 Sta-
the feed of fresh medium into the fermenter and the draw-off 21 44 .. 42 00 7 6
9.9 6.92
8.S4 1:: {ti;-
of the fermented material from the fermenter at equal rates 3 4.25
6
8
5
4.7 4.04 ...
... phase
of 5 per cent per hour were started. This rate was main-
4
5
6
4.50
4.92
5.00
... 2.1
0.6
0.7
1.24
0.11
0.10
182
183
5
10
tained for only one hour and the rate was then increased to 10 7 4.85 i:i 0.6 0.10 196 10
per cent, for the sugar concentration indicated that the 8 4.75 3 0.6 0.10 15
9 4.75 3 0.7 0.12 ii9 15
system could accommodate the 5 per cent per hour easily. 10
11
4.65
4.60
4
4
1.1
1.1
0.50
0.41
...
160
15
13
The 10 per cent rate was held for the next two hours. The 12 4.30 7 0.7 0.32 ... 13
sugar concentration in the fermented medium during this 13 .... ... 0.7 0.57 165 13
period was very low. The cell count remained constant. B . Initial Yeast Count, Approx. 350,000,000 Cells/Cc.
The rate was therefore increased to 15 per cent per hour, and ...
... E {?&
-;
0 5.40 10.3 10.40
this rate was held for three hours. However, under the
1 3.30 15 6.6 5.12 ...
350
2 4.80 5 1.1 0.26
experimental conditions this rate appeared to be slightly too
rapid. The sugar concentration slowly increased and, after
2,
3.5
5.20
4.80
.5.. 0.9
0.7
0.16
0.11
36?
350
phase
20
20
3 hours a t this rate, had risen to about 0.5 gram per 100 cc. 2:; 2:; 0.6
0.6
0.10
0.10
...
325
20
26
Simultaneously the cell count dropped to about 160,000,000
per cc., indicating further that the rate was too rapid. The
7":8 :. 5 4j:;:. 6 5 5"3 0.6
0.6
1.8
0.32
0.58
1.33
340
310
163
27
34
35
rate was then decreased to 13 per cent; it appears that the 9.5 4.65 .... ..... ... 35
system could probably have operated a t 13 per cent per hour, C. Initial Yeast Count, Approx. 350,000,000 CelWCc.; Throughput Rate
Constant
since the amount of sugar and the cell count in the fermenter
medium remained constant a t a desirable level with the 13
0 6.30 ... 10.6 10.52