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Alcoholic

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.

Necessary conditions such as temperature,


agitation, pH, and cell count have been de-
termined for the development of a continu-
QUS fermentation process. A single-vessel W H E N THE FAST, COXTIWJOUS PROCESS I S FULLY
DEVELOPED,
THE MODERN BATCH FERMENTER SHOWN ABOVECAN B E CON-
continuous fermentation system has been VERTED T O A CONTINUOUS UXIT REPLACING A NUMBEROF
developed. BATCH
FERMENTERS
With the system and the proper condi-
tions molasses mashes containing 12-13
grams per 100 cc. of sugar can be fermented
of water, and lowering the pH t o 4.0-4.5 by the addition of
to completion in a 5-7 hour cycle. Yeast acid. Although molasses generally contains most of the
propagation is maintained so that inocula- nutrient substances required for fermentation, ammonium
tion is required only at the beginning of a salts, such as phosphates or sulfates, may be added t o the
run. mash to supply nitrogen or phosphates in which the molasses
The principal value of this process is the may be deficient. The prepared mash is inoculated with a
suitable strain of yeast grown on molasses mash. The
extensive reduction in equipment require- inoculum usually represents 4 to 6 per cent of the total volume
ments. of the main mash. The temperature of fermentation ranges
from 21.1" to 32.2" C., or slightly higher.
Most of the industrial alcohol companies modify the mash-
ing and fermenting procedures according t o their experience
H E success of any commercial process used in the and plant facilities. Therefore the above described procedure

T manufacture of ethyl alcohol by fermentation depends


on its adherence to certain fundamental factors vitally
related to the activities of the yeast cell. These factors in-
is to be considered, at best, a very general picture of existing
methods.
It was intended to develop an alcoholic fermentation proc-
volve the use of an optimum concentration of sugar, an ess which would be continuous and in which sugar concentra-
optimum pH, and an optimum temperature; the addition tions normally used in the batch methods would be fermented
of supplementary substances to the medium if it is deficient to completion in an appreciably shorter time. The con-
in any essential constituent; the inhibition of bacterial tinuous system was to involve a minimum number of fermen-
growth; the use of a vigorous strain of yeast with high alcohol tation vessels.
tolerance; and the maintenance of anaerobic conditions There are many reports in the literature describing con-
during fermentation proper. All of the above factors are tinuous processes for alcohol fermentation. Most of them
dependent on the nature of the raw material to be fermented. are from Russian investigators, but their systems involve
Beverage alchohol, except rum and wines, is produced from many phases, usually three to nine fermentation vessels.
grains, while industrial alcohol is produced principally from Also, the time of fermentation ranges from 34 to 48 hours,
molasses in this country ( 2 ) . depending on the material fermented and other experimental
The preparation of molasses, usually blackstrap, for conditions. I n 1941, Alzola patented a continuous process
fermentation requires adjustment of the sugar concentration for molasses fermentation, but his system also requires a
t o approximately 12 per cent or slightly higher by the addition number of fermentation vessels for continuous operation ( I ) .
1406
November, 1942 INDUSTRIAL AND ENGINEERING CHEMISTRY 1407

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

throughout the fermentation period. Besides


adjusting the pH, the ammonia is a source of
nitrogen for the yeast. The fermentation was
maintained at 32.2" C. (90" F.). This tem-
perature was found to give the fastest fermen-
tation without inhibition of the reproductive
activity of the cells. The initial cell count
ranged from 150-350 million per cc. As would
be expected, the fermentation was faster with
RUBBER STOPPER
the higher cell count. Mechanical agitation
or agitation with 1.8 to 4.8 liters of carbon
dioxide per minute per liter of medium has been
found necessary for rapid fermentation. Agita-
tion accelerates the fermentation by providing ADDITION T u e e FOR
SUOAR SOLUTION
better diffusion of nutrients and carbohydrate.
A diagram of the laboratory unit employed D R A W OFF

for the continuous fermentation system is


shown in Figure 1. It consisted of a single L l W l D LEVEL

fermenter, a wide-mouthed Pyrex jar or flask.


A two-fermenter system was initially em-
ployed successfully, but operations with one
fermenter were easier to control and produced
just as successful results. Inoculated fermen-
tation medium was placed inside the jar. ALOXITP BALL
Carbon dioxide was introduced through B
glass tube and dispersed by one-inch Aloxite
spheres. As stated before, mechanical agi- FERMENTeR
tation could be successfully substituted for
carbon dioxide, Another glass tube provided FIGURE 1. LABORATORY UNIT FOR CONTINUOUS FERMENTATION
1408 INDUSTRIAL A N D ENGINEERING CHEMISTRY Vol. 34, No. 11

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

per cent per hour rate.


The results of a similar experiment appear in Table IIB.
1
2
3
4
4.00
4.20
4.80
4.65
15
10
5
5
8.1
4.0
1.4
1.4
6.24
2.39
0.15
0.20
355
...
*..
365
350
{f::
tion-
phase
25
25
The rate of throughput for the system was determined with 5 4.70 3 1.2 0.25 357 25
6 4.70 3 1.1 0.32 351 25
an initial cell count of approximately 350 rather than 150 7 4.70 3 1.0 0.22 327 26
million per cc. These data indicate that 10 grams sugar per 8
0
4.80
4.75
3
3
1.0
1.0
0 37
0.13
334
321
25
25
100 cc. of medium were almost completely fermented in 2.5 10 4 70 3 1.0 0.11 331 25
to 3 hours. Therefore, after 2.5 hours of fermentation the
November, 1942 INDUSTRIAL AND ENGINEERING CHEMISTRY 1409

The three types tested in the continuous fermentation system


TABLE
111. RAPIDCONTINUOUS FERMENTATION
OF MOLASSES were Cuban blackstrap, refined, and beet molasses. The
3 N
Nn40n,
Reducing Yeast
Sugar, G./ Million
Throughput,
%
' Total
types and amounts of supplements necessary for the fermen-
Hours pH Cc. Balling 100 Go. Cells/Co. Vol./Hr. tation of the three kinds of molasses were determined. Cuban
A . Cuban Blackstrap, No Supplement blackstrap required no supplement, the refined molasses
0 4.92 18.0 12.02 required the addition of 75 mg. (NH&S04 per 100 grams of
1 4.80 5'' 17.3 10.27 396 molasses, and the beet molasses required 100 mg. of (NH&-
2 4.75 2.5 15.4 8.15 .,.
3
4
4.85
4.80
2
3
12.9
9.8
5.60
2.56
...
550 phase
HPOl per 100 grams.
5 4.70 3 8.3 1.03 ... 19 The rate of throughput for the one-fermenter system as
6 4.80 3 8.3 1.14 545 25 determined with Cuban blackstrap molasses is shown in
7 4.70 3 8.7 1.24 460 25
Table IIIA. The initial cell count of approximately 350
8
9
4.80
4.68 ...
2 9.0
9.1
1.25
1.31
417
515
25
25 million cells per cc. was obtained by aerating large flasks
E . Refined Molasses 4- 75 Mg. (NH4)&04/100 Grams containing 18' Balling molasses a t 28.8' C. for 18 hours.
0 5.10 ... 18.0 13.45 ... The cells were separated by centrifugation and resuspended
1
2
3
4.80
4.75
4.75
3
5
5
17.1
15.4
13.5
.....
9.87
4.11
...
315
... in the fermentation medium, which was of the same com-
4 4.56 8 11.4 ..... ... phase position and pH as the aeration medium. The other experi-
5 4.80 5 9.3 3.95 515 15 mental conditions, such as pH, temperature, and agitation,
6
7
4.80
4.60
5
7
7.2
6.4
1.69
1.12
...
580
15
20 were the same as for the work previously described.
8 4.70 6 6.5 ..... 580 20 A period of 5 hours was required to ferment 91.4 per cent of
9
10
4.80
4.83
5
5
6.9
7.1 .....
1.51 540
508
20
20 the sugar in the medium. This is essentially complete
C. Beet Molasses 4- 100 Mg. (NH4)sHP04/100 Grams fermentation of this material. The cell count had increased
0 4.50 ...
... 18.5
15.6
10.15
8.81
350
400
from the initial 350 to 550 million per cc. The continuous
1
2
3
4.45
4.50
4.45
...
... 12.9
10.2
6.10
3.77
... phase of the fermentation was then started at a 19 per cent
per hour rate of feed and withdrawal. Since this rate was
ii6 phase
4
5
4.35
4.50
4.48
...
2
...
7.6
6.8
6.8
1.07
0.53 ... 15
15
satisfactory, it was increased to 25 per cent. At this point
6
7 4.50 ... 7.2
e....
0.85 iA6 15 the sugar concentration slowly increased and the cell count
7.4 0.62 15 began to decrease, indicating too rapid a rate. The optimum
8
9
4.40
4.52 ...
1
... 7.2
7.2
0.87
0.76
ii6
360
15
15 rate of throughput for Cuban blackstrap under the above
10
10"
4.50
4.50 ...
... 7.2 0.76
3ii
15 described experimental conditions lies between 19 and 25
iia
120
4.50
4.50 ... 6.9
7.1
0.76
0.82 ... 15
15 per cent per hour.
a Mechanical agitation. Table IIIB presents data from a similar experiment in
which refined molasses was fermented. The addition of 5
grams of malt sprouts per 100 grams of refined molasses in
addition to ammonium sulfate was necessary to secure a large
in the draw-off was practically negligible, and the high cell cell crop for inoculum. This batch of refined molasses seemed
count was maintained. The rate was then steadily increased to lack certain necessary growth and fermentation factors for
to 26, 27, 34, and 35 per cent per hour. However, when the yeast. Other experimental conditions were the same as for
sugar analyses were made at a later time they indicated that the preceding experiment.
the 26-27 per cent per hour was slightly too high for suitable About 6 hours were required for the system to complete the
operation. stationary phase. This may have been due in part to the
Table IIC indicates the results of another similar experi- fact that the refined molasses contained more sugar than did the
ment except that the rate of feed and withdrawal was main- Cuban blackstrap. Refined molasses (18"Balling) was equiva-
tained a t 25 per cent per hour throughout the period of con- lent to about 13.5 per cent reducing sugar, while 18' Balling
tinuous operation. It was desired to determine whether the Cuban blackstrap was equivalent to approximately 12 per cent
experimental conditions employed and this rate would yield reducing sugar. At the completion of the stationary phase
successful results during a long operating period. The 91.6 per cent of the sugar was fermented, and the cell count
results confirm those of Experiment B (Table 11). The had increased from the original 315 to 515 million per cc.
medium containing 10 grams sugar per 100 cc. was 98.5 per Rate of feed and withdrawal was started at 15 and later
cent fermented in about 3 hours during the stationary phase. increased to 20 per cent per hour. The latter rate appeared
The continuous phase was started at this point. Proof of too rapid for the system under the experimental conditions.
the feasibility of the 25 per cent per hour rate is found in the A rate of throughput between 15 and 20 per cent per hour or
following facts: The sugar level in the fermeqted material a 5-7 hour cycle appears to be satisfactory.
remained between 0.1 and 0.4 gram per 100 cc. during the A third experiment of this type was run on fortified beet
entire 7-hour period of continuous operation, and the cell molasses (Table IIIC). On aeration it also provided a good
count was maintained a t its high initial figure. medium for the growth of yeast for inoculum. Conditions
were the same as those employed in the experiments with
Fermentation of Molasses Cuban blackstrap and refined molasses, except that toward
the end of the run mechanical agitation was substituted for
Since this system appeared to work satisfactorily on carbon dioxide agitation in order to determine the effect of
partially synthetic medium, it was decided to attempt the this type of agitation on the rate of throughput.
continuous fermentation of materials such as molasses, About 5 hours were required for the completion of the
Previous experimental work in these laboratories had in- stationary phase of the fermentation. The sugar was 94.7
volved only standard batch fermentations of various kinds per cent fermented a t this time. The cell count had in-
of molasses, but certain facts obtained were applicable to the creased from an initial 350 to about 530 million per cc.
fast, continuous fermentation work on molasses. The Rate of feed and withdrawal was begun at 15 per cent per
Seagram No. 1 yeast strain was not suitable for molasses hour. At first it appeared that this rate of throughput
fermentation, but the Seagram No. 90 strain, another variety would be too rapid, as the sugar increased slightly and the
of Saccharomyces cerm'siae Hansen (a Java molasses distillery cell count dropped. However, after the system reached an
yeast, American Type Culture No. 4125) was satisfactory. equilibrium point, the 15 per cent per hour rate appeared to
1410 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 Vol. 34, No. 11

be satisfactory. Substitution of mechanical agitation for Literature Cited


carbon dioxide agitation produced no noticeable effect.
Data on experimental work in which alcohol yields were (1) Alzola, Francisco, Mem. 14th conf. a n d , Asoc. tec. uzucar. Cuba,
1940,326-6.
determined on molasses continuously fermented in the ( 2 ) Prescott, 9. c., and ~ u n n C.
, G., “Industrial Miorobiology”,
system for a period as long as 31 hours have not been re- 1940.
ported, but it can be stated that the alcohol yields obtained (3) Stiles, H. R., Peterson, W. H., and Fred, E. B., J . Bact., 12, 428-
with the continuous system were comparable to those ob- 35 (1926).
tained’ from standard 50-hour batch fermentations Of the PRESENTED before the Division of Sugar Chemistry and Technology a t the
same material. 103rd hfeeting of the AMERICAN CHEMICAL SOCIETY, Memphis, Tenn

National Survival through Science


HARRY N. HOLMES
Oberlin College, Oberlin, Ohio

H E S I speak oi Peninsula had so little quinine that their resistance t o the


national sur\i\ sl Japs was tragically weakened by malaria. When the mos-
tlirougli science, I quito carrying yellow fever interfered with the digging of the
refer not only to the vit.11 Panama Canal, it was too much. General Gorgas and his
aid of science in ninniiig medical staff got rid of the mosquito.
this nar, but ilso to it, At this moment we learn of many scattered cases of the
great service in the difii- terrible black plague in the West, a plague with a high percent-
cult years that follon-. To age of mortality for which there is as yet no known cure,
the peqsimiits 11 ho believe Fleas, carried by rats, squirrels, and rabbits, transmit the dis-
that the Allies will lose and ease. Birds spread Rocky Mountain spotted fever.
thst tlic rnited States nil1 The tide of medical battle ebbs and flows, but the “men
finally be forced, by eco- in white” always gain ground. They even make side
nomic strangulation, to forays against laziness, once a sin, and prove that much of it
yield to Hitler’s orders I is caused by infected teeth, hookworm, malaria, hay fever, a
am coninelled to sav that deficiency of vitamins C and B1, and by other understandable
HARRYN. HOLMES under such t h r o t“tl i n g troubles. We hesitate to call this a new distinction between
our chief hoDe of survival virtue and vice.
as a free nation \vi11 lie Famines force modern improvements in irrigation and flood
in the resourcefulness of our scientists. control, stimulate the attack on crop diseases, force extension
The profound influence on our civilization of anesthetics of transportation, and may lead t o planned redistribution of
and antiseptics, the steam engine, the electric dynamo and populations and to birth control. ‘(Modern science”, said
motor, the telegraph, telephone, and wireless, the cotton gin, Vice President Wallace, “when devoted whole-heartedly to
portland cement, the pig iron furnace and steel mill, refrigera- the general welfare has in i t potentialities of which we do not
tion, and the motor car convinc:s every thoughtful person dream.”
that this is a scientific civilization. To be truly cultured yau
must have some understanding of the achievements, the T H E present disaster of a World JT7ar calls upon every re-
methods, and the possibilities of scientific research. source, material and mental, if we are t o survive as a free
Centuries ago recovery from great disasters such as plague, people. To say that we were ill prepared in a military way
famine, flood, war, and oppression was slow, fatally slow for is not enough. Our natural resources, which we viewed with
some nations. Medical science can now check pestilence in complacency, had led us to prodigal extravagance. Forests
most of its forms, although it did not check the world epi- disappeared and were seldom replaced. Antiquated farming
demic of virulent flu in 1918 until millions of lives had been methods permitted rains to wash away rich topsoil and de-
lost. The encouraging fact today is that science learns from pleted or ruined great areas by unwise cropping. The plow-
every disaster, be it yellow fever, typhus, bubonic plague, an ing of marginal grasslands of the West helped create the Dust
earthquake, a great flood on the Yellow River in China or on Bowl, now being improved by contour tillage, deeper plowing,
our own Mississippi. and alternate strip cultivation.
Typhus fever has killed 200,000,000 people in Europe and Our most alarming extravagance, it seems, has been in the
Asia during recorded time, and it is again threatening Europe use of mineral wealth. Since 1900 world consumption of
in the war areas. The body louse that carries it is said to mineral resources has exceeded that of all previous ages.
have done more than the Russian winter to defeat Napoleon. This acceleration cannot continue indefinitely. Recovery
We are told that Hitler is in great fear of a typhus epidemic of scrap metal must become part of a carefully planned na-
in his armies. The American and British armies have quan- tional economy. Substitution of products derived from the
tities of an effective serum. soil, such as wood, laminated plywood, and certain plastics,
Malaria, carried by a vicious type of mosquito, weakened can help in conservation of metals.
ancient Greece and Rome, helped bring them t o their fall. Power will not be produced indefinitely from coal and pe-
Recently you have read that MacArthur’s army in the Bataan troleum, so we will ultimately rely upon water power, alcohol

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