engineering

Acetone-Butanol and
Fermentation of Sugars Prrocess
development

SAMUEL C. BEESCH1
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production of acetone and butanol by the Weizmann sugar concentrations utilizable. The isolation of Clostridia ca-
THE process has been described in some detail by Killeft'er (15), pable of utilizing saccharine materials may be accomplished in
Gabriel (8), Gabriel and Crawford (9), and Prescott and Dunn many ways, One consists of the introduction of a small amount
(i?7). The original Weizmann organism (Clostridium acetobutyli- of soil, manure, roots of leguminous plants, cereals, decayed wood,
cum) was isolated with a view to breaking down starch to solvents. cornstalks, sewage, or river bottom mud into a sterile 4% sugar
Later investigators found it was possible to substitute up to 50% mash (sugar supplied as invert molasses) plus 4% ammonium
of the weight of carbohydrate with molasses. This procedure, sulfate, 5% calcium carbonate, and 0.3% phosphorus pentoxide
however, lengthened the fermentation cycle abnormally. (amounts based on the weight of the sugar). This mash is
In 1936, the first commercial application of a new fermentation usually tubed in 25-ml. quantities, using tubes 200 X 25 mm. in
utilizing molasses or sugar as the sole source of carbohydrate was size. These inoculated mashes are then subjected to heat shock
discovered. This fermentation proved to be more economical by placing the tubes in boiling water for 2 minutes. The heat
from a commercial standpoint for the following reasons: shock destroys most vegetative forms leaving the spores. The
tubes are cooled to 87° F. and then are placed in a desiccator
1. The mash is sterilized at lower temperature and is much
easier to handle. partly filled with moistened wheat or oats. A vacuum of about
2. The ratio of solvents is more desirable in that less ethanol is 29 inches is pulled on the desiccator and it is then placed in an
produced, with a corresponding increase in butanol. incubator at 87° F. for 24 to 48 hours. Normally, in 24 hours the
3. The fermentation is run at 87° F. instead of 98° F., provid- tubes begin to evolve gas, and the color of the mash turns to
ing a less favorable temperature for contaminating organisms.
4. Tanks and equipment are much easier to clean with water light tan. It is difficult to tell by the smell whether butyl or-
and steam, and less blockage of the stills occurs. ganisms are present, although they usually have a sweet butylic
5. Any residual sugar left in the mash can be utilized rapidly
by yeasts with the production of ethanol. This is of value only
when the fermentor has been contaminated in the early stages
and recooking is not desirable.
6. Molasses is normally cheaper than grains or starches.
7. High concentrations of sugar can be fermented with corre-
Table I. Names and Patent Numbers of Various
spondingly high yields in short periods of time. Saccharolytic Bacteria
U. S. Patent
Since this discovery in 1936, research on the fermentation has Names of Bacteria No. Patentee
progressed rapidly, with the isolation of numerous strains of
Bacillus saccharobutylicum-beta 1,725,083 Izsak (18)
organisms capable of carrying out this fermentation. The fol- Clostridium saccharobutylicum- 1,908,361 Izsak et al. (14)
gamma
lowing data are presented in an effort to bring up to date this Clostridium saccharobutyl-acetoni-
modern aspect of the acetone-butanol fermentation. Bacillus technicus
1,922,921 Loughlin (19)
Prescott et al. (28)
1,933,683
Clostridium viscifaciens 2,017,572 Sherman et al. (29)
Clostridium saccharo-acetobutylicum-
Microorganisms Used beta and gamma
Clostridium propyl butylicum
2,050,219 Arzberger (2)
2,063,448 Legg et al. (17)
Clostridium inverto-acetobutylicum 2,073,125 Stiles (80)
The cultures commonly used in the acetone-butanol fermenta- Clostridium saccharo-acetobutylicum 2,089,522 Woodruff et al. (88)
tion of sugars and sugary mashes are members of the Clostridium Clostridium saccharobutyl-isopropyl-
acetonicum 2,096,377 Loughlin (20)
genus. Each company engaged in the manufacture of acetone Clostridium saccharo-acetobutylicum-
and butanol by fermentation has numerous cultures which will alpha 2,110,109 McCoy (22)
Bacillus tetryl 2,113,472 Arroyo (1)
ferment sugary mashes. The majority of the cultures are used in P-bacillus
Clostridium propyl butylicum-alpha
2,123,078 Muller (24)
Muller (25)
2,132,039
patented processes. Various names are assigned to the organisms Clostridium saccharobutyl-acetonicum-
and their morphological, cultural, physiological, and biochemical liquefaciens 2,139,108 Arzberger (8)
Clostridium saccharobutyl-acetonicum-
reactions are described according to the descriptive chart of the liquefaciens-gamma and delta 2,139,111 Carnarius et al. (6)
Hall (10)
Bacillus butacone 2,147,487
Society of American Bacteriologists; other distinguishing charac- Clostridium celerifactor 2,169,246 Hildebrandt et al. (12)
teristics may also be listed. Numerous names are given in the Clostridium granulobacter acetobutyl-
icum 2,195,629 Muller (26)
patent literature for cultures falling into this group (Table I). Clostridium saccharobutyl-isopropyl-
acetonicum-beta 2,219,426 Loughlin (21)
The saccharolytic microorganisms are all spore-forming rods. Clostridium butylo-butyricum 2,386,374 Weizmann (82)
Their inactive stage consists of a rod containing a spore or a free Clostridium madisonii 2,398,837 McCoy (28)
Clostridium amylo-saccharobutyl-pro-
spore by itself. They are characterized by different fermenta- pylicum
Clostridium saccharo-acetoperbutyli-
2,420,998 Beesch et al. (5)
tions of carbohydrates, different ratios of solvents, and different 2,439,791 Beesch (4)
1
Present address, Chas. Pfizer & Co., Brooklyn 6, N. Y.
1672
 1678 INDUSTRIAL AND ENGINEERING CHEMISTRY Vol. 44, No. 7

odor. Some cultures give off hydrogen sulfide from the am-
monium sulfate; others smell butyric and some, highly pro-
teolytic. The cultures that appear to have fermented well are
plated out on a special nutrient agar; the plates then are incu-
bated anaerobically for 48 to 72 hours at 87° F. Upon observa-
tion of the plates numerous typical colonies are picked off and
carried through a series of tests for solvent production, types of
solvents and ratios produced, nutrient requirements, etc. The
best solvent-yielding cultures are allowed to sporulate by incuba-
tion for about 4 days, after which the culture is placed on a sterile
sand-soil carbonate mixture and dried at 87° F. for several days.
This is the stock culture which remains viable for many years if
properly stored. It is quite evident that not every sample of
material tested will contain suitable saccharolytic bacteria for the
production of solvents, but natural sources containing them are
not rare.
plated out on a special nutrient agar and the usual procedure for
picking colonies, testing, etc., is carried out. Still another
method of isolation is to take samples of various soils, roots, wood,
cereals, etc., and introduce small amounts into a potato glucose
mash. Then, these mixtures are heat-shocked for 1 to 3 minutes
in boiling -water, cooled, and incubated at 87° F. This type of
mash not only provides excellent anaerobic conditions, but the
combination of the potato starch molecule plus glucose affords an
excellent medium for the germination of spores of the saccharo-
lytic bacteria. After 20 to 24 hours’ incubation, the tube cultures
are transferred to flasks containing 4% sugar supplied as invert or
blackstrap molasses, 5% ammonium sulfate, 6% calcium car-
bonate, and 0.3% phosphorus pentoxide, based on the weight of
the sugar. These flasks are incubated for 24 hours at 87° F. and
transferred to several quantitative molasses flasks containing the
usual percentages of salts but with an increased sugar concentra-

Table II. Ratios of Solvents Produced by Various Saccharolytic Bacteria

Sol vent Ratios, %
u. s. Butyl Ethvl Isopropyl
Patent No. Names of Bacteria Substrate alcohol alcohol Acetone alcohol
1,725,083 Bacillus saccharobu- Inverted molasses, 75 3 35
tylicum—beta CaCOa
1,908,361 Clostridium saccharo- Blackstrap molasses, 65-80 18-34 1-2
butylicum-gamma CaCOa
1,922,921 Clostridium saccharo- Blackstrap molasses, 64 36
butyl-acetonicum corn gluten, and
(NH4)2S04
2,017,572 Clostridium viscifa- Inverted molasses. 66 3 31
ciens CaCOs
2,050,219 Clostridium saccharo- Cane molasses; de- 68-73 1-3 26-32
acetobutylicum—beta graded protein such as
and gamma ammonia, steep water
or distillery slop
2,063,448 Clostridium propyl bu- Inverted molasses, JNHa, 69-70 4-17 14-28 (mixture
tylicum CaCOs isopropyl and
ethyl)
2,073,125 Clostridium invert o- Louisiana molasses (in- 66-70 2-3 27-31
acetobutylicum verted), ammonium
salts or alkalies
2,089,522 Clostridium saccharo- Louisiana molasses, 68-73 1-3 26-32
acetobutylicum (NH4)2S04, and
CaCOs
2,096,377 Clostridium saccharo- Inverted molasses, de- Low pH, 30-38 Trace-10
butyl-isopropyl- graded protein 60-70; 2-20 10-30
acetonicum High
pH,
65-80
2,110,109 Clostridium saccharo- Cuban molasses, 68-73 1-3 26-32
acet-obutylicum- (NH4)2SÓ4, and glu-
alpha ten meal
2,113,472 BaciUus tetryl 74 6 20
2,132,039 Clostridium propyl bu- Inverted molasses, 65-70 3-4 5-10 16-20
tylicum-alpha (NH4)2S02, CaCOs,
KaHPÓí, and MgS04
2,139,108 Clostridium saccharo- Blackstrap molasses, 58-74 2-6 24-36
butyl-acetonicum- (NH4)2S04, CaCOa,
liquefaciens-gamma and P20&
and delta
2,139,111 Clostridium saccharo- Cuban molasses, 60-69 3-4.5 26-35
butyl-acetonicum- (NH4)2S04, CaCOa.
liquefaciens-gamma and P20&
and delta
2,147,487 Bacillus butacone Blackstrap molasses, 65 28
animal and vegetable
protein
2,169,246 Clostridium celerifac- Inverted molasses, am- 60 2 38
monia, and CaCOs
2,195,629 Clostridium granulo- Molasses, corn gluten, 60-75 1-10 25-30
bacter acetobutyli- ammonium salts, and
CaCOs
2,219,426 Clostridium saccharo- Cane and beet molasses, 60-85 15-40 0
butyl-isopropyl -
(NH4)sSOi, and
acstonicum-beta CaCOs
2,398,837 Clostridium madisonii Cuban blackstrap, 75-76 4-6 17-20
NH4OH, (NH4)2S04,
and CaCOs
2,420,998 Clostridium amylo- Invert molasses, (NHi)i* 65-72 Trace 2-4 26-32
saccharobutyl-pro- S04, CaCOs, and P2O5
pylxcum or NH4OH and 2
2,439,791 Clostridium saccharo- Invert molasses, (NH4)2- 69-76 2-7 18-25
acetoperbutylicum S04, CaCOa, and P2Oe,
or NH4OH and P2Os

Another method of isolation is to macerate samples of rotten
wood, seeds, or soil in sterile water, allow the solids to settle for a
few minutes, and then utilize the supernatant liquid to inoculate a
mash consisting of 50 grams of degerminated corn meal and 20
grams of dried liver per liter. The inoculated liver medium is
then pasteurized for 10 minutes at 177° F., cooled immediately
to 87° F., and incubated for several days. The culture is then
tion of 5 to 7%. After the quantitative flask cultures have
finished fermenting, one of each is analyzed for total solvents,
percentage of acetone, and yield on sugar. Those cultures which
have given good yields are then plated out, and pure cultures are
obtained. The majority of the acetone-butanol sugar-fermenting
bacteria are much larger in size than the starch-fermenting Cl,
acetobutylicum. Most of them will ferment both sucrose and
July 1952 INDUSTRIAL AND ENGINEERING
glucose, but some require complete inversion of the sugar or
molasses before fermentation takes place. The choice of an
organism for acetone-butanol fermentation depends on the
nature of the raw material being used, the ratio of end product
desired, and other factors. Most acetone-butanol fermentation
plants have numerous different cultures and strains available
which will produce a variety of solvent ratios, some producing
best in an invert mash while others are more efficient in black-
strap mashes. Table II shows the various ratios of solvents pro-
duced by some of the patented processes.
Raw Materials Used
Many different raw materials can be used by the saccharolytic
acetone-butanol bacteria, but the classic materials are either
invert or blackstrap molasses. Invert, or “high test” molasses is
an evaporated sugar cane juice that contains all the original sugar
of the juice but most of it in an inverted form as a result of acid
hydrolysis. Blackstrap molasses is the sirup that is left after re-
covery of the crystalline sugar from the concentrated juice of
sugar cane. The typical chemical analysis expected of these
materials is given in Table III. Other saccharine materials
which can be used are sucrose, glucose, beet molasses, citrus
molasses, hydrol, sulfite waste liquor, inverted starches, or starch-
containing grains. Some of the saccharolytic organisms are ca-
pable of fermenting starch directly under suitable conditions,
producing almost full yields of solvents. Starchy mashes can
also be hydrolyzed with acid or enzymes and fermented with the
saccharolytic bacteria to obtain the better ratio of solvents.
Fermentation Process
The modem fermentation is carried out using either invert or
blackstrap molasses as the source of sugar. The molasses is
mixed with water and steam to give a concentration of 5 to 7%
sugar. If invert molasses is used, a small amount of super-
phosphate or monoammonium phosphate is added (usually 0.3%
based on the weight of the sugar), and the mash is cooked in
continuous cookers or sterilizers such as described and patented
by Carnarius (7). Using this process, the mash is heated with a
steam injector and pumped under pressure through a series of
from four to six elongated, vertical pressure-detention tanks of
13,000-gallon capacity, moving downward slowly in each of these
along a spiral path. When leaving the last tank the mash is
sterile. The following procedure is usually followed:
The entire cooking system is filled with mash and the system is
recirculated by means of pumping. A temperature of 225 F. is
°
maintained for about 60 minutes, at which time the mash is con-
tinuously withdrawn with the addition of fresh mash to the sys-
tem. The cooked mash is pumped through sterile lines to a
series of double-pipe coolers where the temperature is reduced to
88° to 90° F. If it is desired to cook a higher concentration of
sugar (8 to 9%), dilution water is added at 180° F. before the
mash is pumped to the coolers. As in the acetone-butanol fer-
mentation of starches all lines, coolers, valves, and tanks are
thoroughly steamed and sterilized before use. The cooled mash
is pumped into a final fermentor of 60,000- to 500,000-gallon
capacity. At the same time the final fermentor is being filled, a
culture from a 24-hour culture tank is also being added to the
fermentor. There are two ways of filling the final fermentor.
One method is to add the culture and partially fill the fermentation
tank with sterilized mash; then after a period of a few hours, add
the rest of the mash. This method is known as a “deferred
filling.” The other, and most commonly applied method, is to
fill the tank in one filling, adding the culture at the start. The
advantage of using a deferred filling is that the culture multiplies
manyfold before the remainder of the mash is added, thus speed-
ing up the final fermentation and tending to suppress contami-
nants.
The handling of the culture or seed is very critical. The plant
bacteriologist must have on hand a pure stock culture of bacteria
in the spore stage. The first step is to remove some of these
spores and add them to a sterilized potato glucose medium. The
CHEMISTRY 1679
latter is then heat-shocked by placing it in boiling water for 90
seconds, removing, and cooling immediately to 87° F. The heat
shock stimulates the spore, causing germination and, at the same
time, eliminates the w'eaker spores. After the heat shock the cul-
ture is incubated at 87° F. for 20 to 24 hours. At the end of 20
to 24 hours the culture is transferred aseptically to 600 ml. of
sterilized molasses mash of the following composition: 4% sugar
(supplied in the form of invert molasses), 5% ammonium sulfate,
6% calcium carbonate, and 0.2% phosphorus pentoxide (supplied
in the form of superphosphate). The amounts of chemicals are
all based on the weight of the sugar used. After transfer the cul-
ture is plated out to detect aerobic contaminants. The medium
is allowed to ferment for 20 to 24 hours at which time it is trans-
Table III. Typical Analysis of Some Fermentable
Saccharine Materials
Invert Blackstrap Citrus Wheat
Molasses, Molasses, Molasses, Hydrol,
% % % %
Total invert sugar 75.6 57.8 48.8 54.6
Sucrose 25.9 35.0 20.7
Total sugar 74.2 56.0 47.7 54*6
Reducing sugar 48.37 21.0 27.0 53.5
Protein 0,81 2.2 4.1 3.6
Ash 1.75 8.0 3.2 0.9
ferred to a 4000-ml. Erlenmeyer flask containing 3000 ml. of the
same type mash used in the preceding stage. Usually, about 3%
inoculum is used. After this flask has been allowed to incubate
for 20 to 24 hours it is used to inoculate a 5000-gallon culture
tank, providing, of course, that no contamination has been de-
tected in any of the previous stages and the fermentation has
appeared to be normal.
The 5000-gallon tank is filled with about 4000 gallons of
molasses mash of a 6% sugar concentration with addition of the
same percentage of chemicals as used in the laboratory stages.
The 5000-gallon tank is inoculated aseptically with the entire
contents of the 4-liter flask, and the tank is agitated for 5 minutes
to mix the culture thoroughly. The tank is then maintained under
15 pounds of sterile fermentation gas pressure to keep outside
contaminants from coming in and to create an anaerobic condi-
tion in the tank until the fermentation can maintain its own
pressure. A close watch is maintained over the seed tank for
changes in pH, acid, and brix, and the temperature is maintained
at 87° F. by circulation of water or steam in the jacket of the
tank. The titratable acidity starts low and builds up to a peak in
approximately 18 hours; then in a normal fermentation, it starts
to fall off. This point is known as the “break.”
After the peak acidity has been reached and declines, the fer-
menter is ready for transfer—usually after 20 to 26 hours of
fermentation. The fermenter is transferred aseptically into the
final fermentation tank of 60,000 to 500,000 gallons which is
filled with sterile mash. These final fermentation tanks are
usually of a pressure type, withstanding 15 to 25 pounds of steam
pressure, and are usually either cylindrical, spherical, or spheroid
in shape. The tanks are fitted with gage glasses, temperature
recorders, vacuum breakers, safety relief valves, and a manhole
cover for use in cleaning. Various percentages of inoculum are
used ranging from 0.5 to 3.0%. In the final fermentor, am-
monium hydroxide is usually added as the source of nitrogen and
as a neutralization agent. Other nitrogen compounds such as
ammonium sulfate, monoammonium phosphate, ammonium
nitrate, and other alkaline substances such as lime or calcium
carbonate can be used for additional neutralization of the acids
formed. Ammonia is preferred for such reasons as ease in
handling, cost, rapid adjustment of pH, and the fact that it does
not have to be sterilized. Ordinarily, ammonia is added on the
basis of the sugar present and 1 to 1.4% NEE is required. The
ammonia requirement varies with the type of mash and depends
1680 INDUSTRIAL AND ENGINEERING
Figure 1. Flow Diagram of Typical Modern Acetone-Butanol Fermentation Using Sugar Products
on whether or not stillage has been recycled. Blackstrap molasses
requires no phosphate and less ammonia than does beet molasses.
The ammonia usually is added to maintain the pH at 5.6 after
the fermentation has reached about 16 hours of age. An initial
shot of ammonia may be added to the young mash to raise the
starting pH to 5.7 to 6.7. The acidity which develops drops this
pH to about 5.5 or less, whereas the rest of the ammonia is fed in
over a period of about 8 to 10 hours. In the normal fermentation,
all the ammonia is added within 18 to 24 hours. Table IV shows
a typical final fermenter mash with addition of ammonia and
other fermentation features utilizing the culture Clostridium
saccharo-acetoperbutylicu m.
Various percentages of ethyl grain stillage, butyl grain, or
molasses stillages may be used in preparation of the final mash.
These stillages supply additional nutrients and buffer the fermen-
tation. A better yield is obtained when stillage is used; less
foaming is noticed in the fermenters, and the buffer action in-
creases the amount of ammonia that can be added at one time.
The ratio of the solvents is changed, however, and more acetone
may be produced. More acetone is also produced by raising the
temperature or by using blackstrap instead of invert molasses.
Figure 1 is a flow diagram for the modern acetone-butanol
fermentation of sugars.
CHEMISTRY Vol. 44, No.
Conditions for Fermentation
The optimum temperature condition for the acetone-butanol
fermentation of sugars using saccharolytic bacteria is 87° F. A
range of 84° to 92° F. results in a fairly normal fermentation but
with changes in solvent ratio and losses of acetone at the higher
temperature unless precautions are taken to scrub the gases with
water to remove the solvents. The organisms are strict anaerobes
and produce their best yield under anaerobic conditions. A slight
head consisting of foam, proteinaceous materials, cells, and some
slime, may be formed in the molasses fermentation. It may be
advisable to maintain sterile carbon dioxide gas pressure on the
top of the fermentation until such a head and pressure are built up
in the tank. The pH may vary from 5.0 to 7.0 by the saccharolytic
organisms. The usual starting pH is 5.5 to 6.5 and the final, 5.2
to 6.2.
Nitrogen Requirements
The majority of the saccharolytic acetone-butanol bacteria re-
quire degraded protein nitrogen for optimum fermentation. De-
graded protein nitrogen includes intermediate degradation products
such as polypeptides, amino acids, and the final hydrolytic product,
ammonia, and its salts. Ammonia or an ammonium salt has been
July 1952 INDUSTRIAL AND ENGINEERING CHEMISTRY 1681

content. Different strains of bacteria may be found to differ

Table IV. Acetone-Butanol Fermentation of Bla slightly in their tendency to produce undue acidity or in their
Molasses susceptibility to alkali in the neutralization process employed for
Age, Gas Rate, NH40H J maintaining pH.
Hours pH Brix Units (28%), It has been found that the saccharolytic acetone-butanol bac-
1 70 teria also require various growth factors. Teodoro and Miekelson
2 5.55 Si 7
3
8.6
(SI) found in studying three different strains that two of these re-
4 5.45
5 quired only biotin for maximum growth and the third needed
6 5.45 8.6 *2 both biotin and p-aminobenzoic acid. Thiamine, riboflavin,
7 8
8 5.40 8.5 10
11
pyrodoxine, pantothenic acid, nicotinamide, and inositol were
9
10 5.25 8.0 12 *30 without effect.
11 13
12 5.15 8.0 14 ¿0
13
14 5,30 7.8
15
15
40
50
Contamination Problems
15 15 50
16 5.20 7.8 15 The contamination problem is of equal importance in the
17 16
18 5.30 7.5 17 *60 acetone-butanol fermentation of sugars as it is in the fermentation
19 18 70 of starches. The maintenance of sterile conditions throughout the
20 5.35 7.3 19 80
22 5.35
5.30
6.9
6.9
20
20
110
160
plant is necessary. All lines, valves, pumps, coolers, and tanks
24
26 5.65 6.5 21 must be thoroughly clean and sterile. The same contaminants
5.65 6.5
28
30 5.55 6.4
21
22 responsible for the downfall of the acetone-butanol fermentation
32 5.55 of starches predominate in the fermentation of sugars. These
34 5.30 é'.i lactic acid bacteria (such as Lactobacillus leichmanni, a high acid
36 5.35 5.6
38 5.35 5.2
40 5.35 4.6 forming organism; Lactobacillus mannitopoeum·; Lactobacillus
42 5.35 4.1 gracile; aiid Lactobacillus intermedium) react in the same way to
44 5,40 3.4
46 5.35 3.3 cut the yield of solvents. They also frequently cause a thickening
48 5.40 3.0 to take place in the mash. Bacteriophages affecting the sugar-
Fermentation Data
fermenting cultures are also encountered. The author has never
Volume of mash, gal. 200,000 found a bacteriophage that would affect both .the starch and
Concentration of sugar, % 6
Culture (Clostridium saccharo-aaeloperbutylicum), % 2 sugar-fermenting bacteria. The bacteriophages found in the
Total NHtOH (28%), gal. 730
Total mixed solvents, g./l. 18.57 acetone-butanol fermentation of sugars differ from those found in
Acetone, % 20.93 Cl. acetobutylicum fermentations in that they are usually de-
-Butyl alcohol, % 74.70
Ethyl alcohol, % 4.37 stroyed in 48 hours’ exposure to room temperature and light.
"Yield of total solvents from sugar, % 31.0
Residual sugar, g,/100 ml. 0.45
Residual ammonia, p.p.ro. 11
Fermentation temp., 0 F. 87
Table VI. Effect of Addition of Ethyl Stillage to Acetone-
Butanol Fermentation of Blackstrap Molasses
Ethyl Total Yield on
found to give satisfactory results but it usually is preferred to use Stillage Neutral- Solvent3, Acetone, Sugar*·,
Flask Added, % ization G./L. % %
ammonia plus a higher form of nitrogenous material such as yeast
A 100 Salts* 15.83 29.2 33.3
water, steep water, or distillation slop in the fermentation mash in B 60 Salts 15.28 27.9 31.9
order to secure a maximum yield of solvents. The majority of C
D
40 Salts 13.78 29.5 28.8
Salts 13.43 27.5 28.2
these bacteria also require phosphate nutrients as do other types of E 100 1.3% NHs 15.23 26.5 32.4
F 60 1.3% NHs 14.92 28.1 31.3
bacteria. Many natural sources of carbohydrate contain suf- G 40 1.3% NHs 14.32 28.2 30.0
ficient amounts of phosphates but, in case of deficiency, this may H None 1.3% NHs 13.43 29.2 29.0
be supplied in the form of calcium acid phosphate, superphos- 0
Average results of two flasks.
Concentration of sugar, 5%
phate or monoammonium phosphate, or any other form of soluble 6
5% (NH4)aS04, 6% CaCOs, 0.3% P2O5, based on weight of sugar.
phosphate. In general, 0.2 to 0.4% of phosphorus pentoxide is
required based on the weight of the carbohydrate used. If am-
monium salts are used, 4 to 6% ammonium sulfate or its equiva- The only means of preservation of these bacteriophages is by re-
lent is necessary, based on the weight of the carbohydrate used. duced temperatures, preferably sealed in tubes at a pH of 6.9 to
If ammonium salts are used it is necessary to neutralize the excess 7.1. How the bacteriophage enters the fermentation process is a
acidity and maintain the proper pH with calcium carbonate— matter of wide speculation. Numerous theories have been de-
usually about 5 to 7% based on the weight of the carbohydrate is veloped, none of which has been proved. The bacteriophages
sufficient. If ammonia is used, 1 to 2% NH3 is sufficient, depend- affecting the acetone-butanol organisms have been found in the
ing on the composition of the mash and on the alkali or buffer soil, in rivers, in the air, and in some of the raw materials used.
In other words, they are widely dispersed. The presence of a bac-
teriophage usually becomes evident at approximately the eigh-
Table V, Material Balance in Acetone-Butanol teenth hour after inoculation.
Fermentation of Blackstrap Molasses The only immediate recourse to a bacteriophage attack is to
1001b. blackstrap molasses containing, lb.
substitute at once an immune strain. Most industrial fermenta-
Starting material:
Total solids tion plants keep on hand a large number of strains, new isolations,
Sucrose and invert sugar 57.0
Protein 3.1 and previously immunized organisms which can be tested rapidly
Ash 6.2 and substituted in the plant for the organism which has been
Fermentation data: Culture of saccharolytic bacteria used with 1.0%
NHs added in form of ammonium hydroxide affected. Various methods of selecting immune strains of the
Yields, lb.
Butyl alcohol 11.5 organism have been tried. Some methods have been patented—
Acetone 4.9
Puthyl alcohol 0.5 Legg (16) and Legg and Walton (18)—showing a process for
Carbon dioxide 32.1
0.8
selecting immune strains by repeated subculturing of the organism
Hydrogen in the presence of small amounts of the bacteriophage. Others
Dry feed containing (6 lb. protein, 6 lb. ash) 28.6
have found that by plating out the original strain and selecting
1682 INDUSTRIAL AND ENGINEERING CHEMISTRY Vol. 44, No. 7

various colonies they may isolate immune strains, or by placing
a nonimmune culture on a plot of open, unsterile soil and reisolat-
ing the culture 6 months later, they may have an immune strain.
The last procedure is the subject of a patent by Hanson (11).
It has been the author’s experience that any culture which has
been properly immunized by suitable means loses some of its
for example, gives higher acetone ratios than Cuban molasses.
The use of distillation slop generally increases the acetone ratio
and ammonia neutralization tends to give higher acetone ratios
than those obtained using calcium carbonate neutralization. The
use of higher temperatures also increases the acetone ratio. The
stillage obtained after distillation of the solvents has become a

Table VII. Feed Values Recoverable from Acetone-Butanol Fermentation of Blackstrap

and Invert Molasses
100 LbMVIolasses
Dry Matter
Kind of in Stillage. Dry feed, Protein. Analysis (Dry Basis), % Riboflavin,
Fractions of Stillage Molasses % lb. lb. Protein i Fat Ash y/O.
Total solids evaporation Invert 1.15 17.7 6.5 36.8 0.31 12.1 52
whole stillage Blackstrap 2.71 28.6 6.0 20.8 0.14 23.2 38
Solids recoverable by Invert 0.17 2.6 2.0 77.6 2.00 4.8 49
centrifuge Blackstrap 0.22 2.3 1.3 57.5 2.60 7.2 27
Dried effluent from cen- Invert 0.96 14,8 4.1 28,1 0.0 13.4 54
trifnge Blackstrap 2.50 26.3 4.2 16.0 0.0 25.9 37

activity. The fermentation is slightly slower, a somewhat lower
concentration of mash is fermented, and the solvent yield is
slightly less than was previously obtained by the nonimmune
strain.
The acetone-butanol fermentation of sugars by Cl. saccharo-
acetoperbutylicum is inhibited by the presence of 2.0 p.p.m. of
copper ion and completely inhibited by 5.0 p.p.m. of copper ion.
The maximum tolerated limit lies between 1.0 and 2.0 p.p.m. of
copper ion, varying for different strains. The addition of alumi-
num, tin, iron, nickel, zinc, manganese, lead, cobalt, cadmium,
chromium, thorium, thallium, and uranium ions at 50-p.p.m.
levels had no effect on the fermentation. The addition of mer-
cury from 7 to 50 p.p.m. delayed the fermentation for 24 hours.
The addition of antimony at 50-p.p.m. level seriously reduced
the fermentation yield.
Yields and Recovery of End Products
major by-product. The normal dried stillage contains various
vitamins: among them, B2 in amounts of 40 to 80 micrograms per
gram.
Up to the present time no one has been able to increase the
riboflavin (vitamin B2) to the levels that have been obtained in
the fermentation of starches with Cl. acetobutylicum. The stillage
is suitable for admixing with other materials for animal feeds.
The feed values recoverable from the acetone-butanol fermenta-
tion of blackstrap and invert molasses are shown in Table VII.
The stillage may also be recycled into the fermentation in order
to supply additional nitrogen and buffer substances. The
molasses stillage has also been used in the manufacture of plastics;
has been concentrated, dried, and burned to an ash high in po-
tassium; and has been used as a binder in foundry work.
Literature Cited
(1) Arroyo, R.. LT. S. Patent 2,113,472 (1938).

The most important products formed in the acetone-butanol (2) Arzberger, C. F„ Ibid., 2,050,219 (1936).
fermentation of sugars are n-butyl alcohol, acetone, ethyl alcohol, (3) Ibid., 2,139,108 (1938).
(4) Beesch, S. C„ Ibid., 2,439,791 (1948).
carbon dioxide, hydrogen, and riboflavin-containing feeds from (5) Beesch, S. C., and Legg, D. A., Ibid., 2,420,998 (1947).
the fermentation residue. (6) Carnarius, E. H., and McCutchan, W. N., Ibid., 2,139,111
In the normal acetone-butanol fermentation of sugars a ratio (1938).
of solvents of 68% butyl, 30% acetone, and 2% ethyl may be ex- (7) Carnarius, E. H., Ibid., 2.423,580 (1947).
(8) Gabriel, C. L., Ind. Eng. Chem., 20, 1063-7 (1928).
pected. Various factors influence the change in these ratios. (9) Gabriel, C. L„ and Crawford, F. M„ Ibid., 22, 1163-5 (1930).
The material to be fermented, the culture or strain of microorgan- (10) Hall, . E., U. S. Patent 2,147,487 (1939).
ism used, temperatures, use of stillage, contaminants present, and (11) Hanson, C. T„ Ibid., 2,085,428 (1937).
the addition of vaiious chemicals all serve to change the normal (12) Hildebrandt, F. M., and Erb, N. J.. Ibid., 2,169,246 (1939).
(13) Izsak, A., Ibid., 1,725,083 (1929).
ratio expected. From the fermentation, a yield of 28 to 33% on (14) Izsak, A., and Funk, F. J., Ibid., 1.908,361 (1933).
the basis of the sugar or approximately 1 pound of mixed solvents (15) Killeffer, D. H., Ind. Eng. Chem., 19, 46-50 (1927).
from 3.6 pounds of invert molasses is normal. In Table V a (16) Legg, D. A., L". S. Patent 1,668,814 (1928).
material balance using blackstrap molasses in the acetone- (17) Legg, D. A., and Stiles, H. R„ Ibid., 2,063,448 (1936).
(18) Legg, D. A., and Walton, M. T.,Ibid., 2,132,358 (1938).
butanol fermentation is shown. The actual yield of solvents (19) Loughlin, J. F„ Ibid., 1,992,921 (1935).
is subject to wide variation. Various samples of molasses of the (20) Ibid., 2,096,377 (1937).
same general type may be found to give different yields with the (21) Ibid., 2,219,426 (1941).
same culture of bacteria. Likewise, different types of molasses (22) McCoy, E. F., Ibid., 2,110,109 (1938).
will often be found to give substantially different yields. For ex- (23) Ibid., 2,398,837 (1946).
(24) Muller, J., Ibid., 2,123,078 (1938).
ample, beet molasses gives higher yields of solvents than any of Ibid., 2,132,039 (1938).
(25)
the types of cane molasses. It has also been found that supple- (26) Ibid., 2,195,629 (1940).
mentary nutrients such as distillation slop from an alcohol fer- (27) Prescott, S. P., and Dunn, C. G., “Industrial Microbiology,” 2nd
mentation of a saccharified grain mash may affect the yield ed., New York, McGraw-Hill Book Co., 1949.
(Table VI). Nutrients of this type tend in general to increase the (28) Prescott, S. P., and Morikawa, K., U. S. Patent 1,933,683 (1933).
(29) Sherman, J. M., and Erb, N. M., Ibid., 2,017,572 (1935).
yield. Similarly, if ammonia is utilized in place of calcium car- Stiles, H. R„ Ibid., 2,073,125 (1937).
(30)
bonate to regulate the pH of the mash throughout the fermenta- Teodoro, R. R., and Mickelson, . N., Arch. Biochem., 6, 471-7
(31)
tion, the yield tends to be increased slightly. The solvent ratio (1945)'.
and yield also depend on a number of factors such as the particu- (32) Weizmann, C., U. S. Patent 2,386,374 (1945).
lar strain of bacteria employed and the composition of the mash. (33) Woodruff, J. C., Stiles, H. R.,
and Legg, D. A., Ibid., 2,089,522
Different ratios of acetone to butanol and ethanol may be ob- (1937).
Received for review July 10, 1951. Accepted April 7, 1952.
tained with different types of molasses. Puerto Rican molasses,