I
Acetone-Butanol Fermentation of Sugars
Ek ;n r;ig rocess development
SAMUEL C. BEESCH' Publicker Industries, Inc;, Philade/phia 2, Pa.
HE production of acetone and butanol by the Weizmann process has been described in some detail by KilleEer ( 1 5 ) , Gabriel (8), Gabriel and Crawford (9), and Prescott and Dunn ( $ 7 ) . The original Weizmann organism (Clostridium acetobutylic i m ) was isolated with a view t o breaking down starch t o solvents. Later investigators found it was possible t o substitute u p t o 50% of the weight of carbohydrate with molasses. This procedure, hciwever, lengthened the fermentation cycle abnormally. In 1936, the &st commercial application of a new fermentation utilizing molasses or sugar as the sole source of carbohydrate wa8 discovered. This fermentation proved t o be more economical from a commercial standpoint for the following reasons:
T
1. The mash is sterilized a t lower temperature and is much easier t o handle. 2. The ratio of solvents is more desirable in that less ethanol is produced, with a corresponding increase in butanol. 3. The fermentation is run a t 87" F. instead of 98' F., providing a less favorable temperature for contaminating organisms. 4. Tanks and equipment are much easier t o clean with water and steam, and less blockage of the stills occurs. 5. Any residual sugar left in t h e 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 correspondingly high yields in short periods of time.
Since this discovery in 1936, research on the fermentation has progressed rapidly, with the isolation of numerous strains of organisms capable of carrying out this fermentation. The following data are presented in an effort to bring up to date this modern aspect of the acetone-butanol fermentation. Microorganisms Used The cultures commonly used in the acetone-butanol fermentation of sugars and sugary mashes are members of the Clostridium genus. Each company engaged in the manufacture of acetone and butanol by fermentation has numerous cultures which will ferment sugary mashes. The majority of the cultures are used in patented processes. Various names are assigned to the organisms and their morphological, cultural, physiological, and biochemical reactions are described according to the descriptive chart of the Society of American Bacteriologists; other distinguishing characteristics may also be listed. Numerous names are given in the patent literature for cultures falling into this group (Table I). The saccharolytic microorganisms are all spore-forming rods. Their inactive stage consists of a rod containing a spore or a free spore by itself. They are characterized by different fermentations of carbohydrates, different ratios of solvents, and different 1
sugar concentrations utilizable. The isolation of Clostridia (sapable of utilizing saccharine materials may be accomplished in many ways. One consists of the introduction of a small amount of soil, manure, roots of leguminous plants, cereals, decayed wood, cornstalks, sewage, or river bottom mud into a sterile 4% sugar mash (sugar supplied as invert molasses) plus 4% ammonium sulfate, 5% calcium carbonate, and 0.3% phosphorus pentoxide (amounts based on the weight of the sugar). This mash is usually tubed in 25-ml. quantities, using tubes 200 X 25 mm. in size. These inoculated mashes are then subjected t o heat shock by placing t h e tubes in boiling water for 2 minutes. The heat shock destroys most, vegetative forms leaving t h e spores. The tubes are cooled t o 87" F. and then are placed in a desiccator partly filled with moistened wheat or oats. A vacuum of about 29 inches is pulled on the desiccator and it is then placed in an incubator a t 87" F. for 24 to 48 hours. Normally, in 24 hours the tubes begin to evolve gas, and the color of the mash turns to light tan. It is difficult to tell by the smell whether butyl organisms are present, although they usually have a sweet butylic
Table I.
Names and Patent Numbers of Various Saccharolytic Bacteria
Names of Bacteria Bacillus saccharobutylicum-beta
Clo8t r i d i u m saccharobutulicumgam,mp Clostradzum saccharobutul-acetonicum Bacillus tech n icus Clostridium viscifaciens Clostridium aacc~aro-acetobutylicumbeta and gamma Clostridium propyl but ylicum Clostridium in!,erto-acetobutylicum Clostridium saccharo-acetobutylicun, Clostridium saccharobutyl-isopropylacetonicurn Clostridium saccharo-acetobutvlicum-
1,908,361
Pat ent ee
Izsak (19) Izsak et al. (14) Loughlin (19) Prescott et al. (88) Sherman et al. (89)
2,050.219 2,063,448 2,073,126 2,089,522
Arzberger (8) Legg et al. (17) Stiles (90) Woodruff et 02. (98)
2,090,377
Loughlin ($0)
2,110,109 2,113,472 2,123,078 2,132,039
McCoy (88) Arroyo (1) Muller (24) Muller (86)
2,139,108
Arsberger ( 9 )
2,13O.111 2,147,487 2,169,246
Hildebrandt et al.
2,196,629
Muller (28)
2,219,426 2,386,874 2,398,837
Loughlin (8f) Weizmann (32) McCoy (8s)
pylicum
2,420.998
Beesch et
cum
2,439,791
Beesch (4)
Bacillus tetrtl P-bacillus Clostridzum propyl butylicum-alpho Clostridium saccharobutyl-acetonicum1 iquefaciens Clostridium saccharobutyl-acetonicu mliguefaciens-gamma and delta Bacillus butacone Clostridium celerifactor Clostridium granulobacter acetobutylicum Clostridium 8accharObutyl-i8opropylacetonicum-beta Clostridium butylo-bvtyrdcum Clostridium madisonii c10stridium amylo-aacchatobu8tyl-pro-
Clostvidium saccharo-acetoperbutl/li-
1622
1,725,083
1,922,921 1,933,683 2,017,572
alpha
Present address, Chas. Pfizer & Co., Brooklyn 6, N. Y.
U. 9. Patent No.
H Carnarius ~ l (l1 0 ) et al. (6)
41.
(6)
(fa)
INDUSTRIAL AND ENGINEERING CHEMISTRY
1678
odor. Some cultures give off hydrogen sulfide from the ammonium sulfate; others smell butyric and some, highly proteolytic. The cultures t h a t appear tJo have fermented well are plated out on a special nutrient agar; the plates then are incubated anaerobically for 48 to 7 2 hours a t 87' F. Upon observation 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 allom ed to sporulate by incubation for about 4 days, after .iT-hichthe culture is placed on a sterile sand-soil carbonate mixture and dried a t 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.
Table II.
u. s.
Patent No. 1,726,083 1,908,361 1,922,921
Names of Bacteria Bacillus saccharobutulicum-beta C l G t r i d i u m saccharohut yl icum-gamma Clostridium saccharobutyl-acetonicum
Closiridium uiscifaczens Clostridium saccharoacatobufylicum-beta a n d gamma
2,063,448
Clostridium propyl butylicum
2,073,125
Clostridium inuertoacetobu&glicum
2,089,522 2,096,377
2,110,109 2,113,472 2,132,039 2,139,108
2,139,111
2,147,487 2,169,246 2,195,629
2,398.837 2,420,998 2,439,791
Substrate Inverted molasses, CaCOa Blaokytrap molasses, CaC08 Blackstrap molasses, corn gluten, a n d (NHai~Soa Ini-erted molasses. CaC03 Cane molasses; deeraded Drotein such as ammonia, steep water or di-tillery slop Inverted molasses, " a , CaCOz
Solvent Ratios, % - _ _Butyl Ethyl Isopropyl alcohol alcohol Acetone alcohol 75 3 35 65-80
18-34
64
36
G6 68-73
69-70
Louisiana molasses Jin66-70 verted), ammonium salts or alkalies Clostridium saccharo- Louisiana molasses, 68-73 (h-He)nSOa,and acetobutylicum CaC03 Clostridium saccharo- Inverted molasses, de- 'Low pH, graded protein butyl-isopropyl60-70; High acetonicum ?HI 65-80 68-73 Clostrzdium saccharo- Cuban molasses. (NIIdsSOe, a n d gluaceto butyl i c u m ten meal alpha 74 Baci'lus tetiu? 65-70 Clontridium propyl butylicum-alpha
Clostridium saccharohutyl-acetmicumliquefaciens-gamma and dslta Clostridium sacrharobut yl-acet onicumliquefaciens-gamma a n d delta Bacillus hutacone
Clostridium saccharobut yl-isopropylacetonicum-beta Clostridium madisonii
... 1-3
...
3
31
.....
4-17
14-28 (mixture isopropyl a n d ethyl)
27-31
1-3
26-32
.....
30-38 2-20
Trace-10 10-30
. . I
6 3-4
20 5-10
58-74
2-6
24-36
Cuban molasses, (XHe)&O4, CaCOa. a n d PzOs
6049
3-4.5
26-35
Blackstrap molasses, animal arid vegetable Drotein
E5
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 &germinated corn meal and 20 grams of dried liver per liter. The inoculated liver medium is then pasteurized for 10 minutes a t 177" F., cooled immediately to 87' F., and incubated for several days. The culture is then
.
I
.
. . I . .
16-20
28
.....
38
60-75
1-10
25-30
..... .....
60-85
...
15-40
0.1-4.0
17-20
.....
60
Clost&ium amulosaccharobutyl-propylicum Clostridium saccharoacetoperbutylicum
.....
2-3
26-32
Cane and beet molasses, HdzSOa, and
1-2
.....
26-32
1-3
Clostridium celarifactor Clostridium granulobacter acetobutylicum
2,219,426
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 a t 87" F. This type of mash not only provides excellent anaerobic conditions, but the combination of the potato starch molecule plus glucoEe affords an excellent medium for the germination of spores of the saccharolytic 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, 670 calciuni carbonate, and 0.3% phosphorus pentoxide, based on the weight of the sugar. These flasks are incubated for 24 hours a t 87' F. and transferred to several quantitative molasses flasks containing the usual percentages of salts but with an increased sugar concentra-
Ratios of Solvents Produced by Various Saccharolytic Bacteria
2,050,219
2,017,572
Vol. 44, No. 7
2
76-76
4-6
66-72
Trace
69-76
2-7
2-4 18-25
26-32
.....
tion of 5 to 770. 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. acetobzitylicurn. Most of them will ferment both Sucrose and
July 1952
INDUSTRIAL AND ENGINEERING CHEMISTRY
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 blackstrap mashes. Table I1 shows the various ratios of solvents produced 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 t h a t is left after recovery of the crystalline sugar from the concentrated juice of sugar cane. The typical chemical analysis expected of these materials is given in Table 111. Other saccharine materials which can be used are sucrose, glucose, beet molasses, citrus molasses, hydrol, sulfite waste liquor, inverted starches, or starchcontaining grains. Some of the saccharolytic organisms are capable 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 modern fermentation is carried out using either invert or blackstrap molasses as the source of sugar. The molasses is mixed with watw and steam to give a concentration of 5 t o 7 % sugar. If invert molasses is used, a small amount of superphosphate 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 endire 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 continuously withdrawn with the addition of fresh mash to the system, The cooked mash is pumped through sterile lines to a series of double-pipe coolers where the temperature is reduced t o 88” t o 90” F. If it is desired to cook a higher concentration of sugar (8 t o 9%), dilution water is added a t 180” F. before the mash is pumped t o the coolers. As in the acetone-butanol fermentation 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 %hour culture tank is also being added to the fermentor. There are two ways of filling the final fermentor. One method is t o 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 speeding up the final fermentation and tending to suppress contaminants. O
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
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 weaker spores. After the heat shock the culture is incubated a t 87’ F. for 20 to 24 hours. At the end of 20 to 24 hours the culture is transferred aseptically t o 600 ml. of sterilized molasses mash of the follow-ing composition: 4y0sugar (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 culture is plated out t o detect aerobic contaminants. The medium is allowed t o ferment for 20 t o 24 hours at which time it is trans-
Table 111.
Typical Analysis of Some Fermentable Saccharine Materials Invert Molasses,
Total invert sugar Sucrose Total sugar Reducing sugar Protein
Ash
Blackstrap Molasses,
%
Citrus Molasses,
Wheat Hydrol,
% 75.6 25.9 74.2 48.37 0.81 1.75
57.8 35.0 56.0 21.0 2.2 8.0
48.8 20.7 47.7 27.0 4.1 3.2
54.6
%
%
5i:6 53.5
3.6 0.9
~~
ferred to a 4000-ml. Erlenmeyer flask containing 3000 ml. of the same type mash used in the preceding stage. Usually, about 3qi’, inoculum is used. After this flask has been allowed to incubate for 20 to 24 hours it is used t o inoculate a 5000-gallon culture tank, providing, of course, t h a t no contamination has been detected in any of the previous stages and the fermentation has appeared t o 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 Pliter flask, and the tank is agitated for 5 minutes t o mix the culture thoroughly. The tank is then maintained under 15 pounds of sterile fermentation gas pressure t o keep outside contaminants from coming in and t o create an anaerobic condition in the tank until the fermentation can maintain its own pressure. close watch is maintained over the seed tank for changes in pH, acid, and brix, and the temperature is maintained a t 87” F. by circulation of water or steam in t h e jacket of the tank. The titratable acidity starts low and builds u p 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 fermenter is ready for transfer-usually after 20 t o 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 t o 3.070. I n the final fermentor, ammonium 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 t h a t it does not have t o be sterilized. Ordinarily, ammonia is added on the basis of the sugar present and 1 to 1.4% N& is required. The ammonia requirement varies with the type of mash and depends
4
INDUSTRIAL AND ENGINEERING CHEMISTRY
July 1952
Table IV. Age, Hours 1
2
Acetone-Butanol Fermentation of Blackstrap Molasses Brix
PH
...
...
8.7
5.55
3
Gas R a t e .
NHlOH Added (28%b), Gal.
.. .. ,. ..
.., ...
2 8
..
Units
4 5
5.45
8 6
6 7 8
5.45
86
5.40
8.5
10 11
5.25
8 0
12 13 14 15 16 17 18 19
5.15
8 0
5.30
7.8
5.20
7 8
5.30
75
5.35 5.35 5.30 5.65 5.65 5.55 5.55 5.30 5.35 5.35 5.35 5.35 5.40 5.35 5.40
7.3 6 9 6.9 6 5 6 5 0 4
15 15 15 15 16 17 18 19 20 20 21 21 22
6.1
..
22 24 26
28 30 32 34 36
38 40 42 44
46 48
10 11
9
20
..
...
5.6
5.2 4.6 4.1 3.4 3.3 3.0
12 13 14
.. ..
..
....
.. .. ..
70
... ... ..
.. ... 30 ... 30 40 50 50
Contamination Problems
...
...
GO 70
80 110 160
...
...
,.. ,..
...
. .
... ... ... ... ..,
...
200,000 6 2 730 18,57 20.93 74.70 4.37 31.0 0.46
’
The contamination problem is of equal importance in the acetone-butanol fermentation of sugars as it is in the fermentation of starches. The maintenance of sterile conditions throughout the plant is necessary. All lines, valves, pumps, coolers, and tanks must be thoroughly clean and sterile. The same contaminants responsible for the downfall of the acetone-butanol fermentation of starches predominate in t h e fermentation of sugars. These lactic acid bacteria (such as Lactobacillus leichmanni, a high acid forming organism; Lactobacillus mannitopoeum; Lactobacillus gracile; and Lactobacillus intermedium) react in the same way to cut the yield of solvents. They also frequently cause a thickening to take place in the mash. Bacteriophages affecting the sugarfermenting cultures are also encountered. The author has never found a bacteriophage t h a t would affect both.the starch and sugar-fermenting bacteria. The bacteriophages found in the acetone-butanol fermentation of sugars differ from those found in C1. acetobzttyEicum fermentations in that they are usually destroyed i n 48 hours’ expoeure to room temperature and light.
11
87
Table VI. Effect of Addition of Ethyl Stillage to AcetoneButanol Fermentation of Blackstrap Molasses
found to give satisfactory results but it usually is preferred to use amnionia plus a higher form of nitrogenous material such as yeast water, steep water, or distillation slop in the fermentation mash in order t o secure a maximum yield of solvents. The majority of these bacteria also require phosphate nutrients as do othertypes of bacteria. Many natural sources of carbohydrate contain sufficient amounts of phosphates but, in Case of deficiency, this may be supplied in t h e form of calcium acid phosphate, superphosphate or monoammonium phosphate, or any other form of soluble phosphate. I n general, 0.2 to 0.4% of phosphorus pentoxide is required based on the weight of the carbohydrate used. If ammonium salts are used, 4 to 6% ammonium sulfate or its equivalent is necessary, based on the weight of the carbohydrate used. If ainmonium salts are used it is necessary to neutralize the excess acidity and maintain the proper pH with calcium carbonateusually about 5 t o 7y0based on the weight of the carbohydrate is sufficient. If ammonia is used, 1 to 2% NHI is sufficient, depending 011 the composition of the mash and on the alkali or buffer
Table V, Material Balance in Acetone-Butanol Fermentation of Blackstrap Molasses Starting material: 100 Ib. blackstrap molasses containing, lb. Total solids Sucrose and invert sugar Protein Ash Fermentation data: Culture of saocharolyti,c bacteria uaed with 1.0% FHa added in form of ammonium liydroxlde Yields, lb. Butyl alcohol Acetone E t h y l alcohol Carbon dioxide Hydrogen D r y feed containing (6 lb. protein, G Ib. ash)
content. Different strains of bacteria may be found t o differ slightly in their tendency to produce undue acidity or in their susceptibility t o alkali in the neutralization process employed for maintaining pH. It has been found t h a t the saccharolytic acetone-butanol bacteria also require various growth factors. Teodoro and Mickelson (31) found in studying three different strains t h a t two of these required only biotin for maximum growth and t h e third needed both biotin and p-aminobenzoic acid. Thiamine, riboflavin, pyrodoxine, pantothenic acid, nicotinamide, and inositol vc-ere without effect
.
Fermentation Data Volume of mash. gal. Concentration of sugar, % Culture (ClOstridhium sUGChUTO-aCelOperbul~~~CUln), To Total “&OH (28%), gal. Total mixed solvents, gJ1. Acetone % n-Butyl ‘alcoIiol, % Ethyl alcohol, 7 Yield of total soyvents from sugar, % Residual sugar, g,/lOO ml. Residual ammonia, p.P.m. Fermentation temp., F.
1681
81.5 57.0 3.1 6.2 11.5 4.9 0.5 32.1 0.8 28.6
Flask A B C
D E
F
G
€I
b C
Ethyl Stillage Added, %
100 60 40 None
100 60 40
Neutralization Saltse Salts Salts Salts 1.3% NH3
:::p E2
Total Solvents,
G./L. 15.83 15.28 13.78 13.43 15.23 14.92 14.32 13.43
Acetone,
Yield on Sugarb,
29.2 27.9 29.5 27.5 26.5 28.1 28.2 29.2
33.3 31.9 28.8 28.2 32.4 31.3 30.0 29.0
%
%
None 1.3% NH: Average results of two flasks. Concentration of sugar 5% 5% (NH4)nSOa, 6% Ca’COa, 0.3% PeOa, based on weight of sugar.
The only means of preservation of these bacteriophages is by reduced temperatures, preferably sealed in tubes at a p H of 6.9 to 7.1. How t h e bacteriophage enters t h e fermentation process is a matter of wide speculation. Numerous theories have been developed, none of which has been proved. The bacteriophages affecting the acetone-butanol organisms have been found in the 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 bacteriophage usually becomes evident a t approximately the eighteenth hour after inoculation. The only immediate recourse to a bacteriophage attack is to substitute a t once an immune strain. Most industrial fermentation plants keep on hand a large number of strains, new isolations, and previously immunized organisms which can be tested rapidly and substituted in the plant for the organism which has been affected. Various methods of selecting immune strains of the organism have been tried. Some methods have been patentedLegg (IO) and Legg and Walton (18)-showing a process for selecting immune strains by repeated subculturing of the organism in the presence of small amounts of the bacteriophage. Others have found t h a t by plating out the original strain and selecting
INDUSTRIAL AND ENGINEERING CHEMISTRY
1682
various colonies they may isolate immune strains, or by placing a nonimmune culture on a plot of open, unsterile soil and reisolating the culture 6 months later, they may have an immune strain. The last procedure is t h e subject of a patent by Hanson ( 2 1 ) . It has been the author's experience t h a t any culture which has been properly immunized by suitable means loses some of its
Table VII.
Vol. 44, No. 7
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 distillatioii of the solvents has become a
Feed Values Recoverable from Acetone-Butanol Fermentation o f Blackstrap and Invert Molasses
Fractions of Stillage Tot.al solids evaporation whole stillage Solids recoverable by centrifuge Dried effluent from centrifuge
Kind of Molasses Invert Blackstrap Invert Blackstrap Invert Blackstrap
D r y Matter in Stillage.
100 Lb. Molasses Gives
Dryieed,, rinalJ'sis
% 1.15 2.71 0.17 0.22 0.96 2.50
activity. The fermentation is slightly sloxer, 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 C1. saccharoacetoperhutylicum 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 aluminum, tin, iron, nickel, zinc, manganrse, lead, cobalt, cadmium, chromium, thorium, thallium, and uranium ions a t 50-p.p.m. levels had no effect on the fermentation. The addition of mercury from 7 to 50 p.p.m. delayed the fermentation for 24 hours. The addition of antimony a t 50-p.p.m. level seriously reduced the fermentation yield.
lb. 17.7 28.6 2.6 2.3 14.8 26.3
Ib. 6.5 6.0 2.0 1.3 4.1 4.2
V
(Dry
F 36.8 20.8
77.6
57.5 28.1 16.0
% m
Riboflavin, ?/G.
0 . 3 1 12.1 0.14 23.2 2.00 4.8 2.60 7.2 0.0 13.4 0.0 25.9
52
38 49 27 54
37
major by-product. The normal dried stillage contams various vitamins: among them, Bzin amounts of 40 to 80 micrograms per gram. Up t o the present time no one has been able to increase the riboflavin (vitamin Bz) t o the levels that have been obtained in the fermentation of starches n ith CI. acetobutylicum. The stillage is suitable for admixing with other materials for animal feeds. The feed values recoverable from the acetone-butanol fermentation of blaclrstrap and invert molasses are shown in Table VII. The stillage may also be recycled into the fermentation iri order t o supply additional nitrogen and buffer substances. The molasses stillage has also been used in the manufacture of plastics; has been concentrated, dried, and burned t o an ash high in potassium; and has been used as a binder in foundry work. Literature Cited
Yields and Recovery of End Products
The most important products formed in the acetone-butanol fermentation of sugars are n-butyl alcohol, acetone, ethyl alcohol, cai bon dioxide, hydrogen, and riboflavin-containing feeds fiom the fermentation residue. I n the normal aretone-butanol fermentation of sugars a ratio of solvents of 68% butyl, 3oY0 acetone, and 2% ethyl may be expected. \-arious factors influence the change in these ratios. The material t o be fermented, the culture or strain of micioorganism used, temperaturep, use of stillage, contaminants present, and the addition of various chemicals all serve t o change the normal ratio expected. From the fermentation, a yield of 28 to 33% on the basis of the sugar or approximately 1 pound of mixed solvents from 3.6 pounds of invert molasses is normal. I n Table V a material balance using blackstiap molasses in the acetonebutanol fermentation is shown. The actual yield of solvents is subject t o wide variation. S'aIious samples of molasses of t h e same general type may be found t o give different yields n ith the same culture of bacteria. Likewise, different types of molasses will often be found to give substantially different yields. For example, beet molasses gives higher yields of solvents than any of the types of cane molasses. It has also been found that supplementary nutrients such as distillation slop from an alcohol fermentation of a saccharified grain mash may affect the yield (Table VI). Kutrients of this type tend in general to increase the yield. Similarly, if ammonia is utilized in place of calcium carbonate t o regulate the pH of the mash throughout the fermentation, the yield tends t o be increased slightly. The solvent ratio and yield also depend on a number of factors such as the particular strain of bacteria employed and the composition of the mash. Different ratios of acetone to butanol and ethanol may be obtained with different tgpec: of molasses Purrto Rican molasses,
I I
(30) (31) (32) (33)
Arroyo, R.. U. S.Patent 2,113,472 (1938). Arzberger, C. F., Ibid.,2,050,219 (1936). Ibirl., 2,139,108 (1938). Beesch, S. C., Ibid.,2,439,791 (1948). Beesch, S. C,., and Legg, D. A , Bid.,2,420,998 (1947). Carnarius, E. H., and McCutchan, W. N., I b i d . , 2,139,111 (1938). Carnarius, E. H., Ibid., 2.423,580 (1947). Gabriel, C. L., IND.ENG.CHEM.,20, 1063-7 (1928). Gabriel, C. L., and Crawford, F. M., Ibid., 22, 1163-5 (1930). Hall, H. E., U. S. Patent 2,147,487 (1939). Hanson, C. T., Ibid.,2,085,428 (1937). Hildebrandt, F. M., and Erb, N. J.. Bid., 2,169,246 (1939). Imak, A , , Ibid.,1,725,083 (1929). Izsak,A,, and Funk, F. J., Ibid.,1.908,361 (1933). Killeffer, D. H., IND. ENG.CHEM.,19,46-60 (1927). Legg, D . A , U. S. Pat,ent 1,668,814 (1928). Legg, D. A , and Stiles, H. R., I b i d . , 2,063,448 (1936). Legg, D. A., and Walton, h l . T., Ibid., 2,132,358 (1938). Loughlin, J. F., Ibid., 1,992,921 (1935). I b i d . , 2,096,377 (1937). Ibid.,2,219,426 (1941). McCoy, E. F., I b i d . , 2,110,109 (1938). I b i d . , 2,398,837 (1946). Muller, J., Ibid.,2,123,078 (1938). Ibid..2,132,039 (1938). I b i d . , 2,198,629 (1940). Prescott, S.P., and D u m , C. G., "Industrial Microbiology," 2nd ed., New York, McGraw-Hill Book Co., 1949. Prescott, S.P., and l I o r l h w a , X., U. S.Patent 1,933,683 (1933). Sherman, J. bI., and Erb, S . M., Ibid.,2,017,572 (1935). Stiles, H. R., Ibid.,2,073,125 (1937). Teodoro, R. R., and Mickelson, & N., 'I Arch. . Biochem., 6 , 471-7 (1946). \yeiemann, C., U. S.Patent 2,386,374 (1945). yioodruff, J. C., Stiles, H. R., and Legg, D. A,, Ihd., 2,089,522 (1937).
K C C E I ~ L D foi
remew July 10 1961.
ACCEPTEDApril 7 , 1952.
