REDUCTIVE PROCESSES OF CLOSTRIDIUM BUTYLI- CUM AND THE MECHANISM OF FORMATION OF ISOPROPYL ALCOHOLI
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REDUCTIVE PROCESSES OF CLOSTRIDIUM BUTYLI-
CUM AND THE MECHANISM OF FORMATION
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OF ISOPROPYL ALCOHOLI
A. F. LANGLYKKE, W. H. PETERSON AND E. B. FRED
Departments of Agricultural Chemistry and Agricultural Bacteriology, University of
Wisconsin, Madison, Wisconsin
Received for publication May 3, 1937
Isopropyl alcohol as a fermentation product of certain butyl
alcohol-producing bacteria was first demonstrated by Pring-
sheim (1906). Folpmers (1920) found it among the products
of Granulobacter butylicum (Beijerinck) and Van der Lek (1930)
reported it as a characteristic product of the same species (which
he called Clostridium butylicum (Beijerinck, Donker). Lang-
lykke, Peterson and McCoy (1935) showed that different strains
of this organism vary markedly in the relative production of
isopropyl alcohol and acetone.
Although isopropyl alcohol has long been recognized as a
fermentation product, no direct evidence as to the manner of
its formation has been presented. It is generally assumed to
arise through the hydrogenation of acetone (Van der Lek (1930),
Kluyver (1935), and Osburn (1935)). Such reductive processes
are characteristic of the butyl alcohol bacteria. In the normal
fermentation of carbohydrates butyric acid is first formed and
then reduced to butyl alcohol. Several workers (Reilly et al.
(1920), Speakman (1920), Blanchard and MacDonald (1935))
have shown that added propionic acid and aldehyde are also
reduced to the corresponding alcohol.
1 Supported in part by a grant from the Special Research Fund of the Graduate
School.
443444 A. F. LANGLYKK, W. H. PETERSON AND E. B. FRED
EXPERIMENTAL
Cultures
The cultures used, Clostridium butylicum strains 21 and 46,2
have been described by Langlykke, Peterson and McCoy (1935).
In addition to butyl and ethyl alcohols both produce acetone
and isopropyl alcohol. Strain 21 produces little acetone while
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strain 46 produces both acetone and isopropyl alcohol in ap-
preciable amounts.
Medium
It was desirable to employ a medium containing as little
extraneous organic matter as possible. After a number of trials
a medium composed of 0.07 per cent dibasic ammonium phos-
phate, 0.5 per cent peptone, 0.1 per cent asparagine, about 3
per cent glucose and tap water was selected. Neither peptone
nor asparagine was adequate as the sole source of organic nitrogen.
In order to obtain complete fermentation, ingress of air had to
be avoided. If this was not done, an incomplete, acid fermen-
tation resulted. Entrance of air was prevented by attaching a
mercury seal to the flask. The gas formed in the initial acid
fermentation displaced enough air to permit the reductive phase
of the fermentation to proceed.
Analytical methods
Glucose was determined by the method of Stiles, Peterson and
Fred (1926), and lactic acid by the method of Friedemann and
Graeser (1933) applied to an ether extract of an aliquot of the
culture.
Neutral volatile products were determined on a distillate of
the culture. For this purpose an aliquot of the culture was
made slightly alkaline and about 50 per cent of the liquid was
distilled off and collected under carbon dioxide-free water. The
distillate was analyzed for butyl and ethyl alcohols by the
2 Both strains are called C. butylicum for the present, although Prof. Elizabeth
McCoy of this laboratory believes that, because of variations in certain character-
istics, the two cultures may later have to be classified as different species.REDUCTIVE PROCESSES OF CLOSTRIDIUM BUTYLICUM 445
method of Johnson (1932), acetone was determined by a mod-
ification of Goodwin's method (1920), and isopropyl alcohol
by the oxidation procedure previously reported (Langlykke
et al. (1935)).
For the determination of volatile acids 100 cc. of the culture
were concentrated by distillation to about 40 cc. and then steam
distilled until 500 cc. of distillate had been collected. Butyric
and acetic acids were determined on this distillate by a modifica-
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tion of the procedure of Virtanen and Pulkki (1928).
For the determination of 2,3-butylene glycol an aliquot of the
culture was neutralized with sodium hydroxide, taken up with
plaster of Paris, and extracted with ether for 48 hours. Water
was added to the extract, the ether was distilled off and the
aqueous solution was oxidized to acetaldehyde by acid periodate
(Brockmann and Werkman (1933)). The acetaldehyde was
absorbed and determined as in the lactic acid method of Friede-
mann and Graeser (1933). Acetylmethyl carbinol was deter-
mined directly on the culture by the distillation procedure of
Langlykke and Peterson (1937).
The procedure of Fromageot and Desneulle (1935) was applied
to an ether extract of the culture for the determination of pyruvic
acid. Ceric ammonium sulphate in acid solution was quan-
titatively reduced with the oxidation of one mol of pyruvic acid
to one of acetic acid and one of carbon dioxide. There was 97
per cent recovery of pyruvic acid. When lactic acid was present,
a correction of 0.098 mgm. per milligram of lactic acid was
necessary.
Products of the fermentation
Although there have been many quantitative studies on the
fermentation products of the butyl-alcohol-producing bacteria
little has been done on the identification of these products.
Before beginning quantitative work it was therefore thought
desirable to identify the products of one of these strains.
For this purpose 14 liters of basic medium containing 100
grams of calcium carbonate were inoculated with 400 cc. of a
culture of strain 21 in 6 per cent corn mash. When fermentation
was complete the culture was neutralized with sodium hydroxide446 A. P. tLAGLYI , W. S. PVTERSON AN])E. 13. PRED
and about 30 per cent of the culture was distilled off to recover
the neutral volatile products. These were then concentrated
by repeated distillation, the concentrated material dehydrated
with anhydrous potassium carbonate and fractionated, and the
various fractions studied by the preparation of suitable
derivatives.
By determination of melting points and mixed melting points
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with derivatives of authentic compounds it was established that
n-butyl alcohol, isopropyl alcohol and acetone were produced.
The 3, 5-dinitrobenzoate prepared from the butyl alcohol fraction
melted at 630 to 640C. (authentic m. p., 630C.), and the p-nitro-
phenylhydrazone from the acetone fraction at 1490C. (authen-
tic, 1490 to 1500C.). Isopropyl alcohol was characterized by
oxidizing to acetone and preparing the dibenzalacetone (m.p.,
1110 to 1120C.; authentic, 1100 to 1110C.) by the reaction with
benzaldehyde. Direct evidence of the presence of ethyl alcohol
could not be obtained. If this compound is produced, it is in
such small amounts that a separation could not be effected.
The evidence for the production of ethyl alcohol is admittedly
weak and lies in the fact that a small value for ethyl alcohol is
always obtained in the analysis by the method of Johnson (1932).
Butylene glycol could not be demonstrated in the culture
residue after evaporation. Several other fermentations were
examined for acetylmethyl carbinol and for 2,3-butylene glycol
by the method of Lemoigne as modified by Kluyver et al. (1925),
but the results were uniformly negative.
Reduction of acetone
To determine whether acetone when added to the culture
would be reduced, a number of experiments were carried out.
A series of 750-cc. Erlenmeyer flasks containing the basic medium
were sterilized at 15 pounds pressure for 40 minutes. Sufficient
sterile glucose solution was added to bring the glucose content
to 3 per cent, and then varying quantities of a sterile acetone
solution were added. In each case the total volume was ad-
justed to 500 cc. by addition of sterile water. A two per cent
inoculum of a corn mash culture of the organism was then addedREDUCTIVE PROCESSES OF CLOSTRIDIUM BUTYLICUM 447
to each flask except the controls, the mercury seals were applied,
and the flasks allowed to incubate at 370C. When fermentation
was complete, as evidenced by the cessation of gassing, the
flasks were analyzed for products.
Data for strain 21 are presented in table 1. They indicate
that the added acetone was almost completely converted to
isopropyl alcohol, and that, as the quantity of added acetone
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was increased, there was an increased formation of isopropyl
alcohol from the carbohydrates. This is due in part to the
better fermentation of the carbohydrate when acetone was
added, but probably another factor is involved.
TABLE 1
Reduction of added acetone in association with the fermentation of glucose (strain 21i)
EXPERI- EXPERI- EXPEZR- EXPERI-
MENT 1 mENT 2 MiNT 3 mENT 4
mM mM mM mM
Glucose fermented, per liter ........ ........ 122.7 124.5 129.5 136.1
Acetone added:
Per liter ................................. 0.0 11.3 22.6 33.9
Per 100 mM. glucose fermented ........... 0. 0 9.1 17.5 24.9
Products (based on 100 mM. of glucose
fermented):
Butyric acid .............................. 9.85 8.70 8.25 6.70
Acetic acid ............................... 9.45 8.90 8.65 8.10
Butyl alcohol ............................. 50.5 57.5 54.6 55.8
Ethyl alcohol ............................. 3.3 2.6 2.2 1.5
Acetone ................................. 1.5 2.7 3.6 3.4
Isopropyl alcohol ......................... 16.1 26.9 39.2 51.0
It will be noted that as added acetone was increased, the
ratio of the sum of isopropyl alcohol and acetone formed from
the carbohydrate to the sum of butyl alcohol and butyric acid
increased (from 0.29 in experiment 1 to 0.41 in experiment 4).
In other words, production of three-carbon compounds increases
at the expense of four-carbon compounds in the presence of the
hydrogen acceptor, acetone.
In the case of strain 46 the cultures containing added acetone
had stopped gassing after five days while in those without added
acetone gas was still forming at this time. Therefore the blank448 A. F. LANGLYKKE, W. H. PETERSON AND E. B. FRED
fermentations were incubated for eight days. In spite of these
differences in fermentation periods the results (table 2) show
the general tendencies pointed out for strain 21.
The great differences in the amounts of glucose fermented
prevent direct comparison, but if experiment 4 and the control,
which do not differ greatly in this respect, are compared it will
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be seen that the total production of acetone and isopropyl
alcohol from the substrate is somewhat greater when acetone is
added, though apparently acetone does not function so efficiently
as a hydrogen acceptor for this strain as it does for strain 21.
TABLE 2
Reduction of added acetone in association with the fermentation of glucose (strain46)
EXPERI- EXPERI- IP1ERI- EXPERI-
MENT 1 MzNT 2 MENT 3 MENT 4
mM mM mm mM
Glucose fermented, per liter ....... ......... 69.2 29.3 45.8 74.6
Acetone added:
Per liter ................0.0. ....... 13.1 26.3 39.5
Per 100 mM. glucose fermented . .......... 0. 0 44.6 57.4 53.0
Products (based on 100 mM. of glucose
fermented):
Butyl alcohol ............................. 39.3 35.1 29.9 40.1
Ethyl alcohol ............................. 1.9 2.0 1.7 0.9
Acetone ................................. 5.6 15.4 30.4 26.8
Isopropyl alcohol ......................... 18.8 46.7 54.5 57.6
Fermentation of pyruvic acid
Since the organisms studied could reduce the carbonyl group
in acetone, experiments were conducted to determine whether
this function was general or specific. For instance, if they could
reduce pyruvic acid to lactic, the position of pyruvic acid as an
intermediate in the fermentation mechanism might be doubtful.
A series of 750-cc. Erlenmeyer flasks containing the basic
medium were sterilized at 15 pounds pressure for 40 minutes.
Pyruvic acid which had been twice redistilled under vacuum
from the Eastman technical grade was added aseptically to
sterile water to make a solution of convenient concentration.
Glucose solution, pyruvic acid solution and sterile water wereREDUCTIVE PROCESSES OF CLOSTRIDIUM BUTYLICUM 449
then added aseptically to each flask to give a final volume of
500 cc. and varying glucose and pyruvic acid concentrations as
shown in table 3. An excess of calcium carbonate was added
to each flask which was then inoculated with 2 per cent of a
corn mash culture of strain 21.
After incubation for five days at 370C. the cultures were an-
alyzed with results shown in table 3. The data indicate a slight
increase in the apparent lactic acid production, but the major
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portion of the added pyruvic acid is accounted for in the normal
products of the fermentation. This does not demonstrate,
TABLE 3
Dissimilation of pyruvic acid added to a glucose fermentation (strain 21)
EoERUIMENT EXPERMENT EXIPEIMENT
1 2 3
miM mm M
Glucose fermented, per liter ................... 179.8 149.6 84.4
Pyruvic acid fermented:
Per liter .................................... 0.0 47.7 125.4
Per 100 mM. of glucose fermented ........
.. 0.0 31.9 148.7
Products (based on 100 mM. of glucose fer-
mented):
Butyric acid ................................ 8.8 7.8 32.2
Acetic acid ................................. 17.1 17.7 77.6
Lactic acid ................................. 1.1 1.6 7.1
Butyl alcohol ............................... 44.6 50.6 41.7
Ethyl alcohol ............................... 2.6 3.5 6.2
Acetone ................................... 1.6 2.5 4.0
Isopropyl alcohol ........................... 15.6 30.6 30.4
however, that pyruvic acid is not directly reduced to lactic acid
since this process may occur followed by metabolism of the
lactic acid so formed. (Lactic acid, in the presence of glucose,
is decomposed by strain 21.)
Like acetone, pyruvic acid acts as a hydrogen acceptor, though
this compound is not simply reduced. The ratio of the sum of
isopropyl alcohol and acetone to the sum of butyric acid and
butyl alcohol increased from 0.33 to 0.57 in the second experi-
ment where 47.7 millimols of pyruvic acid were fermented. The
apparent anomaly observed in experiment 3, where the ratio450 A. F. LANGLYKKE, W. H. PETERSON AND E. B. FRED
dropped to 0.47, is due to the high production of acids. If thee
acids, which are also hydrogen acceptors, are not metabolized,
more hydrogen is available for the other normal functions of
the cell.
Reduction of acetylmethyl carbinol
As was previously pointed out, neither acetylnethyl carbinol
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nor its reduction product, 2,3-butylene glycol, appears among
the products of the fermentation by C. butylicum. Hence, added
acetylmethyl carbinol, if directly reduced, might serve for study
TABLE 4
Reduction of added acetylmethyl carbinol in a glucose fermentation (strain 21)
EXPERIWMENT EPERXIMENT EXPRIMENT
1 2 3
m mM mm
Glucose fermented, per liter.. 95.8 69.1 53.8
Acetylmethyl carbinol fermented:
Per liter .0...................................0. 11.7 35.6
Per 100 mM. glucose fermented .............. 0.0 16.9 66.2
Products (based on 100 mM. of glucose fer-
mented):
Butyric acid. ......... ............... 9.7 14.6 19.7.
Acetic acid ................................. 21.1 29.5 26.0
Butyl alcohol ............................... 47.0 32.9 22..8
Ethyl alcohol ............................... 2.9 4.3 14.9
Acetone .................................... 2.0 2.3 3.0
Isopropyl alcohol ............................ 11.7 12.5 15.8
2,3-Butylene glycol.0.0 17.5 66.0
of the effect of a hydrogen acceptor on the course of the fer-
mentation, without the objections attending the use of a hydro-
gen acceptor which also performs other functions.
Essentially the same procedure as in the preceding experiments
was followed. The acetylmethyl carbinol solution was sterilized
by filtration through a Berkefeld filter. The flasks were inocu-
lated with strain 21 and were incubated at 370C. for four days;
gassing had then ceased. The cultures were analyzed with
results reported in table 4.
The figure for acetylmethyl carbinol utilized represents theREDUCTIVE PROCESSES OF CLOSTRIDIUM BUTYLICtM 451
difference between that added and the amount remaining after
fermentation. Actually the reduction was in each case approx-
imately 95 per cent complete. Quantitatively, the yield of
butylene glycol was 103.6 per cent in the case of experiment 2
and 99.7 per cent in experiment 3, based on the acetylmethyl
carbinol utilized.
Although in each instance poor fermentations of the glucose
were obtained with a high proportion of unconverted acetic
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acid, still the effect of the presence of a hydrogen acceptor may
be noted. The ratio of three-carbon to four-carbon products
increased from 0.24 in experiment 1 containing no additions to
0.31 in experent 2 and 0.43 in experiment 3.
DISCUSSION
The behavior of C. butylicum fermentations when a hydrogen
acceptor is present may be explained on the assumption that
both acetone and butyric acid arise from a common precursor,
acetoacetic acid. Johnson, Peterson and Fred (1933), have
shown that the related organism, Clostridium acetobutylicum,
produces a carboxylase which readily causes the formation of
acetone from acetoacetic acid, and it seems reasonable to suppose
that the latter compound may also be hydrogenated to butyric
acid provided that the hydrogen for this reduction is available.
Presence of an acceptor competing for the available hydrogen
for reduction would disturb the balanced reactions and favor
the decarboxylation reaction by which acetone is produced over
the reductive process through which butyric acid arises.
It is true that the behavior noted is not proof that the three-
and four-carbon products come from a common precursor. They
may arise through separate reaction chains which would be
similarly affected by the paucity of hydrogen because of the
presence of a hydrogen acceptor. Thus, butyl alcohol may arise
by condensation of two molecules of acetaldehyde to acetaldol,
followed by rearrangement to butyric acid, and hydrogenation
to butyl alcohol. It is not probable that acetaldol functions as
an intermediate, as it has been shown by Johnson, Peterson
and Fred (1933) and also by Blanchard and McDonald (1935)452 A. F. LANGLYKKE, W. II. PETERSON AND E. B. FRED
that aldol is extremely toxic to the related organism, C. aceto.
butylicum. Bernhauer and Kurschner (1935) agree that aldol
does not figure in the fermentation Mechanism of C. aceto-
butylicum. The assumption that acetoacetic acid is the common
precursor of acetone and butyric acid, and subsequently of
isopropyl and butyl alcohols, merely offers the most convenient
explanation of the phenomena observed.
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The effect of hydrogen acceptors on the course of the fermenta-
tion effected by Lactobacillus lycopersic has been studied by
Nelson and Werkman (1936). In agreement with the results
reported in this investigation these authors found that acetal-
dehyde and acetylmethyl carbinol were readily hydrogenated
by their organism, and that the presence of these hydrogen
acceptors caused an increase in oxidized products at the expense
of reduced products formed from the carbohydrate.
SUMMARY
1. Acetone and acetylmethyl carbinol added to glucose fer-
mentations were reduced by Clostridium butylicum to the cor-
responding alcohols, isopropyl alcohol, and 2,3-butylene glycol.
2. The quantity of acetone reduced varied with different
strains of the organism.
3. Added pyruvic acid was fermented to the same products
as are formed from glucose.
4. Addition of acetone, pyruvic acid or acetylmethyl carbinol
favors the production of isopropyl alcohol and acetone from the
carbohydrate at the expense of butyl alcohol and butyric acid
production.
5. A proposed explanation of the effect of hydrogen acceptors
is that both three- and four-carbon compounds may arise from
a common precursor, acetoacetic acid.
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