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Patented Aug. V10, 1937
2,089,522‘
- UNITED STATES PATENTv OFFICE
2,089,522
PROCESS OF PRODUCING‘BUTYL ALCOHOL
John C. Woodrulf, Hugh R. Stiles, and David A.
Legg, Terre Haute, Ind., assignors to Commer
cial Solvents Corporation, Terre Haute, Ind.,
a corporation of Maryland
‘No Drawing. Application June 12, 1933
Serial No. 675,459
_ '16 Claims. (Cl. 195-44)
5
Our invention relates to the production of
butyl alcohol and other valuable products by the
fermentation of sugar-containing solutions.
such substances as calcium carbonate. So much
More speci?cally, our invention relates to the
confusion exists in the nomenclature and reported
production of normal butyl alcohol, acetone, and
cultural characteristics of the prior art organ
isms of the Clostrz'dium butyricum type that it is
ethyl alcohol by the fermentation of sugar solu
tions by‘means of bacteria designated herein as
tation conditions, especially an accurate control
of the hydrogen ion concentration by means of
_Clostridium saccharo-acetobutylicum. '
impossible to state de?nitely if any of them are
included in the group now designated as Clostrid
It has previously'been known that sugar solu
tions could be fermented by means of organisms
of the Clostridz'um butyricum group with the pro
ium saccharo-acetobutylicum. A certain organism, for'example, may be described by one in 10
vestigator as having properties which would
duction of various products. such as acetic and
bring it within the present classi?cation, where
butyric acids, butyl alcohol, etc. (see for example 'as another investigator may report an organism
Bergey’s Manual of Determinative Bacteriology,
Williams '& Wilkins 00.‘, Baltimore, 1925, pages
326-7). However, the yields of butyl alcohol have
in all cases been so low as to preclude commer
cial utilization of such fermentations. Therefore,
of the same name to have properties’ which would
de?nitely exclude it from this classi?cation. It
is to be understood, therefore, that our inven
tion includes within its scope the use of any of
the prior art ‘bacteria which have in fact the
in spite of the fact that crude sugar solutions
characteristics hereinafter speci?ed, irrespective
represent the cheapest source of raw material,
up to the present time the production of butyl
alcohol has been accomplished only by the fer~
mentation of starch mashes by organisms of the
of any con?icting characteristics which may have 20
type Clostridium acetobutylz'cum (Weizmann).
The organisms of the C'lostridium butyricum
group have never been shown to be fermenting
agents of a commercial character but_have been
discarded as members of that long list of micro-.
been reported in the literature. It is to be fur
ther understood, of course, that our invention re
lates to the use of these organisms only under the
newly devised fermentation conditions to be here
inafter speci?ed, and not to the use of these or
ganisms generally, under any conditions.
The various conditions which we have found
to be essential forthe production of high yields
organisms which produce the desired products,
of solvents from commercial sugar-containing
mashes by organisms of this group are, brie?y,
the presence of a soluble carbohydrate as the
duce acids and neutral end-products of the type source of carbohydrate, the presence of degraded
produced by butyl organisms such as Clostrz'dium protein (including ammonia) as the source of
aoetobutylz'cum (Weizmann) and have a peaked nitrogen, a fermentation temperature of 24° C.
0.”
m) acidity curve for the fermentation of the same
to 40° 0., preferably 29-30“ 0., and the con
general type as thatv produced by the Weizmann trol of the acidity of the mash during the fer
organism. The butyl organisms of the Clcstrz'd
mentation such that the ?nal hydrogen ion
ium acetobutylicum type maintain their‘ optimum ‘ concentration, obtained by the action of the bac
hydrogen ion concentration in the mash without teria, falls within the range (of pH 5.0 to pH 6.2,
40 the necessity of control by means of neutralizing
preferably 5.5-5.85. Of course, other known 40
agents or the like. In fact; it has been shown fermentation conditions which are usually em
that the addition of materials tending to change ployed with any organism of this‘ general type,
the hydrogen ion ‘concentration has a de?nite such as the presence of necessary mineral ele- , deleterious effect upon certain of these organ~ ments (e. g., phosphates and the like), may be
isms.- For example,'Grimbert (Ann. de l’Inst. employed in the usual manner known to those
Past. 7, 353), Du Claux (Ann. de l’Inst. Past. 9, skilled in the art but will not often be necessary
811), and McCoy et al. (Jour. Infect. Dis., 39, with ‘such materials as cane molasses.
‘
457) have shownthat an addition of calcium
An essential element of the fermentation‘of
0 but in such small amounts as not to be econom
ically feasible. _ The bacteria of this group pro
carbonate to the mash produces a marked de
our invention is the control of the hydrogen ion
crease in the yield of butyl alcohol. The natural
assumption has therefore been that any attempts
concentration, so that’the ?nal pH secured by
‘the action of the bacteria falls within the speci
?ed limits. In practically all other known fer
to reduce the hydrogen ion concentration of fer
mentations by organisms of the Clostridium bu
tyricum group would likewise reduce the yields‘
of neutral end-products.
'However, we have discovered that the group
of bacteria herein designated as Clostridium sac
charo-acetobuiylz'cum will produce high yields of
butyl alcohol from commercial sugar-containing
60 mashes if there. are maintained certain fermen
mentations in which neutral end-products are
secured, the adjustment of the initial hydrogen
ion concentration has been considered to be of .
most importance. If this initial hydrogen ion
concentration is adjusted within the operative
limits, the fermentation will proceed normally
and no attention need be paid to the final hydro
gen ion concentration.
However, we have found 60
2
9,089, 522
in the present case that although the initial
hydrogen ion concentration may vary over a
considerable range, the ?nal pH obtained by the
action of the bacteria must fall within de?nite
5 limits if consistent high yields of solvents are to
besecured.
.
‘
tained by continuous or semi-continuous addi- ‘
tion of an alkaline material, such as ammonia,
during the active stage of the fermentation and
until after the “acidity break". However, the
mechanical difficulties or procedures of this na
,ture are well known to those skilled in the art.
Even a slight over-neutralization at any time
during the fermentation will often result in in
We have found that the final pH secured by
the action of the bacteria may be controlled by‘
the introduction vof certain materials into the hibiting further active fermentation for-a period
1o mash at the beginning of the fermentation. ' For of' many. hours or ‘even days. Consequently,
example, we have found that if calcium car
- automatic electrometric titration apparatus is
bonate, barium carbonate,v iron carbonate, or
most .desirable if such a procedure is employed.
‘ other insoluble non-toxic base, is added to the
In any prccedure'of ‘this nature, the pH should.
mash in an amount sufficient to neutralize any
15 free acidity, and an amount in. excess of this
be controlled to approximate that obtained when
to the extent of about 5-'7% on the weight of
the sugar, the ?nal pH of the fermentation will
be found to be within the operative range. Al
though the various materials mentioned may be .
the .specifled amounts of insoluble basic materials ' 15
are employed.
_
-
,_ Furthermore, from the standpoint of simplicity
of operation, we prefer to control the acidity of
the mash during the fermentation by means of
the insoluble materials such as calcium carbonate. 20
We have found that for a wide range of grades of
20 satisfactorily used in our prQcessLcaIcium car
bonate has been found,'in most cases, to be es
pecially well suited for this purpose, and is to ' molasses, approximately 5-_7% of calcium car
be preferred from an economic standpoint. bonate, or the like, on the weight of the sugar in
However, in choosing the material to bevernployed the mash, secures adequate control of the acidity
25 the composition of the medium should be con
such that the ?nal hydrogen ion concentration se 25
sidered and a material chosen which will not give cured by the action-of the bacteria falls within '
rise to an undesirable concentration of, a par
the desired limits. This fact may be seen to
ticular metal ion, even though generally con
obviate the necessity for individual treatment of
sidered to be non-toxic ‘in character.
each sample of molasses unless the ultimate possi-'
30 The amount to be added in any particular case ble yield is desired. In view of the difficulties of
will of course depend to some ‘extent on the such‘a procedure, we prefer to secure the desired
composition of.the mash. For example?a mash pH control by introducing material of the in
containing a substantial amount of phosphates,
or other material having a bu?erin'g action, will
35 require less calcium carbonate than one which
is devoid 'of such materials. Various samples
of calcium carbonate will also differv in respect
to the amount which is necessary to use, due to
the physical properties of the material and also
40 to its chemical properties, as for example, the
presence of substantial amounts of lime. In any
soluble type into the mash before fermentation
begins. The temperature range which we have
found to be essential for the best results in this 35
fermentation is within the limits about 28° C. to
32° C. Growth will occur and sometimes active
fermentation will take place over a, much wider
range, but for consistent high yields of solvents
from commercial sugar-containing mashes the
temperature should be maintained within the
particular case, preliminary fermentations will
range speci?ed and preferably within the nar
‘enable’ one skilled in the art to determine the
rower range 29-30° C.
optimum concentration ,\for, thencalcium car'
45 bonate employed. However, in general it. may
be said that from 3% to 10%. on the weight. of the
With regard to the-necessary nutrients for this
fermentation, it may be said that degraded pro
tein nitrogen is essential. As used here and in
sugar, in excess of that required to neutralize
the appended claims, the term “degraded protein
- the original acidity, will give very satisfactory ' nitrogen" is to be taken as including hydrolytic
degradation products such as polypeptides, amino .
results. The calcium' carbonate or other in
50 soluble-base used should, in’ general, ,be sum
ciently finely divided so that when resting on
the bottom of the fermentation vessel they will
present a considerable surface to the ferment
ing mash. When employing this means of con
55 trolling the hydrogen'ion concentration, ‘undue
agitation should be avoided so as to prevent the
possibility of ?xing too large a percentage of the
acids produced in the early stages of the fer
acids, etc., metabolic degradation products such 50
as urea, etc., and the ?naldegradation product,
ammonia, and its salts. Although ammonia (or
an ammonium compound such as a sulphate, etc.) '
alone has been found to give satisfactory yields of
solvents, it is preferred to use a mixture of am
55
monia and partially degraded protein materials
such‘as yeast water, steep water, and the like,
in {order to consistently secure optimum yields.
mentation, and thus undesirably displacing ‘the - Howevenvery satisfactory results are obtained
60 equilibrium of the fermentation. It should be
de?nitely understood that the purpose‘ of vthe
addition oi‘ the basic materials in this process is
not to neutralize all the acids produced in the
fermentation, but merely to control the hydrogen
65 ion concentration in such a manner that the
when using only partially degraded protein ma
terial as the nitrogen source.
60
For example, ma
terials such as yeast water, steep water, and the
distillery slop from the Clostrz'dium acetobutyl
icum (Weizmann) fermentation have been found
to be satisfactory. Although undegraded pro
final pH secured by the action of the bacteria ‘ tein, such as corn gluten, corn germ meal, and
(and not by the action of neutralizing agents)
the like cannot‘ be utilized as the sole source of
falls within the speci?ed limits.
nitrogen, small amounts of such materials, in
It is to be understood that the invention is addition to ammonia or partially degraded pro
70 not to be limited to the particular means em
‘tein,
sometimes_ produce improved results. Other 70
‘ _
ployed for securing the desired ?nal hydrogen nutrient materials such as mineral elements, e. g.
ion concentration. Any equivalents or modi?ca
phosphates and the like, should be present in
tions which would naturally occur to one skilled small amounts as in the case of other known
in the art may, of course, be employed. For . fermentations.
_
However, if crude sugar solu
75 example, an accurate pH control may be main
tions such as molasses mashes are employed, these 75
3
2,089,522
materials will usually be found to be present in
sumcient amounts. The amount of ammonia or
degraded protein .to be added will, also vary with
II. Biochemical-Continued
B. Nitrogen metabolism I
1. Ability to produce high yields of butyl
_ ‘the raw material used. For example, certain
alcohol and acetone in sugar media
5 samples of molasses may be found to have su?l
containing ammonia as the principal
cient ammonium compounds'and other degraded
protein so that very little more need be added. In
general, it may be said that with cane molasses
source
mashes from 0.7 to, 1.7% of N_H3 as ammonium
10 sulphate on the weight of the sugar or an equiva
' lent amount of other degraded protein, will give
satisfactory results.
-
(
The following is a typical-medium which we
have found to be suitable for laboratory fermenta
_l5 tions:
'
_
.
'
Y
.
of nitrogen.
-
m
source.
'
-
3. Inability to utilize undegraded protein as
sole source of nitrogen.
4. Inability to liquefy' gelatin or to produce
more than slight proteolysis'of milk.
C. Oxygen requirements
1. Anaerobic. .-
-
Molasses medium (Medium 1)
'
2. Ability to utilize degraded protein (in
cluding ammonia) as the sole nitrogen
15
-
D. Temperature range for solvent production
,
' Louisiana molasses is diluted to a sugar con
centration of about 5.5% and to this is added
about 0.3% (NH4)2SO4 and about-0.4% of Y200
.20 mesh calcite, based on the mash volume. This
mash is. sterilized for 30 minutes at 20 l_bs.~pres
A Of course, it is well known to those skilled
' 1. From 24° C. to 40° 0., preferably 29° C.
to 30° C.
E. Hydrogen ion concentration for solvent
production
,
‘
,
"
1. Final pH of 5.0-6.2, preferably 5.4-5.85.
In view. of the uncertainty in the literature
as to methods utilized for certain of the biochemi
in the art that different samples of molasses vary ' cal tests referred to above, we believe it to be de
25 in a numberof respects, such as sugar content, sirable to amplify, somewhat, the characteristics 25
ash content, and the like. These variations brie?y outlined. For example, the fermentation
.naturally change somewhat the mashing pro
characteristics referred to under the heading
cedure in different cases. For example, some “Carbohydrate fermentation" are those charac
samples of molasses may be found to>be lacking teristics determined under optimum conditions,
30 in sufficient mineral elements such as phosphates as for example, in the molasses medium described 30
and the like. Other samples may be found to be > above or in similar media containing other carbo
lacking in partially degraded nitrogenous ma
hydrates. It should be particularly noted that
terial. Also, there are certain unknown factors these and all other fermentation characteristics
present in some types of molasses which make ' described in this speci?cationirelate to the fer
35
it desirable to use lower concentrations than in mentation of a commercial mash, i. e., one which
the case of other more suitable types. In any hasa sugar concentration of the order of 5%.
particular case, one skilled in the art may deter
mine the special requirements, if any, by pre
- liminary fermentations and may then make such
Quite di?erent results are often obtained with
laboratory ‘media containing lower percentages
cedure. However, such changes in mashing pro
of sugar. It should also be noted that fermenta
tion characteristics such as these refer to normal 40
consistent results and not to abnormally low‘ or
cedure for various types of molasses will be neces
high results which may sometimes be obtained
sary only to secure the absolute maximum yield.
with any culture. A typical carbohydrate fermen- ‘
40 changes as are necessary in the mashing pro
Very satisfactory yields can be secured in prac
45 tically all cases bynieans of the procedure out
lined above.
'
_
tation test of an organism falling in this group
is given below as an illustration.
45
‘
The, bacteria‘ which 'have been designated
C'lostridium saccharo-acetobutulicum in our in
vention and which are so designated in the ap
50 pended claims, comprise any bacteria having the
M‘iggum
Composition percent by weight of mmh
50
' following primary characteristics:
I. Morphological
A. Rod-shaped
s5
>
I
"
-
B. spore-forming-eclostri'dia and Plectridia
55
C. Practically indistinguishable from mem
bers of the Clostrldium butzlricum group
II.- Biochemical
,
‘
A. Carbohydrate fermentation
1. Ability to produce fair yields of butyl‘
‘60
60
alcohol and acetone consistently from
starch as the only source‘ of carbohy
0.
0.4% (210 mesh calcite) C8003 ____ __
drate (i. e., corn ‘or other mash con
65
taining starch and suitable nutrients)
2. Ability to produce yields. of butyl alcohol
‘
and acetone‘consistently above 30%/
on the weight of the sugar from 5%
sucrose
media
or
molasses medium
the
uninverted
(Medium 1) . de-.
scribed above.
'
'
3. Ability to produce yields of butyl alcohol
and acetone consistently above 30%
on the weight of the sugar from 5%
glucose media with suitable nutrients,
} or an inverted molasses medium corre- ,
7,5‘
sponding to Medium I.
.
a ______________________ _
‘ 5.
VI ........ -.'
_
a sugar as uninverted molasses
0.25 o (NH|)2S0| ................ -
0.4 a CaCOz (200 mesh calcite).___
' Louisiana molasses at about 20% sugar concentration inverted by
65
heating with sulphuric acid of about 3.6% concentration on the
weight of the sugar ior 40 minutes at 20 lbs. ressure, free ammonia
added to neutralize acid, calcium carbonate t an added and medium
sterilized 30 minutes at 20 lbs. pressure-
I
_ '
. ,With regard to the nitrogen metabolism,v the
undegraded protein materials referred to are such 70
materials as corn gluten and corn germ meal; the
degraded protein referred to comprises such mate
rials as yeast water, steep water, distillery slop,
and urea; and the gelatin liquefaction refers to
incubation on nutrient gelatin containing 2% 75
4
2,089,522‘
was obtained in each case but at the end of 30
CPI days the gelatin was in all cases found to be solid
The temperature and hydrogen ion concentra
tion ranges referred to do not represent the entire
ranges within which growth will occur but repre
sent merely the ranges within which high yields
of solvents may be obtained when operating-under
at 22° C. The proteolysis of milk refers to tests
such as the standard litmus milk test. In litmus
milk, organisms of this group ?rst reduce the
ratios which are given as characteristic of the
organism are those which are normally con
glucose. ,For example, stab cultures on such
medium were incubated at ‘22° C. and shake cul
tures were incubated at 30° 0. Excellent growth
the other conditions speci?ed. Also, the solvent
litmus and then give a somewhat rennet-like acid - sistently obtained under optimum conditions and
do not refer to abnormal ratios which may some
of 30 days. Thefollow'ing example will .illustrate times be secured with any of the cultures. Fur- _'
10 curd which shows only slight digestion at the end
~
thermore, it is to be understood that the char
acteristics speci?ed for these organisms are not
to be taken as limited to the speci?c methods and
data given above. These were given merely by
way of illustration, whereas the characteristics
a test of the type which may be employed to
determine the nitrogen metabolism of the organ
isms:
l5
‘
_
Solvent
31??(Tm v
Com position
of the organisms as claimed in our invention are
yclgl‘gggt'
those given generally in the outline.
sugar)
The organisms of this group are widely dis
tributed in nature and may be isolated from such 20
‘various sources as soil, rotted wood, grain, corn
stalks, river mud, and the like. In view of the
characteristics .listed above, one skilled in the
art may readily isolate these organisms from
F)
“0
5% sucrose _______________________________ __
0.13% KZHPOL,
0.05% NaCl__
VII _______ __
'
0.01% LigSOi.
6.0.
0.25% peptone
.
0.25% corn germ meal
0.5%
25
.
C2003 . _ . _ _ . . _ _ _ _ _ _ _ _ _ _
I such sources by known methods of isolation. Of 25
course, as is apparent to one skilled in the art,
. _ . _ __
5% sucrose ____ ._
swear
.
l
2
4.‘
VIII ----- -~ 0.07”); KQHPOL.
0.l% MLZSOL .._
these organisms cannot be isolated from every
sample of material tested. However, if a number
2“
O 5% CaC O3 ____________________________ __
of diiferent materials are tried, a good culture
5% sucrose ____ __
30
IX ________ __
0 2% (NII4)2SO4
will nearly always be secured.
0.2% corn germ mea
speci?c example is given as illustrative of one of'
0.07% KHgP
0.07% K2I‘IPO
301‘
the methods applicable to this purpose:
0.01% 1\IgSO4
_A number (say at least twenty) of 125 c. 0.
CaCOs...
sucrose in distiller
_
slo
35 X” -------- -- {025% CaC03.; ______
__
.
capacity, conical ?asks each containing 100 c. c. 35
of medium of the following composition are pre
‘
___________ __ }
31-9’
5 a sucrose in distillery 5101)...
XI‘ _______ __ {0.3% (NH4)2SO4 _________________ __
_
pared:
38.0.'
0.4% 02003 _____________________________ _.
* Slop from fermentation of 7.5% corn mash by C'lostriiiium aceto
butylzcum (Weizmann), adjusted to pH 6.1.
40
The following 30
0.2% corn gluten_
It is to be noted that the statements in the
outline regarding the nitrogen requirements of
these organisms refer to the suitability of nitrog
enous nutrients for the production of consistent
4‘ high yields of solvents and not to the ability or
i’ inability to utilize such forms of nitrogen for
growth or slight fermentation. For example,
'
-
Per cent
fCan'e molasses (Ii-5% sugar) ________ __
8-10
(NH4) 2804 _________________________ __
40
CaCOa (200 mesh calcite) __________ __
These ?asks of media are sterilized in the usual _
manner for 30 minutes at 20 lbs. pressure and
while still hot, e. g., 80-90“ C., are each inocu
lated with about 1 gram of soil, mud, com, com 45
stalks, rotted wood, and the like, preferably from
ya ?eld in which some kind of carbohydrate crop
undeg'raded protein such as a mixture of corn
~ has been grown. The ?asks are held at the in
gluten and corn germ will give a slight fermenta
oculating temperature for a short time, c. g., 1 to 50
_ tion, e. g., a solvent yield of one or two percent
“0 and molasses media will give a fair yield, i. e., 2 minutes and then rapidly cooled to 30-32" C.
and inoculated at, this temperature for ?ve days.
up to 20% or so, in some cases, without the
addition of ammonia or other degraded protein
nutrient.
'
.
'~
It is further to be noted that the utilization
' of ammonia is speci?ed as the principal source of
From the flasks showing most active fermenta
tion are next inoulated a number of '10"- x 3/4"
test tubes each containing 15 c. c. of 20% sterile 55
potato mash, using about 3 c. c. of inoculant.
The inoculated tubes are heated in a steamer for
nitrogen, rather than the sole source, for optimum
solvent production. These organisms can utilize about 1% minutes at a temperature of 95—100° C.
ammonia as the sole source of nitrogen, in some and then quickly cooled to 30—32° C. and incu
bated at this temperature for 4-5 days. From 60
cases with optimum solvent yield, but for con
6 O sistent high yields of solvents it is preferred to
these tubes are next selected for further inves
have a small amount of some other degraded tigation those which showed most active fermen
tation, as judged by. the heading up of the mash.
protein material present in addition tov the am
‘Transfers from these tubes are next made to
monia. This additional amount, however, is gen
erally present in such materials as molasses so dilution plates containing a solid medium com 65
that in this case the use of ammonia ‘alone will '
serve to produce optimum yields.
The term “anaerobic” as used in the above
outline, refers to the inability of the organisms to
grow on the surface of nutrient glucose agar when
O
incubated aerobically. The organisms are, how
posed of :
-
‘
.
'
Per cent
Standard nutrient agar _____ _'_ ___________ __ 1.7
(NH4)2SO4
,
0.1
Sucrose as glucose _______________________ _. 2.0
70
The plates are incubated under strictly anaerobic
. ever, capable of developing and producing satis
factory fermentation in deep liquid media when Y conditions, preferably in an atmosphere of car
bon dioxide. After 72 hours incubation colonies
incubated aerobically, due to the anaerobic con
are selected which are slightly raised, opalescent 75
_ ‘ ditions maintained within the medium.
2,039,529
~‘to opaque, cream colored and having substantial—
ly round entire edges. These colonies are used
as inoculant for tubes of 20% potato mash which
are incubated for 5 days at 30-32" C. The re
5 sultant spore tubes may then be used to inoculate
suitable molasses mashes, as herein described,
heat shocking the bacteria at the ?rst transfer
and then retransferring at 24 hour intervals on
the-same, molasses medium. After a number of
10 such transfers, say 5 to 7, quantitative fermenta
tions may be carried out. The cultures which
show yields of 29-36% of total solvents on the
weight of the sugar of uninverted molasses mash
of 5-5.5% sugar concentration, and a solvent ratio
15 of more than 64% normal butyl alcohol‘ (usually
68-73%), more than‘ 18%" acetone v(usually
25-32%)v and about 1-3% ethyl alcohol with no
indication of isopropyl alcohol in more than
traces will probably be the desired-cultures and
20 may be further tested to see whether they con
form with the criteria herein set forth.
' It is to be understood, of course, that the above
isolation procedures are illustrative only and may
be varied in any manner known to those skilled
“ 25 in the art. Furthermore, it is to be understood
‘ that the present invention is not limited to the
use of cultures isolated by this or any other meth
- od; but, as has been previously stated, it includes
within its scope any previously obtained bac
30 teria, from any source, which have the charac
teristics above outlined.
Although the present invention includes within
its scope all bacteria which have in common the
35 characteristics outlined above, irrespective of any
differences in minor characteristics not included
in this outline, it is preferred to use a particular
member of this group'of organisms which has
been designated Clostridium saccharo-aceto
butylicum a. This organism is described below
49 according to the Descriptive Chart of the Society
of American Bacteriologists.
’
Name of organism: C'lostridium saccharo-‘aceto
~
.
butylicum a
45 Source: Soil
I. Morphology-Continued
5. Flagella: Present
Attachment: Peritrichiate
Stain used: Casares Gil '
6. Irregular forms: Many
"l. Staining reactions
Gram vstain:
1 day: Positive
2 days: Positive; some negative
3 days: Negative, some positive
'
8. Iodine stain: Granulose shows in C1
tridia at 48 hours
II. Cultural characteristics
1. Agar stroke
'15
,
Medium used: 2% glucose agar containing
0.1% (NH4)2SO4
>
Incubation temperature: 30° C.
Age: 3 days
L
'
Growth: Moderate (incubated anaerobical
50
>
24 hours at 30° C.
Form: Short and long rods
Arrangement: Single and chains ’
,
Limits -of' length: 2.5-7.0;t; of diameter
.
55
Form: Beaded
Elevation: Raised to convex
Luster: Glistening
I
Optical character: Opaque
Chromogenesis: None to light cream or light
yellow
'
‘
>
’
Odor: Butyrous or butylic '
Consistency: Viscid
a0
Medium: Unchanged
2. Nutrient broth +1% glucose
Incubation temperature: -30° 0.
Age: 2 days
Surface growth: None
35
clouding: vYes
Odor: Slightly butyrous
-
Sediment: Very slight in 2 days, decided in
7 days
'
_3. Gelatin stab
Medium used: 2% glucose gelatin
Incubation temperature: 22° C.
Age: 30 days
Growth: Slight,‘ better at bottom
Line of puncture: Beaded
60
_
Medium used: 20% potato mash; 14 days
,
Stain used: Nigrosin
_
Location of endospores: Central to terminal
Form: ,Ellipsoidal to cylindrical -
Limits; of length: - 3.0-5.0;l; of diameter‘
,
a Size of majority: 4.0x 2.0;;
4. Motility
In broth: ++
_~75
On agar; ++
,
_
_ 1. Temperature relations
'
Fermentation temperature range: 24-40° C
2. Relationto reaction of medium
Size of majority: 6.0 x 2.2;;
1.8-2.2;1.
Edge: Entire
III. Physiology
of diameter ,
-
at 30° C.
65
Internal structure: Finely-granular
Form: Spindled, clavate
3. Endospores: Present
7'0
Medium: ‘Unchanged
4. Agar colonies
Medium used: 2% glucose agar containing
0.1% ,(NH4)2S04
Incubation temperature: 30° C.
Age: 3 days
Elevation: Raised to convex
'
Limits of length: 2.5-1.1.0p;
65
-
Surface; Smooth
'
' sugar; 48 hours at 30° C.
1.6-2.3p
_
Degree of liquefaction in 30 days: None.
. Form: Circular
Medium used: Medium I containing‘ 4%
_
25
Surface: Smooth to contoured
Growth: Slow
1.0-1.3a
Size of majority: 4.0 x 1.0,;v
Ends: Rounded
'2. 'Sporangia: Present
r
20
1?)
Liquefaction: None
I. Morphology
1, Vegetative cells
Medium'used-z Medium I.(descrlbed above);
10
4 days: Negative
Fermentation pH range: 4.0-7.0
3. Chromogenesis
'
65
>
Nutrient gelatin: None to slight crea
Nutrient agar: None to slight cream
Potato: Cream
'
' 4. Production of Indole
Medium: Broth in 1% glucose
Test used: Paradimethylaminobenzaldehyde
Presence:
Absent
1
I
5. Production of hydrogen sul?de
70
r
,
Medium: Glucose lead acetate agar
Presence: Absent
,
75
6
2,089,522
III. Physiology-Continued
6. Relation to oxygen
Medium: 2% glucoseagar containing 0.11%
ammonium sulphate
5
'
Aerobic growth: None
about 4%. Also, the inoculant should be at least
the second generation removed from the spore
state and preferably the ?fth to seventh genera
tion. Of course, in large scale operations this lat
ter may readily be accomplished‘ by the successive
.
Anaerobic growth: Moderate
'
*
Medium: Medium I (described above) deep
tubes
Aerobic growth: Abundant
10
‘
Anaerobic growth: Abundant
'
7. Litmus milk
Reaction: Acid in.1-2 days
Acid curd: Slowly formed, 7-14 days
Peptonization: Slight in' 30 days
15
Reduction of litmus:
taining 0.5% CaCOg. This medium is then rapid
.
Beginning: 1-2 days
End: Uncertain due to oxidation of litmus
by atmosphere above medium
8; Nitrate reductionv
20
Medium: Nitrate-peptone broth
Nitrite: a-naphthylamine-sulfanilic acid
Medium used: 1.0% carbohydrate
sugar, e. g., a 10% cane molasses mash, are nor
0.5% peptone
0.05% KHZPCM
mal butyl alcohol, acetone and ethyl alcohol, the 25
yields usually ranging from 28-36% of total sol
vents on the weight of the sugar. The follow
ing solvent ratios are obtained:
0.05% K24
0.03%. (NI-l4) 2804
0.02% MgSOij
Butyl alcohol“--- above 64%; usually es-isa,
.001% NaCl
.001% M11304
.001% F6504
Ethyl alcohol _____ below
.
Carbohydrate
Acid‘
Corn starch;.................... -.' ..... ..
'Rnf?nnoa
_
Sucrose ________________ ,_
+++
+++
I
45 Xylose____
Mannitol-
_
_
+++
+
Glycerol ________________________________ __
Dulcitol _________________________________ _-_
‘ - negative;
{
I
>
}
~--
}
:
l
'+
i
i
4-..
+
—
—
-
-
+ very slight; '++ moderate;
.50 ++++ abundant.
+
++
+++v
+++
+++
Example I
+++
+++‘
+++
Lactose _________________________________ __
l- 3%
lustrate the process of the present invention:
+++
+
MaltnsnGlnonen.
Levulose__--
Gas"
+++
-_‘__
Dextrin ________________ __
6%; usually
The gases given off during the fermentation con
sist of carbon dioxide and hydrogen in the ratio 35
of COz/Hz of the order of magnitude of 2 to l.
The following speci?c examples will serve to il—
Incubation temperature: 30° C.
Time: 72 hours
40 Soluble starch .................. .-..--
30'
Acetone ________ __ above 18%; usually 26-32%
pH adjusted to 6.3
35
’
The products obtained in the‘ fermentation of
commercial sugar media containing about 5%
9. Carbohydrate fermentation
30
ly cooled to 3l-32° C. and incubated for 24 hours
at that temperature. The culture is then trans 15
ferred to a ?ask containing Type I Medium of
4% sugar concentration, incubated for 24 hours
at 31-32” C. and then transferred to Type I
Medium of 5% sugar concentration. Transfers
are then continued in this last medium until the 20
desired generation is secured.
Gas: Absent
25
transfers required to build up the necessary vol
ume of inoculant. The following procedure has
been found to be eminently satisfactory for secur
ing an active inoculant. A culture is’ allowed to
sporulate for at least five days on 20% potato 10
mash and is then transferred to a ?ask of hot,
e. g., 90-100° C., 4% malt extract medium con
+++ decided;
It isknown to those skilled in the art that
many of the characteristics included in the above
Descriptive Chart are variable and that diifer
vent ratio were found to be as follows:
.
Yield per
cent
on
an or
8
30.0
ent results‘ may be obtained by only slight chang
'
'
~45.
Solvent ratio
B u W]
alcohol
Acetone
’ 69.6
~
Em y I
alcohol
21.5
5.0
2.9
Example II
55 es in the ‘media, age of culture, or fermentation
conditions. This chart, therefore, is included
40
Medium I containing 5.5% sugar was inocu
lated with 4% of a sixth generation culture of
Clostridium saccharo-acetobutylicum a and incu
bated at 30° C. for 68 hours. The yield and sol
55
Medium I "containing 5.5% sugar was inocu
herein as an aid in identification of the bacteria ' lated with 4% of a sixth generation culture of
and not as an absolute limitation. It is be
bacteria isolated from soil and incubated at 30° '1
lieved that with the aid of‘the general outline C. for 68 hours. The yield and solvent ratio were
60 previouslygiven and the above Descriptiverchart,
one skilled in ‘the art can readily identify Clos
tridium saccharo-acetobutylicum a withv cer-"
tainty in spite of slight ‘variations in the “minor
characteristics of v the chart.
In any event, a
65 check can be obtained, by testing 'the culture at
different interval-sand under slightly different
fermentation conditions noting the characteris
tics which appear most consistently.
When carrying out large scale fermentations
.70 with the organism just’described or with any of
found to be as follows:
,
'
.
Solvent ratio
Yield per
centon
su erv
' B 11 :1
y
g
' alcohol
34
12.0
‘
"
'Ethl
V
Acetone
alcohol
as?
2.3
Example III I
‘ the group Clostridium saccharo-acetobutylicum,
' I it is desirable'to take certain precautions with re
60
Medium I containing 5.5% sugar was inocu
gard to the inoculant in order to insure consistent , lated with 5% of a sixth generation culture of
high yields. The amount of inoculant/ used bacteria isolated from soil (a di?erent strain
.75 should be from 2 to 6% by volume, preferably from that of Example 11) and incubated at
65
70
9,089,022
30° C. for 68 hours. The yield ‘and solvent ratio
were found to be as follows:
, 7
nitrogen, such as amino acids, urea, and the'like,
v may be employed. ‘One skilled in the art may
'
readily determine by preliminary fermentations
the optimum concentration of the particular de
graded proteinmaterial whichit is desired to
Solvent ratio
5
Yield per
centon
B t l
S11E81‘
“mm.
71.8
‘23.8
31.7
10
r
employ. The hydrogen ion control may also be
effected by means of materials other than those
speci?cally mentioned. For example, other non
Eth l
u y
alcohol
Y
alcohol
toxic materials which are substantiallywater
4.4 -
insoluble may be used, or soluble materials may
be used if they are added in such a manner as-to
,
Example ‘IV
. Ferment'ations were carried out as in Example
I with the exception that in addition to the deter
15 mination of solvent yield and solvent ratio, the
fermentation gases were‘ collected and analyzed.
" The following results were secured:
.
simulate the effect of the non-soluble materials
in the amounts speci?ed.
In general, it may be said that equivalents and
modi?cations of procedure which would naturally
tion.
'
20
.
Solvent yield
Gas ratio
Solvent ratio
-
percent on
- Sugar
Butyl
G85
yield
‘p6
rcent
percent by volume
,
Ethyl
°
-
Alcohol Mew“ Alcohol sugar, Col‘
34.8 ____________ _.
73.6
25 34.6 ____________ __
73. 6
‘
11*
.
-
20
1. In a process for the production of normal
butyl alcohol, acetone, and ethyl alcohol by sub
jecting a'fermentable mash containing soluble
1.4
54. s
so
34
carbohydrate, as the principal fermentable car—
1. 4
53. O
65 '
35
bohydrate, to the action of a culture of bacteria 25
of the group herein described and designated as
were found to be as follows:
'
'
'
Solvent ratio
Yield per-
B 11 tyl '
alcohol
Acetone
Clostridiuin saccharo-acetobutylz‘cum, the im
provement which comprises supplying nitroge
nous nutrient to the mash in the form of degraded
protein nitrogen, and supplying non-toxic alka 30
line neutralizing agents to the mash throughout
the fermentation to control the aciditythereof
whereby the ?nal hydrogen ion concentration
secured by the action of the bacteria falls within
the range pH 5.0 to pH 6.2.
V
. 2. In a process for the production of normal
'
cent
on
sugar
we claim is:
as. o
Medium VI containing 5.3% sugar was inocu
lated with 4% of a sixth generation culture of the
30 soil bacteria utilized in Example 11 and incubated
at 30° C. for 68 hours. The yield and solvent ratio
r
-
The invention now having been described, what
25. 0
Example V,
30
15
occur to one skilled in the art may be employed
without departing from the scope of our inven
EH1 y]
butyl alcohol, acetone, and ethyl alcohol by sub
alcohol
, jecting a fermentable mash containing soluble
34. a
4o
73. 4 ‘
2L 2
carbohydrate, as the principal fermentable carbo
2. 4
hydrate, to the action of a culture of bacteria of 40
the group herein described and designated as
_
Example VI '
Clostridium saccharo-acetobutylicum, at temper
A medium containing 7% hydrol (4.8% total
carbohydrate, 90% distillery slop (from fermen
45 tation of» 7.5% corn mash‘by Cloctridium aceto
butylicum (Weizmann) neutralized with soda to
pH 6.0), 0.2% (NHQzSQ; and 0.4% CaCOa was
inoculated with 4% of a third generation culture
atures from 24° C. to 40° C., the improvement
of Clostridium saccharo-acetobutylicum a and
50 incubated at 30° C. for 68 hours. , ‘The yield/and
solvent ratio were found to be as'follows: I,
the ?nal hydrogen ion concentration secured by’ the action of the bacteria falls within the range 50
pH 5.0 to pH 6.2.
‘
. 3. In a process for the production of normal
yield’ per
.
55
08%t an
“8' ° Y‘
dim I
Butyl _
4 -
-
~
tralizing agents to the. mash throughout the fer
mentation to control the acidity thereof whereby
butyl alcohol, acetone, and ethyl alcohol by sub
Solvent ratio
I
which comprises supplying nitrogenous nutrient
to the mash in the-form of degraded protein 45
nitrogen, and supplying non-toxic alkaline neu
jecting a fermentable mash containing soluble
carbohydrate, as the principal fermentable car
Ethyl
alcohol
Mew“
alcohol
65. 7
29. 9
4. 4
' bohydrate, to the action of a culture of bacteria
of the group herein described and designated as
33. 9
Clostridium sacchara-ac'etobutylicum, at tem
peratures from 24° C. to 40° C., the improvement
60 ‘It is to be understood, of course, that the ex
amples given above by way of illustration are not
to be taken as limiting our invention to the spe
ci?c/materials or methods employed.
For ex
ample, other sources of soluble carbohydrate may
65 be utilized, as for example wood sugar, beet
molasses, whey, and the like. However, it is pre
ferred to use molasses as the source of carbo
hydrate since this vmaterial is available in the
_ larg'est'quantities, is low priced, and is fairly
. 70 standard in composition. If raw-materials other
which comprises supplying nitrogenous nutrient
60
to the mash in the form of ‘degraded protein
nitrogen, and supplying substantially water in-'
soluble non-toxic alkaline neutralizing agents to
the mash throughout the fermentation to control
the acidity thereof whereby the ?nal hydrogen 65
- ion concentration secured ‘by the action/of the
bacteria falls within the range pH 5.0 to pH 6.2.
than. molasses are utilized, one skilled iiithe art
4. In a process for the production of normal
butyl alcohol, acetone, and ethyl alcohol'by sub- \
jecting a fermentable mash containing soluble 70
carbohydrate, as the principal fermentable car
'may readily make such adjustments in [the com
bohydrate, to the action of a culture of bacteria '
position of the media as are necessary to approxi
- mate the composition illustrated for ' molasses.
v75 Also, various other sources of degraded protein
of the group herein described and designated as
Clostridium saccharo-acetobutylicum, at temper
atures from 24° C. to 40° 0., the improvement
75.
8
2,089,522
' which comprises'supplying nitrogenous nutrient
to the mash in the form of degraded protein ni
trogen, and supplying calcium carbonate to the
mash throughout the fermentation to control the.
.5 acidity thereof whereby the ?nal hydrogen ion
concentration secured by the action of the bac
teria falls within the range pH 5.0 to pH 6.2.
‘
5. In a process for the production of normal
butyl alcohol, acetone, and ethyl alcohol by sub
lO jecting a fermentable mash containing soluble
carbohydrate, as the principal fermentable car
bohydrate, to the action of a culture of bacteria
of vthe group herein described and designated as
Clostridium saccharo-acetobutylicum, at temper
15 atures from 24° C. to 40° C., the improvement
which comprises supplying nitrogenous nutrient
to the mash in the form of degraded protein ni
-trogen, and supplying non-toxic alkaline neu
tralizing agents to the mash throughout the fer
20 mentation to control the acidity thereof whereby
the ?nal hydrogen ion concentration secured by
the action of the bacteria falls within the range
pH 5.5 to pH 5.85.
-
6. In a process for the production of normal
40
Clostrt'dium saccharo-acetobutylicum, at temper
atures from 24° C. to 40° C., the improvement
which comprises supplying nitrogenous nutrient
to the mash in the form of degraded protein ni
trogen, and supplying a neutralizing agent to the
mash throughout the fermentation to control the
acidity thereof, the said neutralizing agent being‘ 10
introduced into the mash in the form of an initial
addition of calcium carbonate in a concentration
of approximately 5% based'on the weight of the
sugar in the mash in excess of that required to ,
15
neutralize the initial acidity of the mash.
10. In a process for theproduction of normal
butyl alcohoL-acetone, and ethyl alcohol by sub
jecting a fermentable mash to the action of a cul
ture of bacteria of the group herein described and
designated as Clostrz‘dium saccharo-acetobutyl 20
team, at temperatures from 24° C.‘to 40° C., the
improvement which comprises supplying the prin
cipal fermentable carbohydrate to the mash in the
form of molasses, supplying nitrogenous nutrient
to the mash in the form of an ammonium com- 25
jecting a fermentable mash containing soluble
pound and supplying non-toxic alkaline neutral
izing agents to the mash throughout the fermenta
tion to control the acidity thereof whereby the
?nal hydrogen ion concentration secured by the
action of the bacteria falls within the range pH 30
5.0 to pH 6.2.
Clostridium saccharmacetobutylicum, at temper
atures from 24° C. to 40° C., the improvement
which comprises supplying nitrogenous nutrient
to the mash in the form of degraded protein ni
trogen, and supplying substantially water insol
subjecting a fermentable mash to the action of
ublelnon-toxic alkaline neutralizing agents to the
mash throughout the fermentation to control the
scribed and designated as C'lostrz'dz'umvsaccharo
acidity thereof whereby the ?nal hydrogen ion
concentration secured by the action of ‘the bac
acetobutylz'cum, at temperatures from 24° C. to
_40° C., the improvement which comprises sup- "
teria falls within the range pH 5.5 to pH 5.85.
7. In a process for the production of normal
plying the principal fermentable carbohydrate to
the mash in the form‘ of molasses, supplying ni 40
butyl alcohol, acetone, and ethyl alcohol by sub
jecting a fermentable mash containing soluble
carbohydrate, as the principal fermentablecar
bohydrate, to the action of a culture of bacteria
45 of the group herein described and designated as
11. In a process for the production of normal
butyl alcohol, acetone, and ethyl alcohol by
a culture of bacteria of the group herein de
trogenous nutrient to the mash in the form of an.
ammonium compound and vsupplying substan
tially water insoluble non-toxic alkaline neu- _
tr'alizing agents to the mash throughout the fer- .
mentationto control the acidity thereof whereby 45
Clostridium saccharo-acetobutylicum, at temper
the ?nal hydrogen ion concentration secured by ' ‘
atures from 24° C. to 40° C.,-the improvement
the action of the bacteria falls within the range
which comprises supplying nitrogenous'nutrient
pH 5.0 to pH 6.2.=
to the mash in the form of degraded protein ni
50 trogen, and supplying calcium carbonate 'to the
mash throughout the fermentation whereby the
?nal hydrogen ion concentration secured by the
action of the bacteria falls within the range pH
5.5 to pH 5.85.
55
of the group herein described and designated as
butyl alcohol, acetone, and ethyl alcohol by sub
carbohydrate, as the principal fermentable car
bohydrate, to the action of a culture of bacteria
of the group herein described and designated as
30
carbohydrate, as ‘the principal fermentable car
bohydrate, to the action of a culture of bacteria '
-
' '
8._In a process for the production‘ of normal
butyl alcohol, acetone, and ethyl alcohol by sub
jecting a fermentable mash containing soluble
.
- »
12. In a process for the production of normal
butyl alcohol, acetone, and ethyl alcohol by sub
jecting a fermentable mash to the action of a
culture of bacteria of the group herein described
and designated as Clostrz'dium saccharo-aceto
- butylicum, at temperatures from 24° C. to 40° C.,
the improvement which comprises supplying the
principal fermentable carbohydrate to the mash
in the form of molasses, supplying nitrogenous
carbohydrate, as the principal fermentable car ' nutrient to'the mash in the form of an ammo
bohydrate, to the action of a ‘culture of bacteria nium compound and supplying calcium carbon
v60 of the group herein described and designated as ate to the mash throughout the fermentation to 60'
Clostrz'dium saccharo-acetobutylimm, at temper-v control the acidity thereof whereby the ?nal
atures from 24° C. to 40° C., the improvement
which comprises supplying nitrogenous nutrient
to the mash in the form of degraded protein m
65 trogen, and supplying a neutralizing agent to the
hydrogen ion concentration secured by the action
of the bacteria falls within the range ‘pH 5.0 to
pH 6.2,
.
'
,
13. In a process for theproduction of normal 65
~ mash throughout the fermentation to control‘ the . . butyl alcohol, acetone, and‘ethyl alcohol by sub
acidity thereof, the said neutralizing agent being jecting a fermentable mash to the action ofja
- introduced into the mash in the form of an initial culture of bacteria of the group herein described
addition of calcium carbonate in a concentration and designated as C'lostridium saccharo-aceto
70 of 3% to 10% based on the weight of the sugar butylicum; at temperatures from 24° C. to 40° .C., 70
‘in the mash in excess of that required to neu- .
'_ tralize the initial acidity of ' the mash.‘
9. In a process for the production of normal
butyl alcohol, acetone, and ethyl alcohol by- sub.
75 jecting a fermentable mash containing soluble
the improvement which comprises supplying the
principal fermentable ‘carbohydrate to the mash
in the form of molasses, supplying nitrogenous
nutrient to the mash in the form ofan ammo
nium compound and supplying substantially W9. 75
9 .
2,089,622
ter insoluble non-toxic alkaline neutralizing
agents to the mash throughout the fermenta
tion to control the acidity thereof whereby the
nium compound and ‘supplying an alkaline neu
tralizing agent to the mash throughout the ter
mentation to control the acidity thereof, the said
?nal hydrogen ion concentrationisecured by, the ' neutralizing agent being introduced in the form
5 action ‘of the bacteria falls within the range of an initial addition of calcium. carbonate in
pH 5.5 to pH 5.85.
-
14. In a process for the production of normal
vbutyl alcohol, acetone, and ethyl alcohol by sub
jecting a fermentable mash to the action oil a
10 culture of bacteria of the group herein described
and designated as Clostridium. saccharo-aceto
butylicum,‘ at temperatures from 24° C. to 40° 0.,
theimprovement which comprises supplying v‘the
principal fermentable carbohydrate to the mash
, a concentration 0! 3% to 10% based on the v
weight of the sugar in the mash ‘in excess oi that
required to neutralize the initial acidity of the
mash.
'
16. In a process for the production of ‘normal 10
butyl alcohol,‘ acetone, and ethyl alcohol by sub
jecting a fermentable mash to the action of a
culture of bacteria of the group herein described
and designated as Olostridium saccharo-acetm
butylicum, at temperatures-from 24° C. to 40° C.,
the improvement which comprises supplying the
nutrient to the mash in the form of an ammo
‘principal
‘Iermentable' carbohydrate to the mash
nium compound and supplying calcium carbon
ate to the mash throughout the fermentation to _ in the form of molasses, supplying nitrogenous
control the acidity thereof whereby the ?nal hy ‘nutrient to the mash in the form of an ammo
, 20 drogen ion concentration secured by the action nium compound and supplying an alkaline neu
of the "bacteria falls within the range pH 5.5 tralizing agent to the mash throughout the ter
mentation to control the acidity thereof, the said
to pH 5.85.
'
15. In a process for the production of normal neutralizing agent being introduced in the form
butyl' alcohol, acetone, and‘ ethyl alcohol by sub-' of an initial addition of calcium carbonate in
I 15 in the form of molasses, supplying nitrogenous
25 jecting a fermentable mash to-the action of a
. culture of bacteria of the group herein described
a concentration of approximately 5% based on
the weight of the sugar in the mesh in excess
of that required to' neutralize the initial acidity '
ands-designated as clostridium sacchcro-aceto
butylicum, at temperatures from 24° C. to 40° C., o! the mash.
the improvement which comprises supplying the
3‘! principal fermentable carbohydrate to the mash
in the i'orm of molasses, supplying nitrogenous
nutrient to the mash in the tom of an ammo
20
JOHN C. WOODRUFF.
HUGH R. STILES.
DAVID A. LEGG.
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