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2,132,039
, Patented Oct. 4, 1938
UNITED STATES PATENT OFFICE
2,132,039
PRODUCTION OF NEUTRAL SOLVENTS BY
'
FERMENTATION
John Miiller, Philadelphia, Pa., assignor to Com
mercial Solvents Corporation, New York, N. ‘iii,
a corporation of Maryland
No Drawing. Application December 9, 1936,
Serial No. 115,003
24 Claims.
The object of my invention is to provide an im
proved process for increasing the yield of solvents
in the butyl alcohol fermentation of such sub
stances as sugar, molasses, or other carbohydrate
5 raw material which may be hydrolyzed to yield
sugar. More speci?cally, my invention deals with
improved operating conditions of wide applica
tion whereby consistently higher yields of so]
I have found that a very satisfactory method
of supplying the alkaline neutralizing agent con
sists in introducing into the original mash a non
toxic insoluble alkaline neutralizing agent such
as calcium carbonate in a concentration slightly
in excess of that required to neutralize any ini
tial acidity. The presence of this insoluble mate
high yields.
rialvthus controls the hydrogen ion concentration
secured by the action of the bacteria by supplying
alkaline neutralizing material throughout the fer 10
mentation.
The organisms to which my process applies
may be classed as essentially sugar fermenting
to the results obtainable with starch fermenting
vents are obtained by means of organisms hereto
10 fore regarded as incapable of producing such
15
(Cl. 195—44)
rather than starch fermenting, since ordinarily
they produce little, if any, fermentation in
starch-containing mashes, such as for example,
the corn meal mash employed in the industrialv
butyl-acetonic fermentation process. The essen
tially starch fermenting bacteria of the Clos
tridium acetobutylicum Weizmann type, which
have been utilized in the past for the commercial
production of butyl alcohol by fermentation are
not in general bene?ted by the use of my process.
Normally, ?nal fermentation products of the
essentially sugar fermenting organisms ,consist of
high proportions of butyric and acetic acids
rather than neutral solvents. However, by effect
ing the fermentations in accordance with my im
proved procedure embodying the novel operating
conditions‘throughout the fermentations herein
after to be described, I am able to produce ?nal
fermentation products consisting primarily of
neutral solvents and only minor amounts, if any,
of butyric and acetic acids.
35
It will be understood that any organism that is
normally essentially a sugar-fermenter rather
than a starch fermenter and which normally
tends to produce acidic end products is within the
scope of my invention. More speci?cally, the or
40 ganisms which are included within the scope of
my invention are those which are capable of pro
ducing consistently higher yields of solvents from
nutrient glucose mashes than from mashes con
sisting solely of grain meal and water and which
45 produce increasing amounts of acidic end prod
ucts throughout the fermentation in the absence
of hydrogen ion control.
My improved process comprises essentially ef
fecting the fermentation of the sugar-containing
50 maslizin the presence of phosphate and ammonia
nutrients and supplying the mash throughout the
fermentation with an alkaline neutralizing agent
to control the hydrogen ion concentration. If
crude raw materials such as molasses are used as
55 the source of carbohydrate for the mash, there
will usually be sufficient phosphorus and a part of
the necessary nitrogenous nutrient present in the
original mash. In such cases it will usually be suf
?cient to incorporate ammonia or an ammonium
60 compound as a supplementary nitrogen source.
. It can readily be demonstrated that, contrary
bacteria of the type Clostrz‘dium acetobutylicum
Weizmann, the sugar fermenting butyl alcohol 15
producing bacteria with which my process is
concerned cannot maintain the optimum hydro
gen ion conditions for solvent production without
the aid of alkaline neutralizing agents. In a
mash in which no such materials are supplied,
the acidity becomes unduly high and the yield
of solvents is markedly decreased. For satisfac
tory solvent production with bacteria of this type
the hydrogen ion concentration should be regu
lated throughout the fermentation such that the
?nal hydrogen ion concentration secured by the
action of the bacteria falls within the range
pH 5.0-6.5. It will be obvious that any given cul
ture of bacteria of this type will have an optimum
?nal pH range which can readily be determined 30
by preliminary experiments. In general it may
be said that satisfactory yields may be obtained
if the acidity is regulated throughout the fermen
tation such that the ?nal hydrogen ion concen
tration secured by the action of the bacteria falls 35
Within the range pH 5.0-6.5, but that the ulti- '
mate possibleyields can only be secured within
a narrower ?nal pH range for any given culture
of bacteria.
‘
'
As has been previously pointed out, the ?nal 40
hydrogen ion concentration secured by the action
of the bacteria can be suitably regulated by in
corporating in the initial mash a non-toxic in
soluble alkaline neutralizing agent in a concen
tration slightly in excess of that required to neu 45
tralize any initial acidity. For example, it has
been found that'if calcium carbonate, or other
equivalent material such as for example, barium
carbonate, iron carbonate, or other Water in
soluble non-toxic base, is added to the mash, in
an amount su?icient to neutralize any free acid
ity, and an amount in excess of this to the ex
tent of about 6-8% calculated on the weight of
the sugar, the ?nal pH of the fermentation will
be found to fall within the operative range. Al 55
though the various materials mentioned may be
satisfactorily used in this process, calcium car
bonate has been found, in most cases, to be espe
cially well suited for this purpose, and is to be
preferred from an economic standpoint. How 60
2,132,039
2
ever, in choosing the material to be employed, the
composition of the medium should be ‘considered,
and a material chosen which will not give rise to
an undesirable concentration of a particular
metal ion, even though generally considered to be
non-toxic in character.
The amount of calcium carbonate or other
‘nontoxic insoluble base to be added in excess of
that necessary to neutralize the free acidity of
10 the mash will be found to vary somewhat in indi
vidual cases,‘ but in general it may be said to be
from 3.5% to 13% ‘of the weight of the sugar in
the mash. Various samples of these materials
will differ in respect to the amount necessary, due
15 to the physical properties of the material and
also to its chemical properties, as for example,
the presence of substantial ‘amounts of lime.
However, in any case, a preliminary fermenta
tion will enable one skilled in the art to deter
mine the optimum concentration for the calcium
carbonate employed. It should be de?nitely
understood that the purpose of the addition of
basic materials in this process is not to neutralize
all of the acids produced in the fermentation,
but merely to control the hydrogen ion concen
tration in such a manner that the ?nal pH se
cured by the action of the bacteria (and not by
the action of neutralizing agents) falls within
the speci?ed limits.
It is to be understood that my invention is
30
not to be limited to the particular means em
tial for optimum results. As has previously been
pointed out the phosphorus requirements will
usually be satis?ed by the phosphatecontent of
natural raw materials utilized as sources as
carbohydrate for the mash. In the event that
phosphates are not supplied in this manner a
small amount of ammonium, sodium, calcium, or
other‘ phosphate, may be incorporated in the
mash, for'example, in a concentration of about
2.5% based on the sugar content of the mash. v10
The ammonia nitrogen may be supplied in the
form of free ammonia or an ammonium salt such
as ammonium sulphate, ammonium phosphate,
or the like. It will be evident that ammonia is
a very highly degraded form of nitrogenous nu
trient and that equivalents of this material in the
form of other degraded nitrogenous materials
may also be employed. The amount of ammonia,
or its equivalent, to be added to any given mash
will, of. course, vary to some extent with the ‘raw 20
materials used. For example, certain samples
of molasses may be found to have sufllcient am
monium compounds, or other degraded protein,
so that very little more, or perhaps none, need
be added.
In general, it may be said that with
cane molasses mashes from 0.5 to 1.0% of am
monia, or its equivalent, calculated on the weight
of the sugar will give satisfactory results.
The following represents a typical mash pre
pared in accordance with the present invention: 30
To 1000 gallons of mash, having a concentration
ployed for securing the desired ?nal hydrogen
ion concentration. Any equivalents or modi?ca
of from 4 to 6% of sugar such as glucose, are
added about 8 pounds of ammonium sulphate, ~
tions which would naturally occur to one skilled
in the art may, of course, be employed. For ex
ample, an accurate pH control may be main—
tained by continuous or semi-continuous addition
of a soluble alkaline material, such as ammonia,
?nely-divided calcium carbonate. The presence
of this excess of. calcium carbonate should givev
during the active stage of the fermentation and
40 until after the “acidity break.” However, the
mechanical difficulties of 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
45 inhibiting further active fermentation for a pe
riod of many hours or even days. Consequently,
automatic
electrometric
titration
apparatus
would be most desirable if such a procedure were
to be employed. In any procedure of this nature,
50 the pH should be controlled to approximate that
obtained when the speci?ed amounts of insoluble
basic materials are employed. In view of the
difficulties of such procedures, I prefer to ‘secure
the desired pH control by introducing materials
55 of the insoluble type into the mash before fer
mentation begins.
Furthermore, from the standpoint of simplicity
of operation, I prefer to control the acidity of the
mash during the fermentation by means of the
60 insoluble materials such as calcium carbonate.
It has been found that for a wide range of
grades of molasses, approximately 6-8% of cal
cium carbonate, or the like, calculated on the
weight of sugar in the mash, secures adequate
65 control of the acidity such that the final hy
drogen ion concentration secured by the action
of the bacteria falls within the desired limits.
This fact may be seen to obviate the necessity
for individual treatment of each sample of mo
70 lasses unless the ultimate possible yield is desired.
With regard to the special nutrients supplied
to the mash in my improved process it may be
said that both phosphorus and ammonia nitro
gen, or its equivalent, in the form of relatively
75 highly degraded nitrogenous materials are essen-g
about 10 pounds of ammonium, sodium, calcium,
or other phosphate, and about 20 pounds of 35
rise to an initial hydrogen ion concentration of
the order of pH 6.2 to 5.0.
_
This mash is particularly adapted for fermen 40
tation with bacteria of the type herein designated
as Clostridium propyl butyZicum-alpha and the
present invention may be suitably illustrated by
the use of this organism.
When inoculating a
mash of the above type with Clostridium prom/l
ImtyZicum-alpha and allowing fermentation to
take place, solvents yields of 25% to 30%, or even
higher, based on the weight of the sugar in the
mash can be consistently obtained.
,
The Clostridz'um prom/Z butylium-alpha may be
more accurately described by the following char
acteristics:
-
I. Morphological:
A. Rod-shaped
v
.
B. Spore-forming clostridia and plectridia
C. Practically indistinguishable morpho
logically from members of the Clos
tridium butyricum group
'
H. Cultural:
A. Nutrient agar stroke; no growth aerobi
cally or anaerobically
B. ‘Glucose nutrient agar slants; no growth
aerobically but considerable growth
anaerobically moist, raised, usually
white to cream colored; higher alco
hol odor changing to butyrous after
.
exposure to air
I
-
C. Colonies on glucose‘ agar; substantially
round, raised, usually white to cream
colored; higher alcohol odor changing
to butyrous after exposure to air
D. Potato slant; moderate growth, usually
white to cream colored; butyrous odor
3
-2,132,os9
" III. Biochemical:
A. Carbohydrate fermentation:
'
1. Inability to produce appreciable
yields of solvents from starch 'as
the only source of carbohydrate
2. Inability to produce appreciable
yields of solvents from sucrose as
the only source of carbohydrate
3. Inability to consistently produce
10
15
Clostridium prop'yl butylz'cum-alpha is a mem
ber of the group of bacteria which has been desig
nated Clostridium propyl b'utylz'cum and which
comprises a relatively large group of organisms
readily identi?ed by means of the primary char
acteristics listed below. So much confusion exists »
in the nomenclature and reported cultural char
acteristics of the prior art organisms, particularly
of the Clostridium butyricum type, that itis im
possible to state de?nitely if any of them are in
yields of solvents greater than
20% calculated on the weight of
cluded in the group now designated as Clostridium
sugar from uninverted molasses
prom/Z butylicum. It is to be understood, there
4. Ability to produce high yields of
fore, that this group of organisms includes within
solvents from glucose or inverted
molasses
its scope any such prior art bacteria as well as any
newly isolated cultures which have, in fact, the 15
5. Ability to ferment carbohydrates following primary characteristics:
1. Morphological:
as evidenced by production of
'
A. Rod-shaped
acid and/or'gas:
Corn starch _________ __
,
'
B. Spore-forming-Clostridia and Plectridia
C. Practically indistinguishable from mem
1
Soluble starch _______ __
-
Dextrin_ ____________ __
-
Raf?nose_____.. ______ _.
-
Sucrose__- ___________ __
3xx
Maltose_______l_._______ 4xxx
30
Glucose ____ __- _______ __
5xxxx
'Laevulose ____________ _.
xxxx
Xylose ______________ __
xx
Dulcltol _____________ __
—
Mannitol ____________ __
-
Glycerol _____________ _. Lactose ______________ _.
bers of the Clostridium‘ butyricum
group
H. Biochemical:
A. Carbohydrate fermentation:
l. Inability to produce appreciable 25
yields of butyl and isopropyl al
cohols from starch as the only
source of carbohydrate
2. Inability to produce appreciable
yields of butyl and isopropyl al
2x
cohols from sucrose as the only
xxx
source of carbohydrate
2 x very slight.
3 xx moderate.
‘ xxx decided.
5 xxxx abundant.
the sugar from uninverted m0
lasses
B. Nitrogen metabolism:
\1. ‘Ability to produce high yields of
4.' Ability to produce high yields of
butyl and isopropyl alcohols
solvents in sugar media contain
ing ammonia as the principal
source of nitrogen
2. Ability to utilize degraded protein ‘
(including ammonia) as sole ni
trogen source
4
from glucose or inverted mo
B. Nitrogen metabolism: '
1. Ability to produce high yields of
butyl and isopropyl alcohols in
'
sugar media containing ammo
nia as the principal source of ni
tein as sole source of nitrogen
4. Inability to liquefy gelatin
,
slight proteolysis of milk
(including ammonia)v as sole ni
trogen source
C. , Oxygen requirements:
1. Anaerobic
D. Temperature range for solvent produc
'
E. Hydrogen ion concentration for solvent .
production:
1. Final pH of‘5.0-6.5, preferably 5.8
6.1.
The most satisfactory yields, when employing
. Clostridium propyl butylicum-alpha, are obtained
from the monohexoses. The disaccharides such
as sucrose or sucrose-containingmaterials black
65 strap molasses are attacked relatively slowly and
hence require inversion by the action of invertase,
acid, or other means before they are suitable for
fermentation on an industrial-scale. From the
monohexoses or invert sugars, I am able to obtain
70 with C'lostridium propyl butyZicum-alpha by my
new process yields‘ of 25%-30% of total solvents,
the composition of which being of the order of
65%-70% n-butyl alcohol, 25%-“15% isopropyl al
cohol, and the remainder acetone together with a
76 small amount of ethyl alcohol.
50
3. Inability to utilize undegraded pro
tein as sole source of nitrogen
.4..Inability to liquefy gelatin or to
tion:
1. From 25'’ C. to 36° 0., preferably
28° C. to 32° C.
45
trogen
2. Ability to utilize degraded protein
5. Inability to- produce more ‘than
50,
40
lasses
3. Inability to utilize undegraded pro
45
'
3. Inability to consistently produce
yields of solvents greater than
35
20% calculated on the weight of
1 — negative.
40
2,0
'
,
produce more than very slight
proteolysis of milk
55
C. Oxygen requirements:
1. Anaerobic
D. Temperature range for solvent produc
tion:
1. From 25° C. to 36° 0., preferably 60
29° C. to 31° C. '_
E. Hydrogen ion concentration for solvent
production:
1. Final
pH of 5.0-6.5, preferably
65
5.8-6.1.
The above outline is believed to be su?icient to
enable one skilled in the art to identify the or
g'anisms in question.
A complete characteriza
tion such as that of the descriptive chart of the 70
Society of American Bacteriologists' would not
only be unnecessary, but would be confusing since
different members of this group of organisms
would Vary in a number of minor particulars
having no bearing upon the present case. All 75
4
2,132,039
organisms having in common the above charac
materials as corn gluten and corn germ meal,
teristics come within this group of bacteria ir
the degraded protein referred to comprises such
materials as yeast ‘water, steep water, and urea,
and the gelatin liquefaction refers to tests such
respective of further properties which they may
possess.
as incubation on nutrient gelatin containing 2%
glucose. For example, stab cultures on such me
dium were incubated at 22° C. and shake cul
tures were incubated at 30° C. Excellent growth
was obtained‘ in each case but at the end of 30
days the gelatin was in all cases found to be
solid 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 litmus and then give a somewhat rennet-like
In view of the uncertainty in the literature as
to methods utilized for certain of the biochemical
tests referred to above, it is desirable to amplify,
somewhat, the characteristics brie?y outlined.
For example, the fermentation characteristics
referred to under the heading “Carbohydrate fer
mentation” are those characteristics determined
under optimum conditions, as for example, in the
inverted molasses medium‘ described above or in
similar media containing other carbohydrates.
Quite different results may be obtained with lab
oratory media containing lower percentages of
sugar. It should also be noted that fermenta
tion characteristics such as these refer to normal
consistent results and not to abnormally low or
20 high results which may sometimes be obtained
with any culture. A typical carbohydrate fer
mentation test of an organism falling in this
acid curd which shows only slight digestion at the 15
end of 30 days. The following example illustrates
the type of test which may be made to determine
the nitrogen requirements of the organisms:
.
0'
(percent
calculated
on sugar)
INVERTED MOLASSES Memoir (MEDIUM- 1)
IX ______ __ 5.0% invert sugar, 0.15% KaHPO4,0.1%
25.0
X _______ __ 5.0% glucose, 0.6% CaCO; in 10% yeast
34
‘(NHOgSOh 0.05% MgSO4, 0.5% 0800:.
Cuban molasses at about 20% sugar concentra
tion is inverted by heating with sulphuric acid
equivalent to 5% on the weight of the sugar for
40 minutes at 20 lbs. pressure. At the conclusion
of the inversion, about 0.7% of ammonia on the
730 weight of the sugar is added and subsequently
sufficient ?nely-divided calcium carbonate is in
troduced to neutralize the remaining free acidity.
An excess of calcium carbonate amounting to
35 about 6% on the weight of the sugar is then
water.
25
'
XI ______ __ 5.0% invert sugar, 0.15% KzHPOi, 0.05%
2.0
30
MgSO;, 0.3% corn germ, 0.3% corn
gluten, 0l5% 09.00:.
'
It is to be noted that the utilization of am
monia is speci?ed as the principal source of nitro 35
gen rather than the sole source for optimum
solvent production. These organisms can uti
lize ammonia as the sole source of nitrogen, in
some cases with optimum solvent yield, but for
consistent high yields of solvents it is preferred
to have a small amount of some other degraded
protein material present in addition to the am
monia. This additional amount, however, will
usually be present in such material as molasses
so that the use of ammonia alone will serve to 45
introduced and the mash is diluted to a sugar
concentration of about 5% and sterilized for 30
vminutes at 20 lbs. pressure. It might be desir
able to reduce the steam pressure and increase
40 the time of inversion and sterilization so as to
avoid caramelization of the sugar due to local
overheating. In this case 2 hours at 5 lbs. pres
sure is roughly equivalent to 40 minutes at 20 lbs.
A typical yield in this‘ type of medium is 30%-32%
of solvents calculated on the weight of the sugar
in the mash.
20
Solveaat
yie
M‘igmm ., Composition percent by weight of mash
group is given below as an illustration.
25
v10
produce optimum yields.
'
The term “anaerobic” as used in the above
outline, refers to the inability of the organisms
solvleéit '
-
50
yie
M‘iéiéum
Composition percent by weight of mash
‘
percent
'
of carbo
hydrate
v
to grow on the surface of nutrient glucose agar
when incubated aerobically. The organisms, are,
however, capable of developing and producing
satisfactory fermentation in deep liquid medium
when incubated aerobically due to the anaerobic
conditions which they maintain within the me
dium.
111 ______ ._ 20% potato mash, 0.1% (Nrrmsoi ........ .. Trace.
.The temperature and hydrogen ion concentra
tion ranges referred to do not represent the en
IV ______ __ 25% potato mesh, 20% yeast water, 0.6%
2.6
tire ranges within which growth will occur but
represent merely the ranges within which high
yields of solvents may be obtained when oper
00 V _______ __ 5.0% glucose, 0.2% (NHmSoi, 0.3% (NHm26.0
HPO4, 0.05% NHlCl. 0.05% MgSO4. 0.0%
ating under the other conditions speci?ed. Also,
C8003, initial pH adjusted to 6.4.
the solvent ratios which are given as characteris
tic of the organism are those which are normally
VI ______ __ 5%sucrose. 0.1% (NH4)2SO4,0.15% KzHPOr,
3.2
consistently obtained under optimum conditions
0.05% MgSO4, 0.5% QaCOa, initial pH
adjusted to 6.1.
and do not refer to abnormal ratios which may
sometimes be secured with any of the cultures.
VII _____ -. 5.0% so ar as inverted Cuban molasses,
30.7
Furthermore, it is to be understood that the char
0.04%
H; as ammonium sulfate, 0.5%
.
acteristics speci?ed for these organisms are not
CaCOa.
to be taken as limited to the speci?c methods
and data given above. These were given merely 70
VIII ____ -_ 5.5% sugar as uninvertcd molasses, 0.04%
9.0
NH; as ammonium sulfate. 0.5% CaC‘Os.
by way of illustration, whereas the characteris
tics of the organisms as claimed in the present
With regard to the nitrogen metabolism, the invention are those given generally in the out
undegraded
protein materials referred to are such line. '
75
II _______ _.
5% corn mash _____________________________ ..
08003.
.
~
Trace.
5
2,132,039
The organisms of this group are'widely distrib
of these bacteria may be stored in the usual
manner in the form of spore cultures, but unless
uted in nature and may be isolated from such.
various sources as soil, rotted wood, grain, corn
the spore cultures are stored on dried sterile soil
or some highly bu?ered medium, they should be
stalks, river mud, and the like. In‘ view of the
characteristics listed above, one skilled in the art
transferred every 10 days to Medium I contain
ing 3-5% sugar and allowed to germinate.
may isolate these organisms from such sources
‘by known methods of isolation. Of course, as is
apparent to one skilled in the art, these organ
isms cannot be isolated from every sample of ma;
terial tested. However, if a number of different
materials are tried, a good culture will nearly
always be secured. The following speci?c ex
. 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 in the art. Furthermore, it is to 10
be understood that the present invention is not
limited to the use of cultures isolated by this
or any other method; but, as has been previously
ample is given as illustrative of one of the meth
15
stated, it includes within its scope any previously
ods applicable to this purpose:
A large number of ?asks, say twenty each 0
the following media are prepared:
‘
obtained bacteria from any source which have 15
-
the characteristics herein outlined.
When utilizing bacteria of this group for large
M‘igglm
Composition, percent by weight of mash
20
‘I _________ .. - (As described above).
XII ______ __ 3.0% glucose, 0.1% (NHl); S04, 0.15% (NHOZHPO‘,
0.05% N1I4Cl, 0.05% MgSO4, 0.3% CaCOa, pH ad
25
justed to 6.0.
These media are sterilized in the usual man
ner and while still hot, e. g., 80—85° C., are inocu
lated with samples of soil, mud, corn, corn stalks,
30 rotted Wood, and the like. The flasks are held at
the inoculating temperature for a short time,
c. g., 1 to 3 minutes, and are then rapidly cooled
to 32° C. and incubated at this temperature. The
cultures evidencing the strongest fermentation at
the end of 48 hours are chosen for further in
vestigation and are allowed to sporulate for at
least 5 days at 32° C. The cultures are then
transferred to ?asks of Medium I while the lat
ter is still hot, e. g., 95-400° C. ‘After not more
than one minute at this temperature, they are
cooled rapidly to 32° C. and incubated at this
temperature. This procedure may then bere
peated a number of times to further enrich the y
cultures, but as a rule, at the end of the third
45 transfer a number of cultures will show su?'lcient
activity to warrant quantitative determination of
scale fermentations, it is necessary to take cer
tain precautions with regard to the inoculant
in order to insure consistent high yields. The 20
amount of inoculant used should be from 2-5%>
by volume, preferably 3-4%. Also the inoculant
'should be at least the second generation removed
from the spore state and preferably the fourth
to sixth generation. Of course in large scale n
- operations this latter may readily be accom
plished by the successive transfers required to
build up the necessary volume of inoculant. The,
transfers may be made at 24 hours on medium
of the type of Medium I containing 3-5% of 30
sugar.
.
The products obtained in the fermentation of
commercial sugar media containing about 5%
sugar, e. g., a 10% inverted molasses mash, are
normal butyl alcohol, isopropyl ‘alcohol, ethyl 35
alcohol, and acetone, the yields usually ranging
from 28-32% of total solvents calculated on the
Weight of the. sugar. The following solvent ratios
are obtained:
40
Butyl alcohol—above 60%; usually 65-70%
Isopropyl alcohol--above 15%; usually 16—20%
Ethyl alcohol—below 10%; usually 3-4%
Acetone—below 15%; usually 5-10%
The gases given off during, the fermentation con 45
sist of carbon dioxide and hydrogen in a ratio of
the solvents. These cultures which show fair CO2/H2 of the order of magnitude of 3/1.
yields, for example yields of 15% or over, on the
The following are speci?c examples of fermen
weight of the sugar, mayjithen be tested on Me-- ' ' tations employing organisms of the group. Clos
dium
I
and
Medium
26111
,‘(which
is
the
same
me
trz'dz'um prom/l butylicum:
I
50
dium as Medium 1, but containing uninverted
molasses) or the cultures may be plated at'this
Example I
stage if desired. In the former case, those cul
Medium I containing 5% sugar was inoculated .
tures which show good yields on Medium I and
with 3% of the fourth generation of a culture
poor
yields
on
Medium
XIII
are
thenlchosen
for
55
obtained from rotted wood and incubated at
further quantitative fermentations. If the re
sults of these fermentations show high yields of ‘30-32? C.’ for 72 hours. The yield and solvent
ratio were then determined and found to be as
solvents with proportions of butyl alcohol, iso
propyl alcohol, ethyl alcohol, and acetone within
60 the limits speci?ed below, the desired cultures
have probably been obtained. These may then be
further puri?ed by plating if desired.
If the cultures'are plated after the ?rst quan
titative fermentation, this-may be done’ in the
follows:
'
60
Solvent ratio '
Yiflrlgi liericent
on c a e on
Sugar
usual manner utilizing such media as standard
glucose-yeast water agar, standard nutrient agar
containing 2.0%, glucose and 0.1% (NH4)2SQ4,
and the like. These plates mayv then be incu
bated anaerobically at 32° C. and after growth
70 is evidenced, colonies may be tested quantita
, tively on Medium I after several 24 hour trans-.
fers on similar medium. The desired cultures
may then-be chosen on ‘the basis of the quanti
tative results. Further plating for selection of
75 good strains may be made if desired. Cultures
.
28. 8
Ethyl and
Butyl
.
-
alcohol 1551232?! Acetone
66. 3
_
22. 9
1()_ 8
Example II
Medium I containing 5.0% sugar is'inoculated
with about 3% of the fourth generation ofa cul
ture obtained from a colony in plating a culture
obtained from rotted wood. The fermentation
was then carried out in the usual manner at
65
2,182,039
6
30-32° C. ' The yield and solvent ratio were found
to be as follows:
Solvent ratio
‘share:
1
sugar
31.1
10
...
2,1135%! 15231332? Acetone
68.3
25.8
5-9
solvents from a nutrient glucose mash, than from
a mash consisting solely of grain meal and wa
ter, and which produce increasing amounts of
acidic end products throughout the fermentation
in the absence of hydrogen ion control, the fol
lowing examples are cited which were carried out
by fellow workers to whom I had previously dis
closed my process, with various specific types of
such bacteria selected by them.
The same operating conditions that apply to
the group C'lostridium prom/l butylicum and spe
ci?cally to Clostridium propyl butylicum-alpha
Example III
also apply to these organisms, as for example,
Medium I containing 5% sugar was inoculated
15 with the fourth generation of a culture obtained
from a green corn stalk and the fermentation
carried out in the usual manner. The following
yield and solvent ratio were obtained:
Clostr‘idium inverto-aceto-butylicum.'
Bacteria designated as clost?dium inverto
aceto-butylicum comprises any bacteria having
the following primary characteristics:
I. Morphological:
'
A. Rod-shaped
Yieldi péegxui
ca on a e
'
.
B. Spore-forming-Clostridia
Solvent ratio
and Plec 20
, tridia
on
sugar
Butyl
alcohol
Ethyl and
iso propyl
alcohol
, Acetone
_
71. 9
20. O
group
25
II. Biochemical:
A. Carbohydrate fermentation:
25
32. 2
'
C. Practically indistinguishable from mem
bers of the Clostridium butyricum
7. 1
1. Inability to produce appreciable
yields of butyl alcohol and ace
Example IV
30
Medium VII containing 5% corn sugar was
. inoculated with the fourth generation of a cul
ture obtained from rotted wood and the fermen
tation carried out in the usual manner.
The
following yield and solvent ratio were obtained:
Solvent ratio
Yiklilgi Dericent
cac
ate
sugar
40
on
Butyl
Ethyl
and
.
75.6
2()_8
alcohol
tone from starch as the only 30
source of carbohydrate
2. Inability to produce appreciable
yields of butyl alcohol and ace
tone from sucrose as the only
source of carbohydrate
3. Inability to consistently produce 35
yields of solvents greater than
20% calculated on the weight of
the sugar from vuninverted mo
lasses
4. Ability to produce high yields of 40
‘
‘5510352131 AGBLODQ
butyl alcohol and acetone from
27.3
glucose or inverted molasses
3.6
Example V
Medium I containing 5% sugar was inoculated
with the fourth. generation of a culture obtained
from rotted wood and the fermentation carried
out in the usual manner. The following yield
50
and solvent ratio were obtained:
B. Nitrogen metabolism:
1. Ability to produce high yields of
butyl alcohol and acetone in 45
sugar media containing ammonia
as the principal source of nitro
gen
2. Ability to utilize degraded protein
(including ammonia) as sole ni
trogen source
50
i
3. Inability to utilize undegraded pro
tein as sole source of nitrogen
Solvent ratio
altars‘ B H
Sugar
aléll?i?
30.2
73.9
4. Inability tovliquefy gelatin or to
produce more than very slight
Ethyland
isglycigiligyl Acetone
'
20.8
4.7
60
tion:
In this fermentation the pH of the mash was
determined, at intervals ‘throughout the fer
mentation, and at the completion of the fer-,
mentation after an elapsed time of 63 hours. The
65
proteolysis of milk.
C. Oxygen requirements:
1. Anaerobic
D. Temperature range for solvent produc 60
1. From 25" C. to 36° 0., preferably
following pH values were obtained:
I
29° C. to 31° C.
E. Hydrogen ion concentration for solvent
production:
_____
-
5.59
40 hours ________________________________ __. 5.70
46 hours ________________________________ __ 5.98
63 hours _______________________________ __ 5.85
‘
1. Final pH of 5.0-6.5, preferably
5.7-6.1
20-hours.v
.
.
The following speci?c example illustrates the
use of the above organism in my process:
Example VI
As further illustrating the general applicability
Medium I containing 5% sugar was inoculated
of my process to fermentation of sugar meshes
with butyl alcohol producing bacteria of the class
with 2.5 of a culture of Clostridium inverte
aceto-butylz‘cum obtained from a rotted corn
capable of producing consistently higher yields of
stalk and incubated at 31° C. for 68 hours. The
7(1
7
2,132,039
yield and solvent ratio were found to be as fol
lows:
Example VII
I
A mash similar in composition to Medium I
but containing 5.5% uninverted'sucrose in place
Solvent ratio
of inverted sucrose, was inoculated with 4% of a
Yield, por
ccnt on
sugar
B
uty
1
alcohol
Acetone
> sixth generation culture of a member of the group
E h 1
t y
Clostridium saccharoacetobutylz'cum and incu—
alcohol
bated at 30° C. for 68 hours.
The yield and sol
vent ratio were found to be as follows:
28. 8
70. 3
24. 2
5. 5
- _10
Solvent ratio
Yield. per
My process has likewise been satisfactorily ap
plied to improving the yields produced by bac
cent on
sugar
B
uty
1
..
F h 1
at v
alcohol
Auto”
alcohol
69.6
27. 5
2.9
_ teria of the group Clostrz'dium sacc'haro-aceto
butylz'cum.
Bacteria of this group comprise any
bacteria having the following primary charac
teristics:
I. Morphological:
A. Rod-shaped
B. Spore-forming—Clostridia
tridia
and
Plec
'
C. Practically indistinguishable from mem
bers of the clostrz'dz'um butyricum
group
It is to- be understood, of course, that my in
vention is not limited in its application to any of
the particular cultures speci?ed above by way of
illustration. In general, it may be said that my
improved process is applicable to all butyl al
cohol producing bacteria of the class which are
capable of producing consistently higher yields
of solvents from nutrient glucose mashes than
II. Biochemical:
butyl alcohol and acetone consist
from mashes consisting solely of grain meal and
water, and which produce increasing amounts of
acidic end products throughout the fermentation
ently from starch as the only
source of carbohydrate (i. e; corn
or other mash containing starch
produce ?nal fermantation products consisting 30
primarily of neutral solvents when the fermenta
A. Carbohydrate fermentation:
1.-Ability to produce fair yields of
'
36.0
and suitable nutrients)
in the absence of hydrogen ion control, but which
tion is effected in the presence of phosphates, de
graded protein nitrogen, such as ammonia, and
alcohol and acetone consistently .the acidity is regulated throughout the fermenta
above 30% on the weight of the tion whereby the ?nal hydrogen ion concentra
sugar from 5% sucrose media or tion secured by the action of the bacteria falls
an 'uninverted molasses medium within the range pH 5.0 to 6.5; By the term
nutrient glucose mashes, in this connection, is
of the character of Medium I de
scribed above except for the fact meant mashes such as Media V, X, and )HI above.
It will, of course, be evident to one skilled in
that it contains uninverted in
the art that all cultures of butyl alcohol pro
stead of inverted sugar
3. Ability to produce yields of butyl ducing bacteria will not ferment different types
alcohol and acetone consistently of sugars to the same degree, and that my process
above 30% on the weight of the could not enable a particular culture of bacteria
sugar from 5% glucose media to ferment an otherwise unfermentable sugar. It
may be said, however, that my process will make
with suitable nutrients, or on in
2. Ability to produce yields of ‘butyl
verted molasses medium corre
possible consistently higher yields of solvents
sponding to Medium I hereinbe
from a readily fermentable sugar than can be ob
tained without its application. It should be un
fore described
-
B. Nitrogen metabolism:
1. Ability to produce high yields of
butyl alcohol and acetone in sug
ar media containing ammonia as
the principal source of nitrogen
2. Ability to utilize degraded protein
(including ammonia) as the sole
source of nitrogen
3. Inability to utilize undegraded pro
60
tein as the sole source of nitrogen
4. Inability to liquefy gelatin or to
produce more than slight prote
olysis of milk
C. Oxygen requirements:
1. Anaerobic
D. Temperature range for solvent produc
tion:
1. Fromv 24° C. to 40° C., preferably
29° c. to 30° 0.
E. Hydrogen ion concentration for solvent
I
r
derstood therefore that my invention is applicable
to any type of sugar mash if the particular bac
teria employed have the necessary enzymes to
convert the sugar to solvents. In general, it may
be said that equivalents and modi?cations of pro
cedure which would naturally occur' to one skilled
in the art, may be employed without departing
from the scope of this invention.
'
This application is a continuation-in-part of
my application Ser. No. 650,036, ?led January 3,
1933, which in turn, is a continuation-in-part of 60
my application. Ser. No. 534,140, ?led April 30,
1931.
'
The invention now having been described what
I claim ‘is:
I
1. In a process for the production of butyl al
cohol by subjecting a fermentable sugar mash to
the action of a culture of butyl alcohol producing
bacteria of the class which are capable of produc
ing consistently higher yields of solvents from a
nutrient glucose mash than from a mash consist
production:
1. Final pH of 5.0-6.2, preferably
5.4-5.85.
The following speci?c example illustrates the
ing solely of grain meal and water and which
produce increasing amounts of acidic end prod
ucts throughout the fermentation in the absence
use of the above organism in my process:
crease the yield of neutral solvents which'com
of hydrogen ion control, the improvement to in- '
75
8
2,132,039
prises providing phosphate and ammonia nutri
ents in the mash, and also supplying non-toxic
alkaline neutralizing agents to the mash through
out the fermentation to control the acidity there
of, whereby the ?nal hydrogen ion concentration
secured by the action of the bacteria falls within
the optimum range for solvent production.
2. In a process for the production of butyl
alcohol by subjecting a fermentable sugar mash
10 to the action of a culture of butyl alcohol pro—
ducing bacteria of the class which are capable of
producing consistently higher yields of solvents
from a nutrient glucose mash than from a mash
consisting solely of grain meal and Water and
which produce increasing amounts of acidic end
products throughout the fermentation in the ab
sence of hydrogen ion control, the improvement to
increase the yield of neutral solvents which com
prises providing phosphate and ammonia nutri
ents in the mash, and also supplying non-toxic
alkaline neutralizing agents to the mash through
out the fermentation to control the acidity there
of, whereby the ?nal hydrogen ion concentration
secured by the action of the bacteria falls within
the range pH 5.0 to pH 6.5.
3. In a process for the production of butyl alco
hol by subjecting a fermentable sugar mash to the
action of ‘a culture of butyl alcohol producing
bacteria of the class which are capable of produc
ing consistently higher yields of solvents from a
v nutrient glucose mash than from a mash consist
ing solely of grain meal and water and which pro
duce increasing amounts of acidic end products
throughout the fermentation in the absence of
hydrogen ion control, the improvement to increase
the yield of neutral solvents which comprises pro
viding phosphate and ammonia nutrients in the
mash, and also supplying substantially water in
soluble non-toxic alkaline neutralizing agents to
40 the mash throughout the fermentation to control
the acidity thereof, whereby the ?nal hydrogen
ion concentration secured by the action of bac
teria falls within the range pH 5.0 to pH 6.5.
4. In a process for the production of butyl alco
45 hol by subjecting a fermentable sugar mash to the
action of a culture of butyl alcohol producing
bacteria of the class which are capable of pro
ducing consistently higher yields of solvents from
a nutrient glucose mash than from a mash con
sisting solely of grain meal and water and which
produce increasing amounts of acidic end prod
ucts throughout the fermentation in the absence
of hydrogen ion control, the improvement to in
crease the yield of neutral solvents which com
prises increasing the yield of neutral solvents by
providing phosphate and ammonia nutrients in
the mash, and also supplying calcium carbonate
to the mash throughout the fermentation to con
trol the acidity thereof, whereby the ?nal hydro
60 gen ion concentration secured by the action of the
bacteria falls within the range pH 5.0 to pH 6.5.
5. In a process for the production of butyl alco
hol by subjecting a fermentable sugar mash to the
action of a culture of butyl alcohol producing bac
teria of the class which are capable of producing
consistently higher yields of solvents from a
nutrient glucose mash than from a mash consist
ing solely of grain meal and water and which
tion to control the acidity thereof, the said neu
tralizing agent being introduced into the mash in
the form of calcium carbonate in a concentration
slightly in excess of that required to neutralize the
initial acidity of the mash.
6. In a process for the production of butyl alco
hol by subjecting a fermentable sugar mash to the
action of a culture of butyl alcohol producing
bacteria of the class which are capable of produc
ing consistently higher yields of solvents from a 10
nutrient glucose mash than from a mash consist
ing solely of grain meal and water and which pro
duce increasing amounts of acidic end products
throughout the fermentation in the absence of
hydrogen ion control, the improvement to increase
the yield of neutral solvents which comprises pro
viding phosphate and ammonia nutrients in the
mash, and also supplying a neutralizing agent to
the mash throughout the fermentation to control
the acidity thereof, the said neutralizing agent 20
being introduced into the mash in the form of an
initial addition of calcium carbonate in a con
centration of 3.5% and 13% based on the weight
of the sugar in the mash in excess of that required
to neutralize the initial acidity of the mash.
25
7. In a process for the production of butyl alco
hol by subjecting a fermentable sugar mash’ to the
action of a culture of butyl alcohol producing bac
teria of the class which are capable of producing
consistently higher yields of solvents from a nutri 30
ent glucose mash than from a mash consisting
solely of grain meal and water and which produce
increasing amounts of acidic end products
throughout the fermentation in the absence of
hydrogen ion control, the improvement to in 35
crease the yield of neutral solvents which com
prises providing phosphate and ammonia nutri
ents in the mash, and also supplying a neutraliz~
ing agent to the mash throughout the fermenta
tion to control the acidity thereof, the said neu 40
tralizing agent being introduced into the mash in
the form of an initial addition of calcium car
bonate in a concentration of 6% to 7% based on
the weight of the sugar in the mash in excess of
that required to neutralize the initial acidity of 45
the mash.
8. A process for the production of normal butyl
alcohol, isopropyl alcohol, ethyl alcohol, and ace
tone, which comprises subjecting a fermentable
carbohydrate mash containing inverted carbo 50
hydrate, as the principal source of fermentable
carbohydrate, and a nutrient material selected
from the group consisting of ammonia, ammo
nium salts, urea, yeast water, and steep water, to
the action of bacteria of the group C'lostridium 55
propyl b-utylicum, at temperatures from 25° to 36°
C., while controlling the acidity of the mash dur
ing the fermentation by means of neutralizing
agents chosen from the group consisting of cal-7
cium carbonate, barium carbonate, and iron car
bonate, whereby the ?nal hydrogen ion concentra
tion secured by the action of the bacteria falls
within the range pH 5.0 to pH 6.5.
9. A process for the production of normal butyl
alcohol, isopropyl alcohol, ethyl alcohol, and ace 65
tone, which comprises subjecting a fermentable
carbohydrate mash containing inverted molasses,
as the principal fermentable carbohydrate, and an
ammonium compound to the action of bacteria of
produce increasing amounts of acidic end prod
ucts throughout the fermentation in the absence the group Clostridium propyl butylicum, at tem 70
of hydrogen ion control, the improvement to peratures from 28° C. to 32° C., while controlling
the acidity of the mash during the fermentation
increase the yield of neutral solvents which com
prises providing phosphate and ammonia nutri-l whereby the ?nal hydrogen ion concentration
secured by the action of the bacteria falls within
ents in the mash, and also supplying a neutraliz
the range pH 5.8 to pH 6.1.
ing agent to the mash throughout the fermenta
9
2,182,089
10. A process for the production of normal
butyl alcohol, isopropyl alcohol, ethyl alcohol, and
acetone, which comprises subjecting a fermen
table- carbohydrate mash containing inverted
carbohydrate, as the principal fermentable car
bohydrate, to the action of bacteria of the group
Clostrz'dium prom/l butylicum, at temperatures
10
the principal fermentable carbohydrate, the im
provement which comprises subjecting said mash
in the presence of degraded protein nitrogen to
the action of a culture of bacteria of the group
identi?able as Clostridum prom/l butylicum by
means of the herein described primary charac
teristics, at temperatures from 25° to 36° C., and
from 25° C. to 36° C.
11. A process for the production of normal
supplying non-toxic alkaline neutralizing agents
butyl alcohol, isopropyl alcohol, ethyl alcohol,
throughout the fermentation, whereby the ?nal 10
and acetone, which comprises subjecting a fer-_
mentable carbohydrate mash containing in
verted carbohydrate, as the principal ferment
able carbohydrate, and degraded protein nitro
15 gen to the action of bacteria of the group Clos
trz‘dium propyl butylicum, at temperatures from
25° C. to 36° C., while controlling the acidity
of'the mash during the fermentation whereby
the ?nal hydrogen ion concentration secured by
20 the action of the bacteria falls within the range
to the mash to control. the acidity thereof
hydrogen ion concentration secured by the action
of vthe bacteria falls within the range pH 5.8 to
, pH 6.1.
>
..
16. In a process for the production of normal
butyl alcohol, isopropyl alcohol, ethyl alcohol
and acetone by the fermentation of a carbo
is
hydrate mash containing inverted molasses as
the principal fermentable carbohydrate, the im
provement which comprises subjecting said mash
in the presence of degraded protein nitrogen to 20
pH 5.0 to pH 6.5.
the action of a culture of bacteria of the group
12. In a process forrthe production of normal - identi?able as Clostridium propyl butylicum by
butyl alcohol, isopropyl alcohol, ethyl alcohol means of the herein described primary charac
and acetone by the fermentation of a carbohy
teristics, at temperatures from 25° C. "to 36° C.,
25. drate mash containing inverted carbohydrate as and supplying substantially. insoluble nontoxic
25
the principal fe'rmentable carbohydrate, the im
alkaline neutralizing agents to} the mash to con
provement which comprises subjecting said mash trol the acidity thereof throughout the fermenta
in the presence of degraded protein nitrogen to tion, whereby the ?nal hydrogen ion concentra
the action of a culture of bacteria of the group , tion secured by the action ofthe bacteria falls
30 identi?ed as Clostridium prom/Z butylicum by within the range pH 5.8 to pH,6.1.
30
means of the herein described primary charac
17. In a process for the production of normal
teristics, at temperatures from 25°C. to 36° C., butyl alcohol, isopropyl alcohol, ethyl alcohol and
and supplying non-toxic alkaline neutralizing acetone by the fermentation 'of a carbohydrate‘
agents to the mash to control‘ the acidity there
mash containing inverted molasses as the princi
35 of throughout the fermentation, whereby the ?nal pal fermentable carbohydrate, the improvement
hydrogen ion’concentration secured by the action which comprises subjecting said mash in the pres
of the bacteria falls within the range pH 5.0 ence of degraded protein nitrogen to the action
to pH. 6.5.
,
of a culture of bacteria of the "group identi?able
13. In a process for the production of normal
40 butyl alcohol, isopropyl alcohol, ethyl alcohol and
acetone by the fermentation of a carbohydrate
mash containing inverted carbohydrate as the
principal fermentable carbohydrate, the improve-.
secured by the action of the bacteria fall within
action of a culture of bacteria of the group iden
the range pH 5.8 to pH 6.1.
of the herein described primary characteristics,
at temperatures from 25° C._ to 36° C., and sup
plying substantially insoluble non-toxic alkaline
neutralizing agents to the mash to control the
acidity thereof throughout the fermentation,
whereby the ?nal hydrogen ion concentration se
cured by the action of the bacteria falls within
' .
.
18. In a process for the'production of butyl
alcohol and other useful products by the fermen
tation of a mono-hexose mash by the action of
herein-described Clostridium prom/l butylicum
alpha, the step Which comprises e?’ecting fer 50
mentation in the presence of ammonia nitrogen,
a phosphate, and aninsoluble non-toxic alkaline
reacting metal carbonate :ina~.;a vconcentration
the range pH 5.0 to pH 6.5.
'
" $14. In a process for the production of normal
slightly in excess of that requiredjto neutralize
butyl alcohol, isopropyl alcohol, ethyl alcohol
19. In a process for the production of butyl
alcohol and other useful .products by the fer
mentation of a mono-hex'os'e mash ‘by the action
and acetone by the fermentation of a carbohydrate
mash containing inverted carbohydrate as the
principal fermentable carbohydrate, the improve
ment which comprises subjecting said mash in
the presence of degraded protein nitrogen to the
action of a culture of bacteria of the group '
65
calcium carbonate to the mash to control the
acidity thereof throughout the fermentation,
whereby the ?nal hydrogen ion concentration
,ment which comprises subjecting said- mash in
the presence of degraded protein nitrogen to the
ti?able as clostridium propyl butylz'cum by means
60
as Clostridium propyl butylicum by means of the
herein described‘ primary characteristics, at tem 40
peratures from 25° C. to 36° C., and supplying
any initial acidity of the mash.~ ' ‘
V)
'
of the herein-described C'lostri ,um propyl ‘butyl so
icum-alpha, the step which mprises'eifecting
fermentation in the prese
mmonia nitrogen, a phosphate, and» an nsoluble non-toxic
identi?able as Clostridium prom/l butylicum by
alkaline reacting metal carbon e in a concentra
means of the herein described primary charac
teristics, at temperatures from 25° C. to 36° C.,
tion of the order of 0.2% bywwei'ghtrbased on the 65
total weight of the 'mesh, in excess of that re
and supplying calcium carbonate to the mash
to‘ control the acidity thereof throughout the
mash.
fermentation, whereby the ?nal hydrogen ion
-70 concentration secured by the action of the
bacteria falls within the range pH 5.0 to pH 6.5.
15. In a process for the production of normal
butyl alcohol, isopropyl alcohol, ethyl alcohol
and acetone by the fermentation of a carbo
75 hydrate. mash containing inverted molasses as
quired to neutralize anyg-initial acidity of the '
, 4
20. In a process for the production of butyl
alcohol and other useful products-by the fer 70
mentation of an inverted fmolasses mash by the
action of the herein-described C'lostridium prom/l
butylicum-alpha, the step which-comprises effect
ing fermentation in the presence of ammonium
sulphate, a phosphate, and an insoluble none
2,132,089
10
toxic alkaline reacting metal carbonate in a con
centration slightly in excess of that required to
neutralize any initial acidity.
21. In a process for the production of butyl
alcohol and other useful products by the fer
vmentation of an inverted molasses mash by the
action of the herein-described Clostridium prom/1'
' butylz'cum-alpha, the step which comprises effect
ing fermentation in the presence of ammonium
10 sulphate, a phosphate, and an insoluble non
toxic alkaline reacting metal carbonate in a con
centration of the order of 0.2% by weight, based
on the total weight of the mash, in excess of that
required to neutralize any initial acidity of the
15
.
mash.
‘
22. In a process for the production of butyl
weight of the mash, in excess of that required
to neutralize any initial acidity of the mash.
23. In a process for the production of butyl
alcohol and other useful products by the ter
mentation of mono-hexose mashes by the action
of butyl alcohol-producing bacteria identi?able?
as Clostridium prom/l butylicum-alpha by means
of the herein described primary characteristics,
the step which comprises effecting fermentation
in the presence of ammonia nitrogen, a phos 10
phate, and an insoluble non-toxic alkaline react
ing metal carbonate in a concentration slightly
in excess of that required to neutralize any initial
acidity of the mash.
24. In a process for the production of butyl 15
alcohol and other useful products by the ter
mentation of mono-hexose mashes by the action
alcohol and other useful products by the ten
of butyl alcohol-producing bacteria identi?able
mentation of an inverted molasses mash by the
as Clostridium prom/l butylicum-alpha by means
action of the herein-described C'lostridium prom/l
butylicum-alpha, the steps which comprise ad
of the herein described primary characteristics,
the step which comprises e?‘ecting fermentation
justing the initial hydrogen ion concentration of
in the presence of ammonia nitrogen, a phos
the mash to a pH of 5.0-6.2, and effecting fer
mentation at a temperature of approximately
32° C. in the presence-of ammonium sulphate, a
phosphate, ‘and an insoluble non-toxic alkaline
reacting metal carbonate in a concentration‘ of
the order of 0.2% by weight, based on the total
phate, and an insoluble non-toxic alkaline react- _
ing metal- carbonate in a concentration of the
order of 0.2% by weight, based on the total weight
of the mash, in excess of that required to neutral
ize any initial acidity of the mash. v
JOHN MULLER.
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