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Патент USA US2122884

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2,122,884
Patented July 5, 1938
UNITED STATES ‘PATENT OFFICE
2,122,884
BUTYL ALCOHOL FERMENTATION
PROCESS ‘
- David A. Legg and Hugh R. Stiles',‘Terre Haute,
‘
Ind., assignors to Commercial Solvents Cor
poration, Terre Haut'e, Ind., a corporation of
Maryland
\
No Drawing. Application July 12, 1934,
Serial No. 734,802
10 Claims. (Cl. 195—44)
,The present invention relates to the utiliza
tion of waste carbohydrate materials, such as
those obtained in the acid hydrolysis of grain or
starch. More particularly, the present invention
5 relates to the utilization of hydrol, the waste re-'
- , sidual material from the corn-sugar industry, as
_
10
high concentrations. It has been possible to sub
stitute small amounts in the fermentation of
maize mashes by means of bacteria of the type
Clostridium acetobutylicum. Such a method,
however, has obvious disadvantages, especially
from the standpoint of utilizing hydrol directly
a source of carbohydrate for fermentation by
at the source of its production, without the ne
means of bacteria of the group C'Zostrz'dium sac
cessity of fermenting large amounts of maize.
charo-acetobutylicum.
- It has also been attempted to utilize hydrol for
'
Hydrol comprises the molasses obtained as the
residual mother liquor from which corn sugar is
crystallized. This material contains about 75%
solids,‘ the major portion of which comprises glu
cose. However, substantial proportions of dextrin
l5 and other intermediate types vof carbohydrates
are present. A typical analysis of hydrol, as usu
ally obtained, is the following:
‘
Per- cent
Glucose _____ _-' __________________________ __ 55
20 Dextrin and other carbohydrates higher than
glucose _
____ __
-___
__
18
Water __________________________________ __ 27
Di?erent samples of hydrol may vary consider
ably from the ?gures shown above, but this anal-_
ysls illustrates, in general, the type of material
under consideration. It is, therefore, to be under
stood that the term “containing substantial con
centrations of higher soluble carbohydrates” as
30 used herein and in the appended claims denotes
concentrations of the order of those found in
2
hydrol. Hydrol is available in relatively large
quantities, and in view of the high carbohydrate
content, many attempts have been made to uti
35 lize it in commercial fermentations, especially in
.
'10
the production of butyl alcohol by means of bac 10
teria of the group Clot-iridium saccharo-aceto
butylicum or other butyl organisms of the type
which readily ferment sugar solutions. However,
it-has been found that low yields were obtained
in such fermentations unless the hydrol were sub 15
jected to further acid hydrolysis or other costly
procedures which overcame the economic advan
tage of utilizing a waste product of this nature.
We have now discovered that hydrol can be
successfully fermented by bacteria of the group
C'Zostridium saccharo-acetobutylicum without the
necessity of any preliminary treatment if cer
tain types of nitrogenous nutrients are employed
in the mash. 'I'he nutrients which we have found
to be suitable for this purpose comprise grain
distillery slops such as grain alcohol distillery
slops or the slop from the butyl-acetonic fermen
tation of grain mashes. These materials are
especially suited for satisfying the de?ciency in
nitrogen in hydrol mashes, being superior to other
proteinaceous materials or even other distillery
slops-such as those from molasses fermentation. _
The advantages of the use of this type of nitrog
enous nutrient may be seen from the following
35
table:
Table I
Ammonium
lgggiglt??'
‘
sulfate con-
ams w a".
centration;
gr r 100 f6
p9
grams per
. ‘
‘
-Supplementary
nitrogenous
nutrient
100 cc.
-
45
. 5. o
,
.
percent
grams per
sugar
100 cc.
-
nutrient;
0. a
40
yield
of total
4. s
4. 9
0. 3
c. a
0. 3
._
Barley melt ______ __
5.0
0. 3
Maize germ meal...
0. 7
24. i
5. 3
0.3
Wheat middlings. -.
0. 7
24. 4
5. l
0.3
Grain alcohol ~dlS
‘30. 0
33. 0
,
s 01vent
tary
5. 4
5. 3
50
,
o‘iticentrm'
on o
supplemen-
' c. 0. whole slop per 100 cc.
‘the butyl alcohol fermentation. However, up to
the present time, no successful method has been
55' devised for fermenting this material in desirably
tillery slop ..... -_
0. 7
7. 7
0.7
0. 7
16.4
21. 5
45
50
_
It may be seen from the above table that, with
the exception of the last in the series, employing
the nutrients of the present invention, none of 55
2
2,122,884
the hydrol ‘fermentations could be said to be suc
cessful from a commercial standpoint.
The general type of fermentation to which our
invention is applicable is illustrated in copending
application U. S. Ser. No. 675,459 by J. C. Wood
ruff, H. R. Stiles, and D. A. Legg, ?led June
12, 1933, now Patent No. 2,089,552, issued August
10, 1937. The organisms employed are bacteria
of the group Clostridium saccharo-acetobutyli
10 cum, which are described at length in said appli
cation. Our process is particularly adapted to
fermentations by means of Clost?clz‘um saccharo-v
acetobutylicum a, which is likewise described in
detail in the copending application now Patent
15 No. 2,089,552. According to the general process
of said application, now Patent No. 2,089,552, the
soluble carbohydrate mashes, containing an am
monium compound, and preferably additional de
graded protein material, are fermented by means
20 of bacteria of the group Clot-iridium saccharo
acetobutylicum while controlling the acidity of
the fermenting mash whereby the ?nal hydrogen
ion concentration secured by the action of the
bacteria falls within the range pH 5.0 to pH
25 6.2. Our present invention follows this general
procedure, utilizing the speci?c combined supple
mentary nitrogenous nutrients herein described.
The grain distillery slops employed in our
process may be utilized in the form of the whole
30 slop or its equivalents, such as concentrated slops,
evaporated feeds, or the like. Screened feeds
and similar materials from which the soluble ma
terial has been removed will not, in general, be
found to be as satisfactory as the materials in
35 cluding all of the degraded nitrogenous sub
stances.
The optimum amount of distillery slop to be
added in any fermentation will depend upon the
amount of other nitrogenous material present
40 in the mash. For example, a mixed beet molas
ses-hydrol mash will require less additional
nitrogenous material than one in which hydrol
comprises substantially all of the fermentable
carbohydrate. However, in general it may be
45 said that from 10-90% by volume of whole slop,
pend to some extent upon the concentration and
state of degradation of the other nitrogenous ma
terials present. However, it may be said that in
general from 0.l-0.5% by wt. of ammonium sul
fate or its equivalent will be found to be satis
factory. Concentrations of 0.2-0.4% will be pref
erable in most cases.
considerable amounts of organic acids. For ex
will usually be found to have a pH of 3.5 to 4.0.
This slop may advantageously be neutralized to
a pH of from 6.0 to 6.4 before incorporating it
into the mash to be employed in the present proc 15
ess. However, if desired, the slop may be in
corporated in the mash in its orginal form, the
hydrogen ion concentration of the entire mash
then being regulated in accordance with the pro
cedure of copending application U. S. Ser. No. 20
675,459 now Patent No. 2,089,552. According
to thisprocedure, the acidity of the fermenting
mash is regulated either by the addition of in
soluble alkaline neutralizing agents at the be
ginning of the fermentation, or by continuous or 25.
semi-continuous neutralization during the fer
mentation, whereby the ?nal hydrogen ion con
centration falls within the range 5.0 to 6.2. Al
though any of these procedures may be employed,
we prefer to neutralize thedistillery slop to a 30
pH of approximately 6.2 by means of an alkali
metal compound such as soda ash, and to in
corporate into the hydrol-slop mash a'slight
excess, e. g. 03-06%, of an insoluble alkaline
neutralizing agent such as calcium carbonate. 35
This latter material may suitably be in‘ very ?ne
ly divided form, 'such as a freshly precipitated
carbonate. If su?lcient alkaline buffering ma
terial is already present in the mash, as for ex
ample in the case of certain meshes containing
high proportions of beet molasses, it may be 40
found to be unnecessary to add alkaline ma
terials at any stage of the process.
The following examples illustrate the process
of the present invention:
distillery slops generally contain considerably
in somewhat smaller amounts in the present
process. From 10-50% of whole slop, based on
55 the total volume of the mash, will generally be
found to be satisfactory. In the case of the
butyl-acetonic slops, on the other hand, from
20-90% will usually be employed. In this case
the slop may even be substituted for all of the.
60 Water in the mash if desired. However, for most
purposes we prefer to utilize from 30-40% by
volume’ of grain alcohol slops or from 70-90%
by volume of butyl-acetonic slop. These con
centrations may advantageously be employed in
65 any mash containing hydrol as a major source
of carbohydrate. Smaller amounts may be suf
ficient in many cases, and the lower economical
' limit may easily be determined by preliminary
fermentations.
In conjunction with the grain distillery slops,
70
ammonia or ammonium‘ salts should be em
ployed in accordance with the procedure of U. S.
Ser. No. 675,459 now Patent No. 2,089,552. The
_ optimum amount of ammonia nitrogen to be em
75 ployed in any particular case will, of course, de
.45
Example I
Grain alcohol
higher concentrations of nitrogenous materials
50 than are present in grain distillery slops vfrom
the butyl-acetonic fermentation. The grain al
cohol slops, therefore, may usually be employed
10
ample, whole slop from grain alcohol distillerles
or its equivalent of the concentrated materials, '
will be found to be satisfactory.
'
The grain slops, especially those fromv yeast
fermentations, will usually be found to contain
A sterile hydrol mash (5.1% total reducing sug
ars) containing 30% by volume of grain alcohol
distillery slop, 0.3% by weight of (NH4)2SO4,
0.07% by weight of K2HPO4, and 0.6% by weight
50
of CaCOs was inoculated with an actively fer
menting culture of Clostridium saccharo-aceto
butylicum a and incubated at 30° C. for 68 hours.
The yield and solvent ratio were found to be as 55
follows:
Yield
Solvent ratio
Percent of
Grams S‘?'
Butyl al~
total sugar
vel?tserper
cohol
33. U
16. 7
I 67. 5
Ethyl 21
Acetone
60
cohol
28. 1
4. 4
65
Example II
‘ ‘A sterile mash containing 5.3% of total reduc
ing sugars (96% hydrol-4% cane molasses), 40%
by volume of grain alcohol distillery slop (ad 70
justed to pH 6.0 with soda ash), 0.2% by weight
of (NH4)2SO4, and 0.3% by weight of precipitated
CaCOa was inoculated with an actively ferment
ing culture of Clostridium saccharo-acetobutyli
cum on and incubated at 30° C. for 68 hours.
The
3
2,122,884
It may be seen that practically theoretical
yields were obtained in Examples I and vIII and
that in the higher concentration mashes of Ex
amples II and IV absolute yields of over 17 grams
per liter were obtained even though the percent
age yield, based on sugar content, decreased.
yield and solvent ratio were found to be as fol
lows:
Yield
10
Solvent ratio
Percent of
Grams sol
total sugar
veggie?“
52.7
17.3
Butyl al-
cohol
Acetone
70.9
The high yield in Example V is be
lieved to be due in part to the presenceof fer
mentable carbohydrates other than reducing sug
. Ethyl al
_ somewhat.
cohol
25.8
ars in beet molasses.
3.3
Example III
Although our invention has been illustrated by
the above speci?c ‘examples, it is to be distinctly
15 sugars) containing 84% by volume of butyl-ace
understood that it is not limited to the particular
materials or procedures described therein. For
example, although our invention is particularly
adapted to the fermentation of hydrol, it will be
evident to those skilled in the art that this process 20
may advantageously be applied to other soluble
carbohydrate materials which contain monose
tonlc slop (from the fermentation of 7.5% maize
mash. by Clostrz'dium acetobutylz'cum (Weiz
mann) neutralized with soda ash to a pH of
6.0), 0.2% by weight of (NH4)2SO4 and 0.4% by
20 weight of CaCOa was inoculated with an actively
fermenting culture of Clostridium saccharo
acetobutylicum oz and incubated at 30° C. for 68
sugars as major constituents and substantial
amounts of higher carbohydrates which are de
hours. The yield and solvent ratio were found
to be as follows:
?cient in suitable nitrogenous nutrientsfor this 25
type of fermentation. Various equivalent ma
terials readily suggest themselves, as for example
wood sugar solutions which contain glucose and
higher polymers, solutions'obtained by the hy
drolysis of corncobs or other pentosan-containing 30
25
Solvent ratio
Percent of
Grams sol
total sugar
vegggerper
33.9
16.1
Butyl al-
cohol
Acetone
Ethyl al
cohol
30
' materials which solutions contain xylose or other
65.7
29.9
4.4
p-entosesugars, together with higher carbohy
drates ,of this series, and other similar solutionsv
containing soluble carbohydrates and de?cient in
suitable nitrogenous nutrients. It will also be
Example IV
35
10
ously obtainable with hydrol mashes.
A sterile hydrol mash (4.8% of total reducing
Yield
All of these values repre
sent extremely satisfactory yields from a com
mercial standpoint, and exceed any yields previ
A sterile hydrol mash (5.4% of total reducing
sugars) containing 93% by volume of butyl-ace
tonic slop (from the fermentation ‘of 7.5% maize
mash by Clostridium acetobutylicum (Weizmann)
40 neutralized with soda ash to a pH of 5.8), 0.2%
by weight of (NI-102304 and 0.4% by weight of
apparent to one skilled in the art that various
mixed mashes other than the particular hydrol
molasses mashes of Examples II and V may suit
ably be employed.
Our process is applicable,
generally, to mashes containing hydrol or an 40
equivalent material as a major component, irre
CaCOa was inoculated with an actively ferment— spective of the remaining carbohydrate content.
The bacteria designated herein as C'lostridium
ing culture of Clostridium saccharo-acetdbutyli
saccharo
acetobutylicum comprise any bacteria
The.
cum .. and incubated at 30° C. for 68 hours.
having the following primary characteristics:
45 yield and solvent ratio were found to be as fol
lows:
I. Morphological.
-
A. Rod-shaped.
Yield
'
B. Spore-forming-Clostridia‘ and Plectridia.
Solvent ratio
C. Practically indistinguishable from mem
Peroent of
totel sugar
Grams sol
Butyl alvel?gerper .
cohol
31.6
17.2
66.2
Acetone
Ethyl al
cohol
31.4
2.4
II. Biochemicah
.
A. Carbohydrate fermentation.
1. Ability to produce fair yields of. butyl 55
alcohol and acetone consistently from
55
starch as the sole source of carbo
hydrate,(i.'e., corn or other mash con
Example V
A sterile mash containing 5.0% of total reduc
'taining starch and suitable'nutrlents)
ing sugars (50% hydrol-50% beet molasses)
containing 16%. by volume of grain alcohol dis
tillery slop and 0.03% by weight of (NHO2HPO4
2. Ability to produce yields of butyl alcohol 00
and acetone consistently above 30%
on ‘the weight'of ‘the sugar from 5%
was inoculated with an actively fermenting cul
ture of Clostridium saccharo-acetobutylicum oz
and incubated at 30° C. for 68 hours. The yield
and solvent ratio were found to‘be as follows:
sucrose media or 5.5% uninverted mo- _
lasses medium containing about 0.3% ,
(NH4):SO4. and about 0.4% of 200
mesh calcite, based on the mash
-
Yield
70 Percent of
total‘sugar
Grsmssol-
vm?gerper
’
Butyl
al-
cohol
Mew“
volume.
34.8
17.5
74.0
22.9
‘
=
3. Ability to produce yields of butyl alcohol
and acetone consistently above 30%
Solvent ratio
on the weight of the sugar from 5%
Etii lal-_
co 0]
glucose media with suitable nutrients,
.
3.1‘
or an inverted molasses medium cor
responding to the uninverted molasses
medium of (2) above.
75
50
bers of the Clostridium butyricum group.
d
2,122,884
B. Nitrogen metabolism.
1. Ability to produce high yields of butyl
alcohol and acetone in sugar media
containing ammonia as the principal
source of. nitrogen.
in molasses mash and the hydrol-slop solution
2. Ability to utilize degraded protein (in
cluding ammonia) as sole nitrogen
source.
'
3. Inability to utilize undegraded protein as
10
sole source of nitrogen;
4. Inability to liquefy gelatin or to produce
more than very slight proteolysis of
milk.
15
C. Oxygen requirements.
1. Anaerobic.
D. Temperature range for solvent production.
1. From 24° C. to 40° C. preferably 29° C.
to 30° C.
E. Hydrogen ion concentration for solvent
20
production.
1. Final pH of 5.0-6.2, preferably 5.4-5.85.
Although the fermentations of the examples
were all carried out by means of Clostridium
saccharo-acetobutylicum a, any of the bacteria
25 of the general group C'Zostridium saccharo-aceto
butylicum may be employed or any other bacteria
having the general characteristics set forth in
copending application U. S. Ser. No. 675,459 now
Patent No. 2,089,552. For example, the chromo
30 genic strains Clostridium saccharo-acetobutyl
icum p and Clostridium saccharo-acetobutylz'cum
'7 described in copending application U. S. Ser.
No. 714,633 by C. F. Arzberger, ?led March 8,
1934, now Patent No. 2,050,219, issued August
35 4, 1936, may suitably be employed in our process.
These bacteria have the following primary char
acteristics:
40
Likewise, various modi?cations of procedure
may be employed. For example, if a mixed mash
is to be employed, such as a hydrol-molasses
mash, the fermentation could suitably be started
added to this mash after fermentation had be
come sufficiently active. Any of the modi?ca
tions applicable to this type of fermentation
which are disclosed in copending application U. S.
Ser. No. 675,459 now Patent No. 2,089,552 may 10
be adapted to the present process. In general it
may be said that any such modi?cations or the
use of any equivalents which would naturally
occur to one skilled in the art may be employed
without departing from the scope of our inven 15
tion.
Our invention now having been described,
what we claim is:
1. In a fermentation of the Clostridium sac
charo-acetobutylicum type in which the ferment
ing medium comprises essentially a soluble carbo
hydrate mash containing monose sugar as a
major component, containing substantial con
centrations of higher soluble carbohydrates, and
de?cient in suitable nitrogenous nutrients for
said type of fermentation, the step which com
prises eifecting the fermentation in the presence
of the combined supplementary nutrient consist
ing of ammonia nitrogen in a concentration
equivalent to from 0.1 to 0.5% by weight of am 30
monium sulfate and in excess of 10% by volume
of grain distillery slop chosen from the group
consisting of slop from a grain mash fermenta
tion‘by means of starch-fermenting butyl alcohol
bacteria and slop from a yeast fermentation of 35
a sacchari?ed grain mash.
2. In a fermentation of the Clostrz'dium sac‘
.
I. Morphological.
A. Rod-shaped.
B. Spore-forming-Clostr'idia and Plectridia.
charo-acetobutylicum type in which the ferment
ing medium comprises essentially a soluble carbo
hydrate mash containing monose sugar as a 40
major component, containing substantial con
bers of the Clostrz'dz'um butyricum‘ centrations of higher soluble carbohydrates, and
de?cient in‘ suitable nitrogenous nutrients for
group.
said type of fermentation, the step which com
II. A. Biochemical.
1. Ability to produce yields of butyl alcohol ' prises effecting the fermentation in the presence 45
and acetone consistently above 30% of the combined supplementary nutrient consist
on the‘ weight of the sugar from 6% ing of from 0.2 to 0.4% by weight of an am
sucrose media or uninverted molasses monium'salt and from 10-50% by volume of grain
mashes of the type described herein. alcohol distillery slop.
C. Practically indistinguishable from mem
to
2. Ability to produce yields of butyl alcohol
and acetone consistently above 30%
on the weight of the sugar from 6%
glucose media with suitable nutrients,
or an inverted molasses mash.
55
B. Nitrogen metabolism.
1. Ability to produce high yields of butyl
alcohol and acetone in sugar media
containing ammonia as the principal
source of nitrogen.
. 2. Ability to utilize degraded protein (in
cluding ammonia) as the sole nitrogen
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.
70
1. Anaerobic.
D. Temperature range for solvent production.
1. From 24° C. to 40° C., preferably 29° C.
to 30° C.
E. Hydrogen ion concentration for solvent
production.
75
1. Final pH of 5.0-6.2, preferably 5.4-5.85.
F. Chromogenesis-orange to red.
3. In a fermentation of the Clostridium sac
charo-acetobutylz‘cum type in‘ which‘ the ferment
ing medium comprises essentially a soluble carbo
50
hydrate mash containing monose sugar as a
major component, containing substantial con
centrations of higher soluble carbohydrates, and
de?cient in suitable nitrogenous nutrients for
said type of fermentation, the step which com
prises e?ecting the fermentation in the presence
of the combined supplementary nutrient consist
ing of from 0.2 to 0.4% by weight of an am 60:
monium salt and from 20-90% by volume of
grain distillery slop from the butyl-acetonic fer
mentation of a grain mash by means of starch
fermenting butyl alcohol bacteria.
4. In a fermentation of an essentially solu 65
ble carbohydrate mash containing hydrol as a
major constituent, by means of bacteria of the
group Clostridium saccharo-acetobutylicum, the
step which comprises effecting the fermentation
in the presence of ammonia nitrogen and grain 70
distillery slop chosen from the‘ group consisting
of slop from a grain mash fermentation by
means of starch-fermenting butyl alcohol bac
teria and slop from a yeast fermentation of a
sacchari?ed grain mash.
'
75
5
2,122,884
5. In a fermentation of an essentially soluble
carbohydrate mash containing hydrol as a major
constituent, by means of'bacteria of the group
carbohydrate mash containing hydrol as a major
constituent, by means of Clostridium'saccharo
wcetobutylz‘cum u, the step which comprises ef
C'lostridium saccharo-acetobutylicum, the step - fecting the fermentation in the presence of am
which comprises effecting the fermentation in the monia nitrogen and grain distillery slop chosen
presence of ammonia nitrogen in a concentration from the group consisting of slop‘from a grain
mash fermentation by means of starch-ferment
equivalent to from 0.1 to 0.5% by weight of am
monium sulfate and in excess of 10% by volume ing butyl alcohol bacteria and slop from a yeast
of grain distillery'slop chosen from the group fermentation of a sacchari?ed grain mash.
9. In a fermentation of an essentially soluble 10
10 consisting of slop from a grain mash fermenta
tion by means of starch-fermenting butyl alcohol carbohydrate mash containing hydrol as a major
bacteria and slop from a yeast fermentation of a constituent, by means of ClOstridz'um saccharo
acetobutylz'cum p, the step which comprises ef
sacchari?ed grain mash.
6. In a fermentation of an essentially soluble‘
15 carbohydrate mash containing hydrol as a major
constituent, by means of bacteria of the group
Clostridium saccharo-acetobutylicum the step
which comprises effecting the fermentation in
the presence of from 0.2 to 0.4% by weight of an
ammonium salt and from 30-40% by volume of
grain alcohol distillery slop.
'7.‘In a fermentation of an essentially soluble
carbohydrate mash containing hydrol as a. major
constituent, by means of bacteria of the group
Clostridium saccharo-acetobutylicum, the step
which comprises e?ecting the fermentation in
the presence of from 0.2 to 0.4% by weight of an
ammonium salt and from. 70-90% by volume of
grain distillery slop from the .butyl-acetonic fer
mentation of a grain mash by means of starch
fermentlng butyl alcohol bacteria.
8. In a fermentation of an essentially soluble
fecting the fermentation in the presence of am
monia nitrogen and grain distillery slop chosen 15
from the group consisting of slop from a grain
mash fermentation by means of starch-ferment
ing butyl alcohol bacteria and slop from a yeast
fermentation of a sacchari?ed grain mash.
10. In a fermentation of an essentially soluble
carbohydrate mash containing hydrol as a major
constituent, by means of Clostfidium saccharo
acetobutylz'cum 'y, the step which comprises ef
fecting the fermentation in the presence of am
monia nitrogen and grain distillery slop chosen‘
from the group consisting of slop from a grain
mash fermentation by means of starch-ferment
ing butyl alcohol bacteria and slop from a yeast
fermentation of a.- sacchari?ed grain mash.
DAVID A. 'LEGG.
HUGH R. S'I'IIES.
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