Note: Descriptions are shown in the official language in which they were submitted.
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Background of the Invention
This invention relates to a method for the production
of alcohol in high yields from cellulosic materials as the sub-
strate.
Heretofore, production of alcohol (ethanol) has been
attempted by a procedure comprising the steps of reacting a
cellulase upon cellulose as the substrate to enzymatically
saccharify the cellulose to glucosel and subsequently separately
causing the resultant glucose to be reacted upon by an alcohol-
producing microorganism to produce alcohol. According to this
conventional method, the conversion of cellulose to glucose by
a cellulase is low and, consequently, large amounts of uncon-
verted cellulosic residue are obtained. There~ore, if the ;
product of such a cellulase treatment of cellulosic materials
is employed as the raw material for alcohol fermentation it is
necessary to separate the glucose from said product, for example,
by filtration. Thus, in addition to the drawback tha`t the
glucose concentration in the saccharified liquid is low due to
the low conversion of cellulose to glucose, the production of
glucose according to the conventional method includes the
possibility that glucose is lost during the aforementioned
separation. Consequently, low yields of alcohol are obtained
by subjecting the saccharified liquid to fermentation.
Summary of the Invention ;
It has now been found that greater yields of alcohol
can be obtained from a cellulosic material when there are
simultaneously reacted under anaerobic conditions the cellulosic
material, a cellulase, and an alcohol-producing microorganism.
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Detailed Description
Although it is not desired to be bound by any theory,
it is presently believed that in the conventional saccharifica-
tion of cellulose to glucose using a cellulase the yields of
glucose are low because the reaction is inhibited by the glucose
formed as well as by cellobiose obtained as a by-product. It is ;~
therefore postulated that the simultaneous presence of an
alcohol-producing microorganism with the cellulose in the
reaction mixture in accordance with the invention results in
the conversion of glucose to alcohol thereby enabling the
enzymatic conversion of cellulose to glucose to proceed further
than would be the case if the glucose were not so converted.
Regardless of any theory, this invention results in the
~acchari~ication reaction proceeding smoothly and in a notable
increase in the overall yield of alcohol from cellulose.
As described above, this invention is characterized
by simultaneously reacting a cellulase and an alcohol-producing
microorganism upon a substrate made up of either cellulose or
a substance composed preponderantly of cellulose.
The cellulosic substrates which are useful as starting
materials for the present invention include purified cellulose,
agriculturally produced materials such as cotton, wood, rice
straw, wheat straw, maize ears (corn cobs) and other substances -
composed preponderantly of cellulose such as newspaper, cor-
rugated paper, rnagazine paper and scrap paper. For these sub-
stances to be used effectively as substrates for the saccharif- ;
ication reaction in the presence of cellulase, it is desirable
to pulverize or disintegrate them. For the hydrolysis of these
cellulosic substrates, use of a commercially available cellulase
will suffice. An enzymatic preparation such as, for example,
Cellulase Onotsuga may be used. A liquid containiny a cellulase,
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E.C.3.2.1 4 namely a culture liquid from a cellulase-producing
microorganism such as, for example, a culture liquid from
Trichoderma viride may also be used.
As the alcohol-producing microorganism to be simul-
taneously used with the cellulase, there can be employed such
microorganisms as, for example, Saccharomyces cerevisiae and
Rhizopus Javanicus which have heretofore been used for the con-
version of glucose into ethanol.
In order for the cellulosic substrate to be simul-
taneously reacted upon by a cellulase and an alcohol-producing
microorganism, an aqueous suspension containing from 1 to 30%
by weight of cellulose or a substance composed predominantly
of cellulose is prepared and thermally sterilized so as to
serve as the substrate, a cellulase (or a cellula3e-containing
liquid) is added to the substrate and at the same time, an
alcohol-producing microorganism cultured in advance is added
thereto so that the reaction will proceed anaerobically at
temperatures of from about 25C. to about 35C.
When the reaction is carried out as described above
as illustrated by a preferred embodiment described below, the
production of alcohol in a yield approximately four times as
high as the described conventional method becomes possible.
The production of alcohol from cellulose by this method has
an additional advantage in that it is carried out in a simple
one-step operation. To be more specific, in contrast with the
conventional method wherein there are involved the two steps
of saccharifying a cellulosic substrate with a cellulase and
separating the saccharification product by filtration and
subsequently subjecting the saccharified liquid filtrate to
alcohol fermentation, the present invention effects the
saccharification reaction of a cellulosic substrate w:ith a
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cellulase and the alcohol fermentation of the glucose formed
by the saccharification reaction simultaneously in one step.
Thus, the operation involved in the present invention is
highly efficient.
Since alcohol is efficiently produced by the present
invention from cellulose or from agriculturally produced
materials or wastes composed preponderantly of cellulose, as
described above, this invention is not only highly advantageous
for the production of alcohol but also for the effective
utilization of cellulosic resources~
Description of_Preferred Embodlments
The present invention will now be described specif-
ically with reference to preferred embodiments which should not
be construed as limiting the spirit and scope of this invention.
Exa~ple 1
A substrate was prepared by suspending 12.5 g of
pulp obtained from wood (having 80% by weight of cellulose
content) in 100 g of water containing in solution 0.25 g
asparagine as a nitrogen source, 0.1 g potassium hydrogen
phosphate ~KH2PO4),0.3 g magnesium sulfate (MgSO~.7H20) and
0.02 g yeast extract. The resultant mixture was adjusted to
pH 4.0 by addition of an acetate buffer and then thermally
sterilized. To the above mixture, there were added 1 g of refined
commercially available cellulase E.C.3.2.1.4 and two platinum
loopfuls of Saccharomyces cerevisiae mycelium directly from an
agar slant thereof, and the mixture was allowed to react at
about 30C. for 96 hours. When the reaction mixture w~s
analyzed at this point, formation of 2 g of alcohol in the
substrate was confirmed. When the reaction was continued
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for a further 96 hours, there were eventually formed 4 g of
alcohol.
By way of comparison, production of alcohol was
carried out by the procedure described below in accordance
with the conventional two-step method. ;
To a substrate suspension of wood pulp in water in
the amounts described above, 1 g of the same refined com-
~ mercially available cellulase was added and the mixture allowed
; to react at 40C. for 96 hours. The amount of reducing sugar
formed in the resultant saccharified liquid was 5 g, 60~ (3 g)
of which was cellobiose and the remaining 2 g was glucose.
When reaction was continued for an additional 96 hours, no
change was observed in the amount of reducing sugar produced.
The saccharified liquid thus produced was mixed with the same
nitrogen source, phosphate and other inorganic salt~ as above
and the resultant mixture was thermally sterilized. Saccharo-
myces cere_isiae was added to the mixture which was then
allowed to ferment. The amount of alcohol formed after com- ~-
pletion of fermentation fell short of 1 g.
In this case, since the glucose content in the
saccharified liquid as the alcohol fermentation substrate was
2 g, the theoretical maximum yield of alcohol would have been
1 g.
ExamE~le 2_
This example illustrates the use in the process of ~
this invention of a cellulase E.C.3.2.1.4 enzyme complex --
elaborated by Trichoderma viride.
In two separate shake flasks Trichoderma viride QM
9414 (ATCC 26,921) was aerobically cultivated at 30 C. for a
period of 6 days. Each flask contained 100 ml of an identical
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conventional nutrient medium containing cellulose powder as
the carbon source. Duriny cultivation the pH was adjusted to
5.4 twice a day. ~t the end of the six day period, the con-
tents of one of the flasks (A) was filtered to obtain a culture
filtrate containing the cellulase enzyme complex; the contents
of the other flask (B) was left unfiltered. Both cellulase
containing materials were then used in the process of this
invention as described below.
Five (5) g each of sterilized cellulose powder (300
mesh, containing 95% by weight of cellulose) were separately
placed aseptically into two sterilized 100 ml flasks. Into
one of these flasks (C) there was added 45 ml of the above
culture filtrate from flask (A). Into the other flask (D)
there was added 45 ml oE the above well-stirred unfi:Ltered
culture broth from flask (B),that is, the entire a~ueous culture
mass without separation of any component thereof (whole culture
broth). Next there was added to each of flasks (C) and (D)
5 ml of a sterilized solution containing the following ingred-
ients:
Asparagine 125 mg
KH2PO4 50 mg
MgSO4-7~l2O 150 mg
Yeast extract 10 mg
Distilled water 5 ml
The resulting mixtures in each flask were adjusted to pH 4.0
as necessary. Thereafter Saccharomyces cerevisiae was added
to each flask, as in Example 1, and the mixture reacted
anaerobically. After reaction for 96 hours, the reaction
mixture in each flask was analyzed. In the mixture from flask
(C) 1.2 g e~hanol was observed; in that from flask (D) 1.4 g.
When the reaction was continued for another 96 hours, the
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mixture from flask (C~ contained 2~0 g ethanol, and that from
flask (D) contained 2.3 g.
Since in the above Example 2, the cellulosic sub-
strate was employed in an amount of 4.75 g cellulose content,
whereas in Example 1 the cellulosic substrate was employed in
an amount of 10 g cellulose content, it will be seen that the
improvements in the amounts of ethanol produced in the respective
examples are of the same order of magnitude. Surprisingly,
however, a comparison of the results obtained using the whole
culture broth [flask (D)] as the cellulase source with the
results obtained using the culture filtrate [flask (C)] as the
cellulase source, shows a marked further improvement in the
amounts of ethanol obtained, 2.3 g as opposed to 2.0 g, an
increase of 15~.
The use o a whole culture broth as the enzyme source
in the enzymatic saccharification of a cellulosic substrate is
described and claimed in copending application Serial Number
572,428, filed April 28, 1975, and assigned to the same assignee
as the present application. The earlier application, incorporated
herein by reference, discloses cultivation of a cellulolytic
microorganism, such as Trichoderma viride, in an a~ueous nutrient
medium in the presence of a cellulosic material in shake flasks
or by submerged culture. The entire aqueous culture mass so
obtained, or an ali~uot thereof, without separation of any
component, is then used as the enzyme source in the saccharif-
ication of cellulose.
It is seen from the foregoing that the present in-
vention achieves production of alcohol in yields remarkably
greater than those obtainable by the conventional two-step
method.
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