CA1055861A - Manufacture of alcohol from cellulosic materials - Google Patents

Manufacture of alcohol from cellulosic materials

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Publication number
CA1055861A
CA1055861A CA235,874A CA235874A CA1055861A CA 1055861 A CA1055861 A CA 1055861A CA 235874 A CA235874 A CA 235874A CA 1055861 A CA1055861 A CA 1055861A
Authority
CA
Canada
Prior art keywords
alcohol
cellulase
glucose
cellulose
cellulosic material
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
CA235,874A
Other languages
French (fr)
Inventor
William F. Gauss
Shuzo Suzuki
Motoyoshi Takagi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BIO RESEARCH CENTER Co
Original Assignee
BIO RESEARCH CENTER Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP49109335A external-priority patent/JPS5839517B2/en
Application filed by BIO RESEARCH CENTER Co filed Critical BIO RESEARCH CENTER Co
Priority claimed from DK111576AA external-priority patent/DK142880B/en
Application granted granted Critical
Publication of CA1055861A publication Critical patent/CA1055861A/en
Expired legal-status Critical Current

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Abstract

MANUFACTURE OF ALCOHOL FROM
CELLULOSIC MATERIALS

Abstract of the Disclosure Alcohol is manufactured from cellulosic materials by a one-step process involving the simultaneous reaction of a cellulosic material, a cellulase and an alcohol-producing microorganism.

Description

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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, : :, .. . . .. ...

1055t~61 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 '~

~055861 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 ,~`;,i ., .
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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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Claims (4)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
1. In the manufacture of alcohol from a cellulosic material, wherein the cellulosic material is enzymatically saccharified to glucose by a separately prepared cellulase and the glucose is fermented in the presence of an alcohol-producing microorganism to obtain alcohol, the improvement which comprises:
simultaneously reacting under anaerobic conditions said cellulosic material, said separately prepared cellulase and said alcohol-producing microorganism, whereby greater yields of alcohol are obtained.
2. The process of Claim 1, wherein the reaction is carried out at a temperature of 25°C. to 35°C.
3. The process of Claim 2, wherein the cellulase is derived from Trichoderma viride and the alcohol-producing microorganism is Saccharomyces cerevisiae.
4. The process of Claim 1, wherein the cellulase is derived from the cultivation of a cellulolytic microorganism in an aqueous nutrient medium in the presence of a cellulosic material and is used in the form of the entire aqueous culture broth obtained in such cultivation.
CA235,874A 1974-09-20 1975-09-19 Manufacture of alcohol from cellulosic materials Expired CA1055861A (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP49109335A JPS5839517B2 (en) 1974-09-20 1974-09-20 Cellulose Scala Alcohol
US05/610,731 US3990944A (en) 1974-09-20 1975-09-08 Manufacture of alcohol from cellulosic materials using plural ferments
DK422375AA DK140764B (en) 1974-09-20 1975-09-19 Process for producing alcohol from cellulosic material.
DK111576AA DK142880B (en) 1975-09-08 1976-03-15 Process for preparing alcohol from cellulose material.

Publications (1)

Publication Number Publication Date
CA1055861A true CA1055861A (en) 1979-06-05

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
CA235,874A Expired CA1055861A (en) 1974-09-20 1975-09-19 Manufacture of alcohol from cellulosic materials

Country Status (1)

Country Link
CA (1) CA1055861A (en)

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