JPS60244294A - Process for semicontinuous production of alcohol in high concentration from cellulose - Google Patents

Abstract

PURPOSE:To produce an alcohol in a high concentration in high yield, by carrying out decomposition of cellulose and fermentation of an alcohol in the first fermenter at the same time, and adding a cellulosic substance to part thereof to carry out saccharifying fermentation. CONSTITUTION:A cellulosic substance, e.g. wood, rice straw, corrugated board paper, cellulolytic enzyme and a microorganism having the ability in alcoholic fermentation are added to the first fermenter to carry out the simultaneous saccharifying fermentation. The resultant fermented KOJI is then partially transferred to the second fermenter, and the cellulosic substance and cellulolytic enzyme are supplied to the first fermenter to carry out the simultaneous saccharifying fermentation. Only the cellulosic substance is added to the second fermenter to carry out the saccharifying fermentation to collect the alcohol from the second fermenter.

Description

DETAILED DESCRIPTION OF THE INVENTION The present HA uses cellulose as a substrate and relates to a method for semi-continuously producing high-concentration alcohol at high yields from cellulose.

Conventionally, the following three methods are known as methods for producing alcohol from cellulosic materials by biological means. [F] A method in which a cellulosic animal cellulose-degrading enzyme is made to act to produce glucose, and then a microorganism capable of producing alcohol (hereinafter referred to as "alcohol-producing microorganism") is made to act on the glucose. . ■ A method using the so-called parallel saccharification and fermentation method (ssy), in which cellulose-degrading enzymes and alcohol-producing microorganisms are simultaneously applied to cellulosic substances, and saccharification and alcohol fermentation are carried out in the same fermenter. ■A method for producing alcohol in one step by allowing cellulose-fermenting bacteria to act on cellulosic substances.

However, method (2) does not involve the saccharification step using cellulose degrading enzymes, so the process is simpler than methods (2) and (2), but the final alcohol concentration is lower! Due to drawbacks such as the slow growth rate of bacteria, it has not yet been put to practical use.

Method (2) allows continuous fermentation using immobilized microorganisms, etc., but product inhibition occurs due to the reaction in which all 1 g ρ course is produced from cellulosic substances, and the cellulose decomposition rate and final decomposition rate are inferior. It is recognized that 'high concentration of glycation'? It has the disadvantage that it is extremely difficult to prepare.

In addition, method (2) has the advantage that saccharification and alcohol fermentation can be performed all at once in the same fermenter, so the reaction system is simplified and the loin saccharification rate and final saccharification rate are increased. In terms of operation, continuous fermentation using immobilized microorganisms is difficult and difficult, and alcohol-producing microorganisms have to be cultured and added in batches.

Under these circumstances, the present inventor conducted intensive research in order to overcome the above-mentioned drawbacks, and in order to overcome the above-mentioned drawbacks, the present inventor conducted the above-mentioned method (1) in which cellulose-degrading elements and alcohol-producing microorganisms are simultaneously applied to cellulosic substances.
In the method, a portion of the fermented mash (V2
~2/3 of the amount) is removed, and the remaining liquid is replenished with the substrate (cellulosic material) corresponding to the amount extracted and all of the cellulolytic enzymes, and the saccharification and fermentation is repeated. Finally, the alcohol-producing microorganisms in the fermented moromi. The holes were found to grow and semi-continuously produce relatively high concentrations of alcohol.

However, with this method, increasing the concentration of cellulosic substances significantly lowers the fluidity of the reaction solution, making semi-continuous operation difficult, and the cellulosic substances suppress the growth of alcohol-producing microorganisms, so replenishment is difficult. The amount of cellulosic material used may be limited.

Therefore, the present inventor conducted further research and used two fermenters, one of which (the first fermenter) carried out simultaneous saccharification and fermentation under conditions suitable for the saccharification of cellulosic substances and the growth of alcohol-producing microorganisms. In the other one (second fermenter),
The present invention has been completed based on the discovery that high-concentration alcohol can be produced semi-continuously by adding only cellulosic substances to the fermented mash obtained in a fermenter and carrying out saccharification and fermentation.

That is, the present invention provides a first fermenter with a cellulosic material,
Simultaneous saccharification and fermentation is performed by adding cellulose-degrading enzymes and microorganisms containing alcoholic fermentation ability, and a portion of the fermented mash is entirely transferred to the second fermenter, and the first fermenter is supplemented with cellulose substances and cellulose-degrading enzymes. Highly concentrated alcohol from cellulose is characterized in that simultaneous saccharification and fermentation are carried out, only cellulosic substances are added to the second fermenter, saccharification and fermentation is carried out, and all alcohol/I/alcohol is collected from the second fermenter. iI/'
This article provides a method for semi-continuously producing fc.

Examples of the cellulosic substance used as a raw material in the present invention include pure cellulose, wood, agricultural and forestry products such as rice straw and bagasse, and substances whose main constituent is cellulose such as newspaper and Dangor paper. It is preferable to use all agricultural and forestry products such as wood, rice straw, bagasse, etc. that undergo physical cleaning or chemical pretreatment such as crushing, crushing, or alkali treatment.

Furthermore, as the cellulolytic enzyme, various commercially available cellulase preparations or enzyme solutions obtained by culturing cellulase-producing bacteria such as Trichoderma reesei can be used.

Furthermore, as alcohol-producing microorganisms, 8acch
aromyces cerevisiol Saccba
rOmycθeuvarum, 8accbaromyc
es forinojensis 6 Which alcohol-producing yeast, Merui is ZymOmOnasmoklili
Alcohol-producing bacteria such as No. 8 can be used, but
In order to efficiently produce alcohol from cellulose,
It is preferable to use all heat-resistant oil or thermophilic oil-producing bacteriaφ.

The reaction conditions in the first fermenter are preferably such that the cellulosic substance is rapidly saccharified by the cellulose-degrading enzyme and the alcohol-producing microorganisms are stably and repeatedly proliferated. Although it varies depending on the temperature, the temperature is 30-40℃
, reaction time 10 to 30 hours, final concentration of fermented mash g V
/V% or less, particularly preferably 5 to 7V/V%.

After fermentation is carried out in this manner, a portion of the fermented mash, preferably 172 to 2/3, is extracted and transferred to a second fermenter.

The remaining liquid in the first fermenter is supplemented with approximately the same amount of cellulosic material as that which was removed, and all of the cellulose decomposing enzymes are added to carry out fermentation. The first fermenter contains yeast extract, ? Nutrient sources such as lipeptone corn staple liquor can also be added.

In the second fermentation tank, a solid quantity of cellulosic material is added in an amount to be saccharified and fermented by the cellulose-degrading enzyme and alcohol-producing microorganism transferred from the first tank, and the entire fermentation is carried out. This fermentation is preferably carried out at 30-40°C for 24-48 hours. In this way, the second fermenter will have an additional 2 to 4 V/
V% alcohol is produced, with a final concentration of approximately 107/V%
A high concentration of alcohol is obtained.

As described above, according to the present invention, when producing alcohol (from cellulosic substances), heptalulose-degrading enzyme and alcohol-producing microorganisms act simultaneously to efficiently produce alcohol from cellulosic substances, and then The semi-continuous operation eliminates the need to produce sake mash at all, and the use of two fermenters makes it possible to produce alcohol with a higher mold content more efficiently than conventional methods. provides a promising industrial method for alcohol production from cellulosic materials as biomass conversion energy.

Next, an example will be given and explained.

Example 1 In this example, a 71v2J capacity fermenter equipped with a stirrer was used.

(In the first fermenter, add commercially available Trichoderma-derived cellulase preparation TAP-6 (Ohno Pharmaceutical Co., Ltd. (copper) 100P), enzyme solution IJ dissolved in 0.05 M citrate buffer, and cellulose. Alkali-treated bagasse (cellulose content 81%) 250? as a sexual substance, yeast extract 3t, malt extract 3F, &lJ Pezoton 5 as a nutritional source
9. Furthermore, yeast (Saccbar) obtained by pre-culture
omyces uvarum ATo(!26602)
Add so that the total is 9108 pieces, and culture temperature 37
The reaction was started at a temperature of 300 rpm and a stirring speed of 300 rpm. 24 hours after the start of preparation, 2 to 3 amounts of the fermented mash was sent to the second fermenter. To the first fermentation tank, 167f of alkali-treated bagasse corresponding to the extracted fermented mash, 66f of nutrients and cellulase preparations were dissolved, and 666m' of large enzyme solution was added, fermentation was carried out in the same manner as above, and this was repeated. I returned it. The results are shown in Table 1. Oshidemeshi and yeast repeatedly multiplied stably, and approximately 77/V of alcohol was stably produced for 25 days.

(i+) Alkali-treated bagasse 6 is added to the fermented mash in the second fermenter.
6 ft was added, and irradiation was carried out at 37° C. and 300 rpm for 48 hours. The results are as shown in Table 1, approximately 10V/
V% alcohol was stably produced.

Table 1 Example 2 An enzyme solution obtained by filtering the culture solution of a cellulase-producing mutant strain of Trichoderma reesei was used as a cellulose-degrading enzyme, and the alkali-treated bagasse was added to the first and second fermenters. Addition amount of 150F (from second time onwards, 150F
00? ) and sat, and the operation was carried out in the same manner as in Example 1, except that the fermentation in the second fermenter was carried out for 24 hours. The results are shown in Table 2), and alcohol of 77/V56 was stably obtained from the second fermenter for 14 days.

Margin below Table 2 that's all

Claims (1)

[Claims] L. Cellulosic substances, cellulolytic enzymes, and microorganisms capable of alcoholic fermentation are added to the first fermenter for simultaneous saccharification and fermentation.
The first fermenter is supplemented with cellulosic substances and cellulolytic enzymes for fermentation, and the second fermenter is supplemented with only cellulosic substances for simultaneous saccharification and fermentation. A method for semi-continuously producing high concentration alcohol from a ce/I/c2-se, which is characterized by collecting I/.