KR101536132B1 - Method for producing microbial inhibitor-free fermentable sugar solutions from lignocellulosic biomass - Google Patents

Abstract

The present invention relates to a method for preparing fermented sugars useful for culturing various industrial fermentation strains from woody biomass, and the method of the present invention is characterized in that the pretreatment of hydrothermal treatment of biomass is carried out at a temperature lower than 100 ° C By washing with an aqueous alkaline solution, it is possible to minimize the amount of acetic acid produced in the subsequent enzymatic saccharification, incidental increase in sugar yield, and no loss of sugar during the washing process.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for producing a biomass raw material fermented sugars having a microbial inhibitor,

The present invention relates to a method for producing a fermented sugar which can be used for microbial fermentation by using woody biomass as a raw material.

Lignocellulosic biomass is composed of a small amount of extractable components that can be extracted with water or an organic solvent and a structural component that is polymerized and does not dissolve in water or organic solvents. Cellulose is one of the structural components of woody biomass, accounting for 25% to 60% of biomass, and is a polymer made by dehydration condensation of glucose. Cellulose in biomass is the main target of acid hydrolysis or enzymatic hydrolysis when fermentable sugar is produced from biomass as raw material but surrounded by other structural components hemicellulose and lignin It is not easily fractionated.

Hemicellulose (15 to 35%), which is one of the structural components of woody biomass, is composed of glucose, galactose, mannose and arabinose bound to the side chain in the Xylen skeleton formed by dehydration condensation of xylose, and glucuronic acid Acids such as acetic acid and acetic acid are linked by an ester bond, so that hydrolysis is relatively easy. On the other hand, lignin (10 to 30%) is a polymer having a complex structure of lignan, which is an aromatic compound, and thus is not easily hydrolyzed by an acid, but exhibits a property of being dissolved in an alkali.

It is common to pretreat biomass biochemically or biologically in order to facilitate the action of fibrin hydrolyzing enzyme when preparing a fermentation sugar by enzymatic saccharification of cellulose by using woody biomass as a raw material. Enzymatic glycosylation of biomass refers to the conversion of cellulase or a mixture of cellulase and hemicellulase into the pretreatment and hydrolysis of cellulose or hemicellulose for a certain period of time to convert it into a sugar solution based on glucose and xylose.

The hydrothermal treatment (autohydrolysis or hydrothermolysis), which is widely applied in the pretreatment of herbaceous biomass, is a simple technology in which biomass and water are sealed in a high-pressure reactor and then reacted at 160 ° C to 220 ° C for a certain period of time. In this hydrothermal pretreatment, hemicellulose in woody biomass is first hydrolyzed and eluted with xylose in the form of xylooligosaccharide in water. Generally, most of the herbaceous and woody biomass has a maximum dissolution rate . Since the concentration of xylose and xylooligosaccharides detected in water is rapidly decreased, the xylose degrades more rapidly than the rate at which hemicellulose in biomass is hydrolyzed and eluted into water, resulting in the production of furfural and other undissociated products This is remarkable. However, the concentration of acetic acid in the pretreatment liquids steadily increases from below 160 ° C to over 220 ° C. Most acetic acid seems to be a hydrolysis product of acetic acid attached to the xylan main chain of hemicellulose by ester bonding.

Generally, hydrothermal treatment is disadvantageous in that the yield of sugar after saccharification of the enzyme is somewhat lower than that of dilute acid pretreatment which mainly uses low concentration of sulfuric acid or hydrochloric acid in the pretreatment. In addition, since hemicellulose, which is left by the incomplete hydrolysis during the pretreatment, is hydrolyzed by the enzyme during the saccharification process, organic acids such as acetic acid are released, so that the saccharide solution contains such an acid.

The fermentation sugar prepared by hydrothermal treatment and enzyme saccharification using lignocellulosic biomass as a raw material can be used as impurities such as furfural, 5-hydroxymethyl-2-furaldehyde (HMF) Phenolic substances and acetic acid. These substances are known to act to inhibit the growth of microorganisms or the production of metabolites, depending on the kind of the fermenting bacteria. It has been reported that, in a study where each substance was added to an artificially prepared sugar solution at different concentrations and the ethanol fermentation broth was tested for growth using mainly yeast, the furfural did not affect ethanol production, whereas HMF had some effect (Jeffrey D. Keating et al., 2006, Biotechnology and Bioengineering , 93 (6), 1196-1206). Phenolic compounds significantly inhibited the growth of yeast, and acetic acid and furfural were reported to have greater inhibitory potency compared to those used alone.

In general, most industrial microorganisms, such as E. coli and Clostridium acetobutylicum , can significantly reduce growth or metabolite production by some impurities in the sugar solution prepared from biomass. Therefore, in order to detoxify various microbial inhibitors contained in sugar solutions prepared from woody biomass, various studies such as excessive lime over-lime and lignin peroxidase-based polymerization Has been performed. Many other researchers have applied various chromatographies using adsorption and partitioning to study the removal of these substances (Villarreal, MLM et al., 2006, Enzyme and Microbial Technology , 40, 17-24). In particular, a technique using adsorption chromatography to remove the acetic acid contained in the sugar solution (Hee-Geun Nam, Sungyong Mun, 2012, Process Biochemistry , 47, 725-734; S. Ranil Wickramasinghe, David L. Grzenia, Desalination , 234, 144-151), a technique using a membrane (David L. Grzenia et al., 2012, Journal of Membrane Science , 415-415, 75-84; Sung-Jae Kim et al., 2012, Process Biochemistry , 2057] and the like are used.

Excess lime utilization technology for detoxification of sugar solutions is widely used because it is the least expensive. This technique dissolves lime by adding lime until the pH of the sugar solution reaches 10, and then it is heated for a certain time at 60 ° C or below. As a result, various impurities such as furfural, HMF, and protein precipitate and can be removed by filtration or precipitation. However, it is common that the process involves loss of hemicellulose sugar.

On the other hand, in order to minimize the amount of the microbial inhibitor that can be contained in the sugar solution, a method of washing the pretreatment product before the glycation of the enzyme is also used. Inbicon, which operates a pilot plant-scale bio-alcohol manufacturing plant, pretreats the biomass by hydrothermal treatment, removes a large amount of microbial inhibitor-containing liquid through solid-liquid separation and then rinses the pretreated solid matter with water (Jan Larsen et al., 2012, Biomass and Bioenergy , 46, 36-45). However, since the microbial inhibitors such as acetic acid and phenolic compounds are released from the pretreater as the enzymatic saccharification and the ethanol fermentation proceeds, further research and development will be required in order to apply this method to the production of fermentation sugars suitable for culture of other fermenting bacteria .

Another method of removing acetic acid from biomass is to add sodium hydroxide to the biomass and heat it to hydrolyze and elute the acetic acid and then remove it by solid-liquid separation and water washing (Cho, DH et al., 2010, Bioresource Technology , 10, 4947-4951). This method is a method that is performed before the dilute acid pretreatment of biomass, and is a method that can be used because it adds an acid to act as a catalyst for pretreatment. That is, the hydrolysis reaction of the hemicellulose main chain by the acid catalyst can not be expected in the biomass obtained by hydrolyzing and removing the acetic acid possessed by hemicellulose by treating the strong alkali in advance, so that if the acid is not artificially added, The pretreatment effect can be expected only by heating. This pretreatment technique is known as pH-controlled liquid hot water pretreatment.

[Non-Patent Document 1] Jeffrey D. Keating et al., 2006, Biotechnology and Bioengineering , 93 (6), 1196-1206

[Non-Patent Document 2] Villarreal, MLM et al., 2006, Enzyme and Microbial Technology , 40, 17-24.

[Non-Patent Document 3] Hee-Geun Nam, Sungyong Mun, 2012, Process Biochemistry , 47, 725-734.

[Non-Patent Document 4] S. Ranil Wickramasinghe, David L. Grzenia, 2008, Desalination , 234, 144-151.

[Non-Patent Document 5] David L. Grzenia et al., 2012, Journal of Membrane Science , 415-415, 75-84.

[Non-Patent Document 6] Sung-Jae Kim et al., 2012, Process Biochemistry , 47, 2051-2057.

[Non-Patent Document 7] Jan Larsen et al., 2012, Biomass and Bioenergy , 46, 36-45.

[Non-Patent Document 8] Cho, DH et al., 2010, Bioresource Technology , 10, 4947-4951.

Accordingly, it is an object of the present invention to provide a biomass pretreatment cleaning technique capable of removing acetic acid from a substance capable of inhibiting the growth of an industrial fermenting microorganism from a pretreatment product of biomass, and maximizing sugar yield by the subsequent enzymatic saccharification will be.

In order to achieve the above object,

1) adding water to the woody biomass crude pulverized material or powder to subject it to hydrothermal treatment, and then subjecting the obtained pretreated material to solid-liquid separation to obtain a solid fraction;

2) adding a hot aqueous alkaline solution to the solid content obtained in step 1) from room temperature to 100 ° C or lower, and then dehydrating the solid content to recover the solid content; And

3) Step of treating the solid fraction obtained in step 2) with cellulose hydrolyzing enzyme to saccharify the enzyme

Wherein the acetic acid is removed from the woody biomass.

The method of the present invention can remove the acetic acid group remaining unreacted in the pretreated solid matter before hydrolysis of the biomass before the saccharification of the enzyme to thereby significantly lower the concentration of acetic acid contained in the saccharide solution as a result of enzyme saccharification, , Especially fermenting sugars capable of culturing microorganisms capable of inhibiting growth by acetic acid can be produced. In addition, the pretreatment effect is enhanced during the removal of the nitric acid group, so that the sugar yield obtained by the enzymatic saccharification is greatly increased, and the impurity content in the sugar solution can be significantly lowered by the washing effect of the pretreatment.

The method for producing a fermented sugar according to the present invention comprises the steps of (i) hydrothermal pretreatment and solid-liquid separation of biomass, (ii) alkaline water washing step, and (iii) enzyme saccharification step, The content of the inhibiting substance is minimized, and as a result, the fermenting sugar can be used throughout fermentation of various industrial microorganisms.

In the present invention, the term "fermented sugar" means sugar which can be used for fermentation of microorganisms, and is distinguished from "sugar solution"

In the method for producing a fermentation sugar in which most of microbial inhibitors such as acetic acid according to the present invention are largely removed, the first step is to prepare a solid content containing a minimum amount of liquid substance by subjecting the pretreatment obtained by hydrothermal treatment of woody biomass to solid- (Hydrothermal treatment and solid-liquid separation step).

The more acidic pre-treatment liquid is removed as much as possible at this stage, the smaller the amount of chemicals required in the subsequent washing step with the aqueous alkaline solution.

The hydrothermal treatment step may be performed according to a method well known in the art, for example, a woody biomass can be hydrothermally pretreated at 160 to 230 ° C for 0.001 to 60 minutes. For separating and removing the acidic liquid product from the pretreated product obtained through the above process, all the solid-liquid separation methods commonly used in the art can be used. Examples thereof include centrifugal separation, rotary dehydration, suction filtration, .

In the second step of the method of the present invention, an alkali aqueous solution is added to the pretreated solid component obtained in the first step to mix the acetic acid in the liquid phase remaining in the pretreated solid component and the acetic acid in an unreacted state in the solid component (Alkaline water washing step) of removing liquid matter containing acetic acid by solid-liquid separation.

The main purpose of the present invention is to separate and remove the remaining acetic acid groups without hydrolysis even after the pretreatment of the biomass, by washing the pretreated solid matter with the alkaline aqueous solution. The alkaline aqueous solution which can be used herein may be selected from the group consisting of a base dissolved or suspended in water, and the base may be selected from the group consisting of calcium hydroxide, potassium hydroxide, sodium hydroxide and a mixture thereof. The biomass pretreatment product , It can be used without any particular limitation as long as it is prepared so as to exhibit alkalinity of pH 11 or more.

In order to remove acetic acid remaining in the pretreated water at this stage, it is preferable to firstly dissolve or suspend the base in water to prepare a highly concentrated aqueous alkaline solution or suspension, and then add the aqueous alkaline solution or suspension to the pretreated solid content. And reaction time are closely related to each other, so that they can be controlled within a certain range.

The alkaline aqueous solution to be added to the pretreatment solid content is prepared by neutralizing acetic acid which is already liberated in the solid component so as to maintain reactivity with acetic acid chemically bonded to hemicellulose, and at the initial concentration at which the pH can be maintained above 11, Is preferably such that the pH is 11.5 to 14 when mixed with water. As the initial concentration of the alkaline aqueous solution is high and the pH is closer to 14, the removal efficiency of the nitric acid group in the pretreated solid is high. However, in order to maximize the enzyme activity in the enzymatic saccharification step after the removal of acetic acid, the amount of water to be used for adjusting the acidity to weak acidity or the amount of acid to be used as the neutralizing agent is increased. Therefore, Is effective in reducing the consumption of water and chemicals.

When the pretreatment solids are washed with an aqueous alkaline solution and then the acetic acid is removed, the acetic acid removal rate increases as the temperature of the reaction system increases. In addition, when the temperature of the reaction system is raised, the concentration of the alkali aqueous solution used can be lowered and the washing time can be reduced. For example, when 1 g of sunflower stem is pretreated by hydrothermal treatment for 5 minutes at 190 ° C for 2 minutes, acetic acid is extracted and removed. In the case of potassium hydroxide aqueous solution of the same saturation concentration, extraction efficiency at 80 ° C Doubled. However, when the temperature of the aqueous alkaline solution exceeds 100 ° C, a special apparatus is required to maintain the pressure. When the pH of the reaction system is 11 or higher at room temperature, the elimination reaction of acetic acid occurs. . In order to reduce reagent consumption and shorten the cleaning time, it is more preferable to select a temperature within this range, for example, 80 ° C to 95 ° C, and when it is treated at this temperature, It is 60 minutes.

The amount of the alkaline aqueous solution to be added to the pretreatment solid content is not particularly limited as long as it can be uniformly mixed with the pretreatment solid content. However, considering the efficiency and cost of removing the already formed acetic acid by solid- It is desirable to adjust the amount. For example, the amount of the alkaline aqueous solution to be added is such that the pretreated product is mixed with an aqueous alkaline solution and reacted, and the volume (about 50% moisture content or more) of the liquid phase of the reaction system can be partially removed by solid-liquid separation such as screw press or centrifugal filtration To an amount corresponding to 20 times the weight of the pretreated solid (for example, an amount of 1 to 20 times based on the total weight of the pretreated solid). Thereafter, if necessary, a washing process using water may be added to further remove the free acetic acid and the alkali aqueous solution from the pretreatment product, but this can be adjusted depending on the usage amount of the aqueous alkaline solution.

In the method of the present invention, the enzyme saccharification step as the third step is an enzyme saccharification step (enzyme saccharification step) by adding a fibrin hydrolyzing enzyme to the pretreated solid matter from which the acetic acid obtained in the second step is largely removed.

This step is not largely different from the method commonly known for converting cellulose and hemicellulose contained in the pretreated biomass into monosaccharides such as glucose and xylose. According to one embodiment of the present invention, an acid is added to a pretreated solid component from which most of the acetic acid has been removed, the pH is adjusted to 4.5 to 5.5, and then a fibrinolytic enzyme is added. The fibrin hydrolyzing enzyme may be selected from the group consisting of Cellic CTec2, Cellic HTec2, and mixtures thereof. After the enzyme is treated, the enzyme is saccharified by stirring at 50 ± 1 ° C and pH 5.0 ± 0.1 for 24 to 72 hours .

The enzyme saccharification material obtained through the enzyme saccharification step may be subjected to centrifugation or the like to produce a fermentation sugar from which acetic acid has been removed.

The fermentation sugar prepared according to the method of the present invention is characterized in that the acetic acid content is reduced to 1/2 or less as compared to the sugar solution prepared without removing acetic acid after the pretreatment. According to one embodiment of the present invention, the yield of acetic acid produced from sunflower stem is reduced from 0.51 g to 0.09 g per 100 g of biomass and from 0.25 g to 0.05 g, / 5 or less (Examples 1 and 2).

The fermentation sugars prepared by the method of the present invention can be produced by pretreatment of biomass and inorganic salts derived from biomass such as acetic acid, furfural, HMF, lignin-decomposing phenolic compounds which can be produced in enzyme saccharification, It is possible to produce E. coli and yeast which are used as universal strains for the production of various chemicals and biofuels, Clostridium acetobutylicum and Clostridium beans which are used for the production of butanol and acetone, rinki (Clostridium beijerinckii), Lactococcus lactis (Lactococcus lactis) that primarily produce lactic acid and Lactobacillus species (Lactobacillus sp.), Corinne tumefaciens glutamicum (Corynebacterium glutamicum) which is mainly used in the production of amino acids, the production of ethanol ( Zymomonas mobilis ), which is commonly used for fermentation of many industrial microorganisms such as Zymomonas mobilis . In particular, it is useful for culturing strains which can respond very sensitively to the concentration of acetic acid at the time of growth. Such strains include yeast ( Saccharomyces cerevisiae , Saccharomyces cerevisiae ).

Accordingly, the present invention provides a method for fermenting a microorganism using a fermentation sugar in which acetic acid is removed, which is produced according to the method of the present invention.

The woody biomass that can be used as a raw material in the method of the present invention includes both herbaceous biomass and woody biomass. Examples of herbaceous biomass include trunks of oil palm, frond, empty fruit bunch, sunflower stem, straw straw, straw straw, corn stalks, reeds, Examples of woody biomass include, but are not limited to, lily trees, willow trees, acacia, eucalyptus, spruce, and the like.

The fermenting sugar according to the present invention can be prepared by hydrolyzing a fermentation sugar by a hydrolysis technique using an acid catalyst such as a hydrothermal treatment to minimize the content of acetic acid in the fermented sugar finally produced, It is useful for preparing a fermentation sugar solution using woody biomass as a raw material since the growth inhibiting substance can be removed.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: Preparation of fermentation sugars made of sunflower stem

Weighing 120 g of the sunflower stem powder, which is known to be a constituent, is weighed into a building and placed in a jar of Parr reactor (Parr Instrument Co. Ltd., USA) for 2 L and distilled water is added to make the weight of the contents 1,500 g Respectively. The reactor was heated and subjected to hydrothermal treatment at 190 ° C for 5 minutes. After the reaction was completed, the reactor bottle was immersed in running water, cooled rapidly, and the contents were transferred to a lightweight bag. The product was dehydrated in a dehydrator (Kyo Soon Lee, Hanil Electric Co., Korea) for 30 minutes. The dewatered solid was placed in a plastic bag, sealed and placed in an autoclave set at a temperature of 90 DEG C and a time of 30 minutes. 2.04 g of calcium hydroxide was added to a beaker (for 5 L), 1,200 ml of distilled water was added, and the mixture was heated in a constant temperature water bath (90 ° C, 10 L, Korea K.K.) and heated. Before the autoclave was settled, the pretreatment was taken out, and the opening of the light gray cylinder was opened. The contents were added to a beaker containing an aqueous solution of calcium hydroxide (pH 12.7) in a constant temperature water bath and stirred for 3 minutes. The contents were all taken out, transferred to a light wood bag, and dehydrated for 5 minutes to remove the aqueous calcium hydroxide solution. The dewatered pretreatment product was immersed in a 5 L beaker containing 1.2 L of distilled water and immersed in water, immersed in water for 1 hour, and dehydrated three times to remove calcium hydroxide. The solids remaining in the gill bag were transferred to a fermenter of a 5 L fermenter (BioTron, Korea) and non-ionized water was added to give a total amount of 740 g. Then, the fermenter was sealed, placed in an autoclave, and sterilized at 121 DEG C for 60 minutes. 12 ml of Cellic CTec2 as a cellulose hydrolyzing enzyme was dissolved in 108 ml of ultrapure water and filtered through a filter system (Corning product, USA) equipped with a 0.22 占 퐉 membrane filter, and then added to the fermentation tank in a clean bench. The fermenter was hydrolyzed for 72 hours with stirring at 150 rpm while maintaining the temperature at 50 1 C and pH 5.0 0.1. A portion of the hydrolyzate was centrifuged to obtain a supernatant. The supernatant was then analyzed by Waters HPLC equipped with a BioRad Aminex HPX-87H column and a refractive index detector, and the yield of glucose and other sugars and acetic acid was calculated Are shown in Table 1 below.

Example 2: Preparation of fermentation sugars made from reed

Weighing 150 g of Korean native reed powder, which is known to be a constituent of the composition, is put into a jar of Parr reactor (Parr Instrument Co. Ltd., USA) for 2 L and distilled water is added to make the content weight 1,500 g Respectively. The reactor was heated and subjected to hydrothermal treatment at 200 ° C for 10 minutes. After the reaction was completed, the reactor bottle was immersed in running water, cooled rapidly, and the contents were transferred to a lightweight bag. The product was dehydrated in a dehydrator (Kyo Soon Lee, Hanil Electric Co., Korea) for 30 minutes. The dewatered solid was placed in a plastic bag, sealed and placed in an autoclave set at a temperature of 90 DEG C and a time of 30 minutes. 5.56 g of calcium hydroxide was added to a constant temperature water bath (10 L, Korea Kogyo Co., Ltd., Korea), 900 ml of distilled water was added, and the mixture was heated to 90 ° C. Before the autoclave was settled, the pretreatment was taken out, and the opening of the light gray cylinder was opened. The contents were added to a beaker containing an aqueous solution of calcium hydroxide (pH 12.7) in a constant temperature water bath and stirred for 3 minutes. The contents were all taken out, transferred to a light wood bag, and dehydrated for 5 minutes to remove the aqueous calcium hydroxide solution. The dewatered pretreatment product was immersed in a 5 L beaker containing 1.2 L of distilled water and immersed in water, immersed in water for 1 hour, and dehydrated three times to remove calcium hydroxide. The solids remaining in the gill bag were transferred to a fermenter of 5 L fermenter (BioTron, Korea) and non-ionized water was added to make the total amount 950 g. Then, the fermenter was sealed, placed in an autoclave, and sterilized at 121 DEG C for 60 minutes. 13.5 ml of Cellic CTec2 and 1.5 ml of Cellic HTec2 were dissolved in 135 ml of ultrapure water and filtered through a filter system (Corning product, USA) equipped with a 0.22 ㎛ membrane filter, and then added to the fermenter in a clean bench. The fermenter was hydrolyzed for 72 hours with stirring at 150 rpm while maintaining the temperature at 50 1 C and pH 5.0 0.1. A portion of the hydrolyzate was centrifuged to obtain a supernatant. The supernatant was then analyzed with a BioRad Aminex HPX-87H column and a Waters HPLC equipped with a refractive index meter, and the yields of glucose and other sugars and acetic acid were calculated and shown in Table 1 .

Comparative Example 1: A solution of sunflower stem raw material prepared by washing hydrothermal treatment

120 g of the sunflower stem powder used in Example 1 was weighed into a building and put in a bottle of a 2 L reactor (Parr reactor; Parr Instrument Co. Ltd., USA), and distilled water was added to make the content weight 1,500 g. The mixture was then subjected to hydrothermal treatment at 190 ° C for 5 minutes. After the reaction was completed, the reactor bottle was immersed in running water, cooled rapidly, and the contents were transferred to a lightweight bag. The product was dehydrated in a dehydrator (Kyo Soon Lee, Hanil Electric Co., Korea) for 30 minutes. The dehydrated solid was immersed in 20 L of boiling water and dehydrated by a dehydrator. It was then dipped in 20 L of warm water and dehydrated again. The solids remaining in the gill bag were transferred to a fermenter of a 5 L fermenter (BioTron, Korea) and non-ionized water was added to give a total amount of 740 g. Then, the fermenter was sealed, placed in an autoclave, and sterilized at 121 DEG C for 60 minutes. 24 ml of Cellic CTec2 as a cellulose hydrolyzing enzyme was dissolved in 96 ml of ultrapure water, filtered through a filter system (Corning product, USA) equipped with a 0.22 占 퐉 membrane filter, and then added to the fermentation tank in a clean bench. The fermenter was hydrolyzed for 72 hours with stirring at 150 rpm while maintaining the temperature at 50 1 C and pH 5.0 0.1. A portion of the hydrolyzate was centrifuged to obtain a supernatant. The supernatant was then analyzed with a BioRad Aminex HPX-87H column and a Waters HPLC equipped with a refractive index meter, and the yields of glucose and other sugars and acetic acid were calculated and shown in Table 1 .

Comparative Example 2: Preparation of a fermentation sugar having a reed as a raw material

Weighing 150 g of Korean native reed powder, which is known to be a constituent of the composition, is put into a jar of Parr reactor (Parr Instrument Co. Ltd., USA) for 2 L and distilled water is added to make the content weight 1,500 g Respectively. The reactor was heated and subjected to hydrothermal treatment at 200 ° C for 10 minutes. After the reaction was completed, the reactor bottle was immersed in running water, cooled rapidly, and the contents were transferred to a lightweight bag. The product was dehydrated in a dehydrator (Kyo Soon Lee, Hanil Electric Co., Korea) for 30 minutes. The dehydrated solid was immersed in 20 L of boiling water and dehydrated by a dehydrator. It was then dipped in 20 L of warm water and dehydrated again. The solids remaining in the gill bag were transferred to a fermenter of 5 L fermenter (BioTron, Korea) and non-ionized water was added to make the total amount 950 g. Then, the fermenter was sealed, placed in an autoclave, and sterilized at 121 DEG C for 60 minutes. 13.5 ml of Cellic CTec2 and 1.5 ml of Cellic HTec2 were dissolved in 135 ml of ultrapure water and filtered through a filter system (Corning product, USA) equipped with a 0.22 ㎛ membrane filter, and then added to the fermenter in a clean bench. The fermenter was hydrolyzed for 72 hours with stirring at 150 rpm while maintaining the temperature at 50 1 C and pH 5.0 0.1. A portion of the hydrolyzate was centrifuged to obtain a supernatant. The supernatant was then analyzed with a BioRad Aminex HPX-87H column and a Waters HPLC equipped with a refractive index meter, and the yields of glucose and other sugars and acetic acid were calculated and shown in Table 1 .

Ingredients Yield (g / 100g biomass) Sunflower stem Reed Example 1 Comparative Example 1 Example 2 Comparative Example 2 glucose 27.0 24.2 34.8 34.2 Other party 4.5 4.5 1.7 1.6 Acetic acid 0.09 0.51 0.05 0.25 HMF 0.0 0.00 0.0 0.0 Furfural 0.0 0.00 0.0 0.0

As shown in Table 1, the acetic acid yield in terms of the saccharide solution prepared by the method of removing acetic acid by washing the hydrothermal pretreatment product of the present invention with an aqueous alkaline solution was 100 g of biomass in Example 1 in which the sunflower stem was a raw material 0.09 g per 1/5 of 0.51 g of Comparative Example 1 obtained by simply washing with boiling water and enzymatic saccharification. The yield of acetic acid per 100 g of biomass was 0.05 g, while that of Comparative Example 2 was 0.25 g, which was one fifth of the acetic acid yield of Comparative Example 2. Therefore, the method of the present invention is very useful for the production of a fermentation sugar for microbial fermentation, since the concentration of acetic acid in the sugar solution can be greatly reduced by washing the biomass hydrothermal treatment with an alkaline aqueous solution at 100 ° C or less.

In addition, the method of the present invention has an effect of improving sugar yield as compared with the hydrothermal washing method in addition to the removal of acetic acid, and it can be confirmed that there is no sugar loss during the acetic acid removal process.

Claims (6)

1) adding water to the woody biomass crude pulverized product or powder and hydrothermally pretreating the product at 160 to 230 ° C for 0.001 to 60 minutes, and then subjecting the obtained pretreated product to solid-liquid separation to obtain a solid fraction;
2) The pH of the mixture is adjusted to 11 or more by adding an aqueous solution of warmed calcium hydroxide, potassium hydroxide, sodium hydroxide or a mixture thereof to the solid content obtained in the step 1) at a temperature ranging from room temperature to 100 ° C or less. After dehydration for 0.001 to 60 minutes, Recovering; And
3) Step of treating the solid fraction obtained in step 2) with cellulose hydrolyzing enzyme to saccharify the enzyme
Wherein the acetic acid is removed from the woody biomass. delete delete The method according to claim 1,
Characterized in that an aqueous solution of said calcium hydroxide, potassium hydroxide, sodium hydroxide or mixtures thereof is used in an amount of 1 to 20 times based on the total weight of solids obtained in step 1). The method according to claim 1,
Wherein the fermentation sugar contains acetic acid in an amount of 1/2 or less of the acetic acid content of the sugar solution prepared without removing acetic acid after the pretreatment. A method for fermenting a microorganism by using a fermentation sugar from which acetic acid has been removed, which is produced according to the method of any one of claims 1, 4 and 5.