KR101216829B1 - Method of manufacturing glucose comprising multistage process - Google Patents

Abstract

The present invention relates to a method for preparing glucose comprising a multi-step process. The glucose production method including the multi-step process according to the present invention improves the saccharification efficiency of the saccharification process by improving the saccharification process of the biomass by vapor decompression process, alkali compound treatment process and multi-stage treatment of the chlorine-based compound to improve the productivity and production efficiency of glucose from the biomass. Can be improved.

Description

Glucose manufacturing method including multi-step process {METHOD OF MANUFACTURING GLUCOSE COMPRISING MULTISTAGE PROCESS}

The present invention relates to a method for preparing glucose comprising a multi-step process.

As of 1998, domestic biomass-based resources amounted to about 12 million tons per year, and the current technology uses 2.3 million tons, which is about 20% of the amount. In addition, the remaining resources of agricultural by-products amount to 4 million tons / year, and the supply potential reaches 1,050,000 tons / year, which is equivalent to 367 billion won in cash. As of the end of 2002, Korea's forest resources amounted to 461,635,000 tons, and there is an urgent need for the use of waste resources that cannot be used as waste remnants and materials produced by the forest cultivation project in the forest or thinning and lumbering. . Therefore, the development of a specific manufacturing process for producing high-purity cellulose from biomass and producing glucose based on the same is required at a low cost in Korea with few resources remaining.

It is an object of the present invention to provide a method for producing glucose comprising a multi-step process.

In order to achieve the above object, the present invention comprises the steps of steam explosion treatment of the biomass raw material; Treating the vapor depleted biomass feedstock with an alkali compound; Treating the biomass raw material treated with the alkali compound with a chlorine compound; And it provides a glucose production method comprising the step of saccharifying the biomass raw material treated with the chlorine-based compound.

The biomass includes bio / microbial organisms including plants generated by photosynthesis of plants and microorganisms that receive solar energy, and animals living on them. Specifically, by-products, wastes, and food wastes from agriculture including various animals and plants , Bio-based industrial wastes, crops grown for biofuel production, and the like. For example, the biomass may be sugars such as sugar cane and sugar beet; Starches such as corn, wheat, rice, cassava, and tapioca; And lignocellulose such as wood waste, agricultural residues such as rice straw or corn stalks. As an example, the biomass may be wood waste such as oak wood, among lignocellulosic systems.

The steam explosion treatment is a process of pretreatment of biomass using high pressure steam. That is, after steaming the biomass in a high pressure vessel containing a high temperature steam for a predetermined time, it may be a method of immediately opening the valve of the high pressure vessel to induce the structure of the molecules in the biomass such as popcorn to open instantly. .

According to one embodiment of the invention, the step of steam decay treatment may be carried out at 213 to 235 ℃ under a pressure of 20 to 30 kg / ㎠. If the pressure is less than 20kg / ㎠ may not be sufficient steam decay treatment, if it exceeds 30kg / ㎠, the efficiency of steam decay treatment due to the increase in pressure can be reduced economically 20 to 30kg / ㎠ Can be carried out under pressure. If the temperature is less than 213 ℃ steam decay treatment may not be sufficient, if it exceeds 235 ℃, the efficiency of steam decay treatment with increasing temperature can be reduced economically can be carried out at 213 to 235 ℃ have. The steam decay treatment time may be about 3 to 10 minutes. For example, the steam decay treatment may be performed at 225 ° C. for 5 minutes at a water vapor pressure of 25 kg / cm 2.

According to one embodiment of the invention, the step of treating with the alkali compound can be treated with an aqueous alkali solution of 1 to 3% by weight concentration.

In the case of treating the alkali compound of less than 0.5% by weight concentration in the step of treating the alkali compound, the effect of the alkaline compound treatment may not be sufficient, when treating the alkali compound in excess of 5% by weight concentration, alkaline compound treatment It is not desirable in terms of environmental pollution or drug recovery, recycling, and outbreaks, as the effect by is not increased. The concentration of the aqueous alkali compound solution in the step of treating the alkali compound may be 0.5 to 5% by weight. Preferably the concentration of the aqueous alkali compound solution may be 1 to 3% by weight.

The alkali compound may include sodium hydroxide (NaOH), potassium hydroxide (KOH), lithium hydroxide (LiOH), ammonium hydroxide (NH ₄ OH) or calcium hydroxide (CaOH) and the like. Preferably, the alkali compound may be sodium hydroxide (NaOH) or potassium hydroxide (KOH). More preferably, the alkali compound may be potassium hydroxide (KOH).

According to one embodiment of the invention, the step of treating with the chlorine-based compound may be treated with an aqueous solution of chlorine-based compound of 1 to 3% by weight.

When treating the chlorine compound of less than 0.5% by weight concentration in the step of treating the chlorine compound, the effect of the chlorine compound treatment may not be sufficient, when treating the chlorine compound in excess of 5% by weight concentration, the chlorine compound treatment It is not desirable in terms of environmental pollution or drug recovery, recycling, and outbreaks, as the effect by is not increased. The concentration of the aqueous solution of chlorine compounds in the step of treating the chlorine compound may be 0.5 to 5% by weight. Preferably the concentration of the aqueous solution of the chlorine compound may be 1 to 3% by weight.

The chlorine-based compound may include sodium hypochlorite (NaClO), sodium chlorite (NaClO ₂ ) or calcium hypochlorite (Ca (ClO) ₂ ) and the like. Preferably the chlorine-based compound may be sodium hypochlorite (NaClO) or sodium chlorite (NaClO ₂ ). More preferably, the chlorine compound may be sodium chlorite (NaClO ₂ ).

According to one embodiment of the present invention, the alkali compound is selected from the group consisting of sodium hydroxide (NaOH), potassium hydroxide (KOH), lithium hydroxide (LiOH), ammonium hydroxide (NH ₄ OH) and calcium hydroxide (CaOH) At least one, and the chlorine-based compound may be any one or more selected from the group consisting of sodium hypochlorite (NaClO), sodium chlorite (NaClO ₂ ) and calcium hypochlorite (Ca (ClO) ₂ ).

According to an embodiment of the present invention, the alkali compound may be potassium hydroxide (KOH), and the chlorine compound may be sodium chlorite (NaClO ₂ ).

The step of saccharification may be carried out by acid saccharification according to a conventional method, but more preferably by enzyme saccharification.

According to one embodiment of the present invention, the step of saccharification may be performed by enzyme saccharification.

"Saccharification" is the process by which the cellulose component is converted into glucose by the action of an enzyme, the process by which cellulase is adsorbed on the reaction surface of the cellulose to convert the cellulose into cellobiose and the resulting cellobiose is β- It can be divided into the process of conversion to glucose by the enzymatic reaction of glucosidase (β-glucosidase).

The enzyme may include cellulase, β-glucosidase, hemicellulase and xylanase. For example, the enzyme may include Celluclast 1.5L (Novozymes, Denmark), Viscozyme L (Novozymes, Denmark) and the like.

The enzyme saccharification process temperature may be 40 to 80 ℃. If the saccharification temperature is less than 40 ℃ may be slow saccharification rate, if the enzyme saccharification process temperature exceeds 80 ℃ may cause enzyme denaturation. Preferably, the enzyme saccharification process temperature may be 45 to 55 ℃.

In order to achieve the above object, the present invention comprises the steps of steam decay treatment of biomass raw material at 213 to 235 ℃ under a pressure of 20 to 30kg / ㎠; Treating a potassium hydroxide (KOH) aqueous solution at a concentration of 1 to 3% by weight to the vapor-expanded biomass raw material; Treating a sodium chlorite (NaClO ₂ ) aqueous solution at a concentration of 1 to 3% by weight to the biomass raw material treated with the potassium hydroxide (KOH) aqueous solution; And saccharifying the biomass raw material treated with the aqueous sodium chlorite (NaClO ₂ ) solution using an enzyme saccharification process.

The glucose production method including the multi-step process according to the present invention improves the saccharification efficiency of the saccharification process by improving the glycosylation efficiency of the biomass by improving the productivity and manufacturing efficiency of the glucose from biomass by multi-stage treatment of steam decay process, alkali compound and chlorine compound. Can be. In addition, it can be usefully used for the process of producing bio ethanol and the like using the glucose.

1 is a photograph showing a raw material (raw material) obtained from a biomass raw material, (a) and (b) shows the oak chips and oak mill grinding.
Figure 2 shows the results of HPLC chromatography of the monosaccharide standard, wherein (a), (b), (c), (d) and (e) are glucose, xylose and galactose, respectively. ), Arabinose and mannose. Here RT represents the retention time (minutes) of each monosaccharide.
3 shows a photograph of a biomass raw material (b) subjected to high pressure steam pretreatment using the high pressure steam pretreatment device (a) and the high pressure steam pretreatment device (a).
Figure 4 is a graph showing the glucose conversion rate according to the chemical treatment of the high-pressure steam pretreated biomass raw material.

Hereinafter, the present invention will be described in detail by way of examples, but the following examples are merely illustrative of the present invention, and the content of the present invention is not limited thereto.

In Examples and Experimental Examples of the present invention, chemical treatment is used to distinguish from the expression "physical treatment" or "biological treatment", and means using a chemical reaction. However, the present invention does not exclude other physical means or methods or biological means or methods in the chemical treatment, so long as the chemical reaction is the main one.

Example

1.Steam explosion pretreatment of biomass raw materials

The biomass raw material used in this example was provided by Yulim Hi-Tech. The raw material received from the company was provided as a residue after high pressure steam treatment. High pressure steam pretreatment conditions were treated for 5 minutes at 225 ° C with a steam pressure of 25 kg / cm ² .

2. Chemical treatment of high pressure steam pretreated biomass feedstock: multistage treatment (1 wt% aqueous KOH solution + 1 wt% NaClO) ₂  Aqueous solution (Example 1), 1 wt% KOH aqueous solution + 3 wt% NaClO ₂ Aqueous solution (Example 2))

The high-pressure steam pretreated biomass raw material was immersed in a 1 wt% KOH aqueous solution at a ratio of solid: liquid (1:20) and stirred at 100 rpm for 3 hours in a shaking incubator set at 30 ° C. After stirring, the mixture was filtered and separated into a residue and a liquid. The residue was washed with 1% glacial acetic acid and distilled water until the pH was 7, and the residue was again dissolved in 1 wt% aqueous NaClO ₂ solution and 3 wt% NaClO ₂ aqueous solution, respectively. 20) It was immersed in a ratio and stirred for 3 hours at 100 rpm in a shake incubator set to 30 ℃. After stirring, the mixture was filtered and separated into a residue and a liquid. The residue was washed with acetone and distilled water and dried at 105 ° C.

3. Enzymatic Glycation

Chemical treatment of biomass raw material with high pressure steam pretreatment (1 wt% aqueous KOH solution + 1 wt% aqueous NaClO ₂ solution ( Example 1), 1 wt% aqueous KOH solution + 3 wt% NaClO ₂₎ Aqueous solution ( Example 2)) and enzyme saccharification.

1 g of the pretreated sample and 50 ml (pH 4.8) of 0.1M sodium citrate buffer were added to the Erlenmeyer flask and heated to 50 ° C, followed by 0.8 ml of commercial enzymes Celluclast 1.5L and Viscozyme L, respectively. , 0.2ml was added and hydrolyzed enzymatic hydrolysis for 96 hours at 50 ℃, 150rpm in a shaker.

Experimental Example  One. Biomass  Chemical composition analysis of raw materials

1. Sample

Oak wood was used as a biomass material, and it was crushed with Willis mill and passed through 20 mesh and uniformized to the size of the sample that did not pass through 80 mesh, and used as a sample for biomass chemical composition analysis. 1 is shown.

2. Method for Analyzing Chemical Composition of Biomass Raw Materials

In order to analyze the chemical composition of the biomass raw material, the following analysis was performed according to the National Renewable Energy Laboratory (NREL) method.

(1) Extract Content (NREL / TP510-42619)

After transferring 2 g of the sample to a cylindrical filter (thimble filter) and extracted with Soxhlet extractor with 200ml of ethanol for 24 hours, the extract content was measured and the degreasing sample was used as a lignin content and carbohydrate content measurement sample. Extract content,% was calculated by the following formula (1).

[Equation 1]

Extract Content (%) = (Weight of Whole Sample-Weight of Whole Sample after Extraction) / Weight of Whole Sample × 100

(2) Mineral Content (NREL / TP510-42622)

2g of the sample in the crucible was placed in the crucible and completely carbonized in an electric furnace at 600 ± 25 ° C. for 24 hours to determine the inorganic content. Inorganic content,% was calculated by the following formula (2).

&Quot; (2) "

Mineral Content (%) = Ash Weight / Full Weight of Sample × 100

(3) Carbohydrate and Lignin Content (NREL / TP510-42618)

Carbohydrate content and lignin content were hydrolyzed at 72 ° C for 30 minutes with 72% H ₂ S0 ₄ using ethanol extracted skim samples, and the hydrolyzate diluted with 4% H ₂ S0 ₄ with distilled water was added at 121 ° C. After autoclaving for hours, the filtrate was measured for carbohydrate content and acid soluble lignin and the residue was measured for acid insoluble lignin. Acid insoluble lignin content, acid soluble lignin content,% were calculated by the following equation.

&Quot; (3) "

Acid insoluble lignin (%) = weight of acid insoluble residue / weight of degreasing sample × 100

&Quot; (4) "

Acid soluble lignin content (%) = [UV absorbance × amount of filtrate × dilution factor] / [ε × degreasing sample weight] × 100

In Equation 4, UV absorbance = absorbance at UV spectrophotometer 240 nm, ε = absorbance at recommended wavelength (L / g? Cm)-25)

(4) Monosaccharide Analysis (NREL / TP510-42623)

Monosaccharide analysis was performed using HPLC (Agilent Technologies, Inc., 1100 series, USA) for carbohydrate content analysis. The column was Aminex HPX-87P (Bio-Rad, Hercules, CA, USA), the mobile phase was distilled water, the flow rate was 0.6 mL / min, the temperature was maintained at 85 ℃. 2 shows the retention time of the standard per monosaccharide.

3. Chemical Composition of Biomass Raw Materials

As shown in Table 1, the chemical composition of the biomass raw material was 44.6% and 14.0% of the carbohydrate cellulose and hemicellulose, respectively, accounting for 58.6% of the total biomass weight. In particular, the potential cellulose content that can be converted into glucose was high (44.6%), but the total content of inactive lignin was 27.5% and ash 2.9%.

Wood-based biomass feedstock was composed of a structure that interferes with the accessibility of enzymes during glucose conversion, which may lead to a decrease in saccharification efficiency. Therefore, physical and chemical pretreatment to increase the accessibility of enzymes was required.

Composition % Dry weight Recovered raw material 100.0 ± 0.0 Extractives 5.7 ± 0.4 Cellulose as glucose 44.6 ± 0.0 ^A hemicellulose (Hemicellulose as) 14.0 Xylose 10.8 ± 0.2 Galactose 2.4 ± 0.1 Arabinose 0.5 ± 0.1 Mannose 0.3 ± 0.1 Acid insoluble lignin 24.2 ± 0.3 Acid soluble lignin 3.3 ± 0.6 Ashes (Ash) 2.9 ± 0.3 Total 94.7

The data in Table 1 above are based on oven dried samples and hemicellulose ^a represents xylose + galactose + arabinose + mannose.

Wood-based biomass feedstock was composed of a structure that interferes with the accessibility of enzymes during glucose conversion, which may lead to a decrease in saccharification efficiency. Therefore, physical and chemical pretreatment to increase the accessibility of enzymes was required.

Experimental Example  2. Biomass  High Pressure Steam Pretreatment Process of Raw Material

Steam explosion pretreatment

The biomass raw material used in this experiment was provided by Yulim Hi-Tech. The raw material received from the company was provided as a residue after high pressure steam treatment, and the high pressure steam pretreatment condition was treated at 225 ° C. for 5 minutes at a steam pressure of 25 kg / cm ² . A photograph of the apparatus used for the high pressure steam pretreatment and the pretreated biomass raw material is shown in FIG. 3.

2. Chemical Composition Analysis of High Pressure Steam Pretreated Biomass Raw Materials

In order to analyze the chemical composition of the biomass raw material subjected to the high pressure steam pretreatment, it was analyzed in the same manner as described in Experiment 1 according to the National Renewable Energy Laboratory (NREL) analysis.

3. Chemical Composition of High Pressure Steam Pretreated Biomass Raw Materials

The chemical composition of the high pressure steam pretreated biomass is shown in Table 2. The amount of biomass recovered after high pressure steam pretreatment was 95%, representing 5% loss. The cellulose content was 52.4% and hemicellulose content 0.4%. Decreased. Relative increase in cellulose content and lignin content is thought to have been removed by hemicellulose in the biomass raw material in the low molecular state by hydrolysis during high temperature pretreatment. In the case of total lignin, an increase of 7.7% increased to 35.2%, thus increasing the cellulose content and removing the hemicellulose and the lignin.

Composition % Dry weight Recovered raw material ^a 95.0 ± 0.0 Extractives 5.3 ± 0.3 Cellulose as glucose 52.4 ± 0.0 Hemicellulose ^b 0.4 Xylose 0.1 ± 0.0 Galactose 0.1 ± 0.1 Arabinose 0.1 ± 0.0 Mannose 0.1 ± 0.0 Acid insoluble lignin 32.0 ± 0.3 Acid soluble lignin 3.2 ± 0.3 Ashes (Ash) 2.4 ± 0.2 Total 95.7

The data in Table 2 above are based on oven dried samples. The pretreatment condition is hydrothermal extraction for 1 hour after treatment at 225 ° C. for 5 minutes with a water vapor pressure of 25 kg / cm ² . The yield of the said ^a residue is shown. Hemicellulose ^b represents xylose + galactose + arabinose + mannose.

Experimental Example  3. Derivation of technology for securing cellulose from pretreated raw materials

1. Chemical treatment

The biomass raw material (residue after high-pressure steam treatment) pretreated by the high pressure steam by the method of Experimental Example 2 was chemically treated using alkali and chlorine system.

(1) Alkali treatment (1-3 wt% NaOH aqueous solution, 1-3 wt% KOH aqueous solution)

In a 1% by weight NaOH, 3% by weight NaOH solution, 1% by weight KOH, and 3% by weight KOH aqueous solution, the biomass raw material subjected to the high-pressure steam pretreatment was immersed in a ratio of solid to liquid (1:20), respectively. It was stirred for 3 hours at 100 rpm in a shaking incubator set at 30 ° C. After stirring, the residue was filtered and separated into a residue and a liquid. The residue was washed with 1% glacial acetic acid solution and distilled water until pH 7 and dried at 105 ° C., and then analyzed for chemical composition.

(2) Chlorine treatment (1-3 wt.% NaClO aqueous solution, 1-3 wt.% NaClO ₂ aqueous solution)

High-pressure steam pre-treated biomass raw materials were immersed in a solid: liquid (1:20) ratio at 30 ° C. in 1 wt% NaClO, 3 wt% NaClO aqueous solution, 1 wt% NaClO ₂ aqueous solution, and 3 wt% NaClO ₂ aqueous solution, respectively. It was stirred for 3 hours at 100 rpm in a set shaker. After stirring, the residue was filtered and separated into a residue and a liquid. The residue was washed with acetone and distilled water, dried at 105 ° C, and analyzed for chemical composition.

(3) Multistage treatment (1 wt% aqueous KOH solution + 1 wt% aqueous NaClO ₂ solution, 1 wt% aqueous KOH solution + 3 wt% NaClO ₂ aqueous solution)

The biomass raw material subjected to high pressure steam pretreatment in a 1 wt% KOH aqueous solution was immersed at a ratio of solid: liquid (1:20) and stirred for 3 hours at 100 rpm in a shake incubator set at 30 ° C. After stirring, the mixture was filtered and separated into a residue and a liquid. The residue was washed with 1% glacial acetic acid solution and distilled water until pH 7 and the residue was further dissolved in 1 wt% NaClO ₂ aqueous solution and 3 wt% NaClO ₂ aqueous solution, respectively: It was immersed in a liquid (1:20) ratio and stirred for 3 hours at 100 rpm in a shake incubator set at 30 ° C. After stirring, the residue was filtered and separated into a residue and a liquid. The residue was washed with acetone and distilled water, dried at 105 ° C, and analyzed for chemical composition.

2. Analysis of chemical composition by chemical treatment

In order to analyze the alkali, chlorine, and multi-stage chemical composition of the biomass raw material subjected to the high pressure steam pretreatment by the method of Experimental Example 2, it was analyzed in the same manner as described in Experimental Example 1 according to the NREL method.

3. Chemical Composition of Pretreated Biomass Raw Materials by Alkali Treatment

(1) NaOH treatment

Table 3 shows the chemical composition of the biomass treated with 1 wt% NaOH and 3 wt% NaOH aqueous solution to the biomass raw material subjected to the high pressure steam pretreatment by the method of Experimental Example 2. When NaOH was treated, the yield of biomass recovered was about 50%, and the content of cellulose was 72.0% and 69.9%, respectively, in 1 wt% NaOH and 3 wt% NaOH aqueous solutions, respectively, showing high cellulose content of about 70%. The cellulose content was higher in the 1 wt% NaOH aqueous solution than the 3 wt% NaOH solution, which was about 19.6% higher than the high pressure steam pretreated biomass. The total lignin content was about 15%, about 17% less than the high pressure steam pretreated biomass, and some of the lignin was extracted.

Composition % Dry weight 1 wt% NaOH 3 wt% NaOH Recovered raw material ^a 51.0 50.2 Extractives 4.8 ± 0.4 5.2 ± 0.2 Cellulose as glucose 72.0 ± 0.1 69.9 ± 0.1 Hemicellulose ^b 0.5 0.2 Xylose - - Galactose 0.5 ± 0.0 0.1 ± 0.0 Arabinose 0.0 ± 0.0 0.1 ± 0.0 Mannose - 0.0 Acid insoluble lignin 14.8 ± 0.4 15.7 ± 0.5 Acid soluble lignin 1.7 ± 0.3 1.7 ± 0.1 Ashes (Ash) 3.6 ± 0.1 3.0 ± 0.1 Total 97.4 95.7

The data in Table 3 above are based on oven dried samples. Pretreatment conditions are 1 wt% NaOH aqueous solution at 30 ° C. and 3 wt% NaOH aqueous solution at 30 ° C., 3 hours, 100 rpm, solid: liquid (1:20) immersion. ^a Shows the yield of the residue. The hemicellulose ^b represents xylose + galactose + arabinose + mannose.

(2) KOH treatment

Table 4 shows the chemical composition of the biomass treated with 1 wt% KOH and 3 wt% KOH in a biomass raw material subjected to high pressure steam pretreatment by the method of Experimental Example 2. The yield of biomass recovered by KOH treatment was about 50%, and the content of cellulose was 1% by weight KOH and 3% by weight KOH, respectively, 75.1% and 75.2%, respectively. The increased hemicellulose content was not detected in the case of 3 wt% KOH and 1.0% in the case of 1 wt% KOH. The total lignin content was about 14%, about 19% less than the high pressure steam pretreated biomass. As there was no significant difference in 1 wt% KOH aqueous solution and 3 wt% KOH aqueous solution, it was judged that 1 wt% KOH aqueous solution was most suitable for the multi-stage treatment.

Composition % Dry weight 1 wt% KOH 3 wt% KOH Recovered raw material ^a 51.7 50.7 Extractives 3.2 ± 0.2 3.4 ± 0.1 Cellulose as glucose 75.1 ± 0.0 75.2 ± 0.0 Hemicellulose ^b 1.0 - Xylose 0.1 ± 0.0 - Galactose 0.7 ± 0.1 - Arabinose 0.1 ± 0.0 - Mannose 0.1 ± 0.0 - Acid insoluble lignin 13.2 ± 0.1 13.0 ± 0.0 Acid soluble lignin 1.6 ± 0.1 1.6 ± 0.2 Ashes (Ash) 2.5 ± 0.3 2.4 ± 0.6 Total 96.6 95.6

The data in Table 5 above are based on oven dried samples. Pretreatment conditions are immersion of 1 wt% KOH aqueous solution at 30 ° C. and 3 wt% KOH aqueous solution at 30 ° C. for 3 hours, 100 rpm, solid: liquid (1:20). ^a Shows the yield of the residue. The hemicellulose ^b represents xylose + galactose + arabinose + mannose.

3. Chemical Composition of Biomass by Chlorine Pretreatment

(1) NaClO treatment

The chemical composition of the biomass treated with 1 wt% NaClO aqueous solution and 3 wt% NaClO aqueous solution in the biomass raw material subjected to high pressure steam pretreatment by the method of Experimental Example 2 is shown in Table 5. The yield of biomass recovered from NaClO treatment was 76.5% for 1 wt% NaClO and 67.4% for 3 wt% NaClO, indicating higher recovery than alkali treatment. The content of cellulose was 50.2% and 50.1% for 1 wt% NaClO and 3% NaClO, respectively. The lignin removal rate was 0.4% and 0.5%, respectively, and the total lignin content was about 37 ~ 39%, which was lower than the alkali treatment.

Composition % Dry weight 1 wt% NaClO 3 wt% NaClO Recovered raw material ^a 76.5 67.4 Extractives 5.7 ± 0.5 5.8 ± 0.1 Cellulose as glucose 50.2 ± 0.2 50.1 ± 0.0 Hemicellulose ^b 0.4 0.5 Xylose 0.1 ± 0.0 0.1 ± 0.0 Galactose 0.2 ± 0.0 0.3 ± 0.3 Arabinose 0.1 ± 0.1 0.0 ± 0.0 Mannose 0.0 ± 0.0 0.1 ± 0.1 Acid insoluble lignin 36.3 ± 0.2 34.3 ± 0.2 Acid soluble lignin 2.6 ± 0.2 2.6 ± 0.1 Ashes (Ash) 3.3 ± 0.2 3.1 ± 0.3 Total 98.5 96.4

The data in Table 5 above are based on oven dried samples. Pretreatment conditions are 1 wt% NaClO aqueous solution at 30 ° C. and 3 wt% NaClO aqueous solution at 30 ° C., 3 hours, 100 rpm, solid: liquid (1:20) immersion. ^a Shows the yield of the residue. The hemicellulose ^b represents xylose + galactose + arabinose + mannose.

(2) NaClO ₂ treatment

The chemical composition of the biomass treated with 1 wt% NaClO ₂ aqueous solution and 3 wt% NaClO ₂ aqueous solution to the biomass raw material subjected to the high pressure steam pretreatment by the method of Experimental Example 2 is shown in Table 6. If the NaClO ₂ processing time of the biomass is recovered yield was 1 weight% NaClO ₂ would have 74.8%, 3 wt% NaClO ₂ when 1 wt% NaClO _2, treated with 50.2% in the case of the indicated high recovery rate. However Compared to cellulose content of the case of 1 wt.% NaClO ₂ for 53.4%, 3 wt% NaClO _2, when treated with a 3 wt% NaClO ₂ as 70.6% exhibited a high cellulose content. The content of hemicellulose was 0.6% and 0.2% for 1 wt% NaClO ₂ and 3 wt% NaClO ₂ , respectively. The total lignin content was 33.1% for 1 wt% NaClO ₂ and 19.3% for 3 wt% NaClO ₂ . Low lignin content was seen upon treatment with 3 wt% NaClO ₂ . 3 wt% NaClO ₂ handling at 1 wt% NaClO ₂ high carbohydrate content when you consider the transition to more recoveries natatjiman glucose when processed 3% by weight of the lignin lower NaClO ₂ processing of high-pressure steam pretreated biomass material It has been found that it may be preferred by chemical treatment.

Composition % Dry weight 1 wt% NaClO ₂ 3 wt% NaClO ₂ Recovered raw material ^a 74.8 50.2 Extractives 6.1 ± 0.1 6.2 ± 0.1 Cellulose as glucose 53.4 ± 0.3 70.6 ± 0.2 Hemicellulose ^b 0.6 0.2 Xylose 0.0 ± 0.0 - Galactose 0.5 ± 0.1 - Arabinose 0.1 ± 0.0 0.1 ± 0.0 Mannose 0.0 ± 0.0 0.1 ± 0.0 Acid insoluble lignin 30.6 ± 0.2 16.4 ± 0.1 Acid soluble lignin 2.5 ± 0.4 2.9 ± 0.3 Ashes (Ash) 2.8 ± 0.5 2.6 ± 0.1 Total 96.0 98.9

The data in Table 6 above are based on oven dried samples. Pretreatment conditions are 1 wt% NaClO ₂ aqueous solution at 30 ° C. and 3 wt% NaClO ₂ aqueous solution at 30 ° C., 3 hours, 100 rpm, solid: liquid (1:20) immersion. ^a Shows the yield of the residue. The hemicellulose ^b represents xylose + galactose + arabinose + mannose.

4. Chemical Composition of Biomass by Multistage Treatment of Alkali and Chlorine

(1) Multistage Treatment of KOH and NaClO ₂

Multistage treatment was performed by selecting the chemical treatment method of 1 wt% KOH aqueous solution and 1 to 3 wt% NaClO ₂ aqueous solution which had high efficiency in treating KOH, NaOH, NaClO, NaClO ₂ .

Table 7 shows the chemical composition of the biomass treated with 1 wt% KOH and 1 to 3 wt% NaClO ₂ aqueous solution to the biomass raw material subjected to the high pressure steam pretreatment by the method of Experimental Example 2. When multi-stage treatment with 1 wt% NaClO ₂ and 1-3 wt% NaClO ₂ aqueous solution, the yield of biomass recovered was about 50%, which was similar to that of 1 wt% KOH. Some of hemicellulose and lignin were extracted, and it was judged that they were not affected by NaClO ₂ treatment.

Cellulose content have 1 weight% KOH, and 1 wt.% NaClO ₂ multi-stage processed with 74.1%, the KOH and the NaClO ₂ solution to 1 weight% KOH and 3 wt% NaClO ₂ 77.2% when the multi-stage process the first stage of treatment Compared to the It was judged that the multistage treatment was affected by the concentration because it showed a similar content or increased content. The total lignin content was about 14% in the case of the multistage treatment, which was similar to the lignin content in the KOH treatment. Therefore, the lignin removal was determined to be affected by alkali.

Composition % Dry weight 1 wt% KOH + 1 wt% NaClO ₂ 1 wt% KOH + 3 wt% NaClO ₂ Recovered raw material ^a 50.7 50.1 Extractives 4.3 ± 0.1 4.6 ± 0.1 Cellulose as glucose 74.1 ± 0.1 77.2 ± 0.1 Hemicellulose ^b 0.3 0.1 Xylose 0.2 ± 0.1 0.1 ± 0.1 Galactose 0.1 ± 0.1 0.0 ± 0.0 Arabinose - - Mannose - - Acid insoluble lignin 13.2 ± 0.1 12.9 ± 0.1 Acid soluble lignin 2.5 ± 0.1 1.4 ± 0.1 Ashes (Ash) 0.8 ± 0.1 0.3 ± 0.1 Total 95.5 96.6

The data in Table 7 above are based on oven dried samples. Pretreatment conditions were 30 ° C., 3 hours, 100 rpm, solid: liquid (1:20) immersion. ^a Shows the yield of the residue. The hemicellulose ^b represents xylose + galactose + arabinose + mannose.

Experimental Example  4. Established glucose conversion technology using enzyme from prepared cellulose

1. Enzymatic Saccharification

Alkaline treatment (1 wt% NaOH, 1 wt% KOH aqueous solution) and chlorine-based treatment (1 to 3 wt% NaClO, 1 to 1) of the biomass raw material subjected to high pressure steam pretreatment and the high pressure steam pretreatment by the method of Experimental Example 2 3 wt% aqueous NaClO ₂ solution), enzyme glycosylation was carried out to evaluate the conversion rate of cellulose prepared by multistage treatment into glucose.

1 g of the pretreated sample and 50 ml (pH 4.8) of 0.1M sodium citrate buffer were added to the Erlenmeyer flask, heated to 50 ° C, and 0.8 ml of commercial enzymes Celluclast 1.5L and Viscozyme L, respectively. , 0.2ml was added and hydrolyzed enzymatic hydrolysis for 96 hours at 50 ℃, 150rpm in a shaker. After completion of the hydrolysis, the residue was filtered to measure the enzyme hydrolysis rate. The filtrate was subjected to monosaccharide analysis using HPLC to measure glucose content and glucose conversion rate.

(1) Delignification

Talignin rate is the percentage of lignin removed.

[Equation 5]

Talignin rate (%) = (Lignin content before pretreatment-Lignin content after pretreatment) / Lignin content before pretreatment × 100

(2) Enzymatic digestibility

Enzyme hydrolysis rate was calculated using the weight of the residue after enzymatic hydrolysis on the injected sample.

&Quot; (6) "

Enzyme hydrolysis rate (%) = (weight of injected sample-weight of residue after enzymatic hydrolysis) / weight of injected sample × 100

(3) glucose conversion

Glucose conversion was calculated using the weight of glucose in the digested solution after enzymatic hydrolysis on the glucose contained in the injected sample.

[Equation 7]

Glucose Conversion Rate (%) = Weight of Glucose in Degradation Solution after Enzymatic Hydrolysis / Weight of Glucose in Sample × 100

2. Monosaccharide Analysis (NREL / TP510-42623)

Monosaccharide analysis was performed using HPLC (Agilent Technologies, Inc., 1100 series, USA) for glucose content analysis. The column was Aminex HPX-87P (Bio-Rad, Hercules, CA, USA), the mobile phase was distilled water, the flow rate was 0.6ml / min, the temperature was maintained at 85 ℃.

3. Analysis of Glucose Conversion of Cellulose Prepared from Biomass and High Pressure Steam Pretreated Biomass

Table 8 shows the glucose content and the sugar conversion rate produced after enzymatic hydrolysis of biomass subjected to high pressure steam pretreatment by the method of biomass and Experimental Example 2.

As the hemicellulose and lignin were hydrolyzed by the small amount of hemicellulose and lignin during high pressure steam pretreatment, the yield of substrate was about 95%, and the cellulose content was 7.8% higher in the biomass pretreated biomass than the biomass. . As the hemicellulose content was decreased, the lignin content was relatively higher than before the high pressure steam pretreatment.

The amount of glucose produced was 1.1 g / l for biomass and 9.1 g / l for high pressure steam pretreatment. A large amount of glucose was produced during high pressure steam pretreatment and the enzyme hydrolysis rate increased 64.9% for high pressure steam pretreatment. The glucose conversion rate on the basis of the high conversion rate was 86.8% at high pressure steam pretreatment.

Raw material Steam exploded Yield of residue (%) - 95 Lignin ^a (%) 27.5 35.2 Talignin rate (%) - - Cellulose (%) 44.6 52.4 Holocellulose ^b (Holocellulose; Cellulose + Hemicellulose) (%) 58.6 52.8 Enzyme hydrolysis rate ^c (%) 12.1 64.9 Glucose (%) 1.1 4.1 Glucose Conversion Rate (%) 12.3 39.1

The data in Table 8 above are based on oven dried samples. Lignin ^a represents acid insoluble lignin plus acid soluble lignin. Holocellulose ^b represents cellulose + hemicellulose. Enzymatic hydrolysis condition ^c was hydrolyzed for 96 hours at pH 4.8, 0.1 M sodium citrate buffer, 50 ° C, 150 rpm.

4. Analysis of Glucose Conversion of Cellulose Prepared from Alkali Pretreated Biomass

Table 9 and Table 10 show the glucose content and the sugar conversion rate of the hydromass produced by the hydrolysis of the high-pressure steam pretreated by the method of Experimental Example 2, respectively, followed by enzyme hydrolysis. .

1 wt% NaOH Yield of residue (%) 51.0 Lignin ^a (%) 16.5 Talignin rate (%) 53.1 Cellulose (%) 72.0 Holocellulose ^b (Holocellulose; Cellulose + Hemicellulose) (%) 72.5 Enzyme hydrolysis rate ^c (%) 59.1 Glucose ^c (%) 9.7 Glucose Conversion Rate (%) 67.4

The data in Table 9 above are based on oven dried samples. Lignin ^a represents acid insoluble lignin plus acid soluble lignin. Holocellulose ^b represents cellulose + hemicellulose. Enzymatic hydrolysis condition ^c was hydrolyzed for 96 hours at pH 4.8, 0.1 M sodium citrate buffer, 50 ° C, 150 rpm.

1 wt% KOH Yield of residue (%) 51.7 Lignin ^a (%) 14.8 Talignin rate (%) 58.0 Cellulose (%) 75.1 Holocellulose ^b (Holocellulose; Cellulose + Hemicellulose) (%) 76.1 Enzyme hydrolysis rate ^c (%) 58.5 Glucose (%) 10.2 Glucose Conversion Rate (%) 67.9

The data in Table 10 above are based on oven dried samples. Lignin ^a represents acid insoluble lignin plus acid soluble lignin. Holocellulose ^b represents cellulose + hemicellulose. Enzymatic hydrolysis condition ^c was hydrolyzed for 96 hours at pH 4.8, 0.1 M sodium citrate buffer, 50 ° C, 150 rpm.

5. Glucose Conversion Analysis of Cellulose Prepared from Chlorine-Pretreated Biomass

(1) NaClO

Table 11 shows the glucose content and the sugar conversion rate of the hydromass produced by enzymatic hydrolysis after treating 1 wt% NaClO and 3 wt% NaClO aqueous solution to the biomass raw material subjected to the high pressure steam pretreatment by the method of Experimental Example 2, respectively.

Yield of recovered substrate was 76.5% for 1 wt% NaClO and 67.4% for 3 wt% NaClO, and the amount of substrate recovered for enzymatic hydrolysis was greatly reduced. As the lignin was relatively increased, the lignin showed an ineffective value in the talignin ratio.

The content of cellulose was less than expected compared to the amount of substrate recovered by about 50% upon treatment with 1 wt% NaClO and 3 wt% NaClO. The amount of glucose produced after enzymatic hydrolysis was 4.8 ~ 4.9g / l, which was lower than that of alkali pretreatment, and the conversion rate of glucose was low at about 48% when 1% NaClO and 3% NaClO were treated. .

Secondary treatment of NaClO in high pressure steam pretreated biomass feedstocks is not suitable as a single pretreatment method when evaluating the amount of substrate recovered but the amount of cellulose, the amount of glucose produced, and the conversion of glucose.

1 wt% NaClO 3 wt% NaClO Yield of residue (%) 76.5 67.4 Lignin ^a (%) 38.9 36.9 Talignin rate (%) - - Cellulose (%) 50.2 50.1 Holocellulose ^b (Holocellulose; cellulose + hemicellulose) (%) 50.6 50.6 Enzyme hydrolysis rate ^c (%) 25.6 26.1 Glucose (%) 4.8 4.9 Glucose Conversion Rate (%) 47.8 48.9

The data in Table 11 above are based on oven dried samples. Lignin ^a represents acid insoluble lignin plus acid soluble lignin. Holocellulose ^b represents cellulose + hemicellulose. Enzymatic hydrolysis condition ^c was hydrolyzed for 96 hours at pH 4.8, 0.1 M sodium citrate buffer, 50 ° C, 150 rpm.

(2) NaClO ₂

Table 12 shows the glucose content and the sugar conversion rate of the hydromass produced by hydrolysis of enzyme by hydrolysis of 1 wt% NaClO ₂ and 3 wt% NaClO ₂ solutions to the biomass raw material subjected to the high pressure steam pretreatment by the method of Experimental Example 2, respectively. . If the amount of the recovered substrate 1 wt% NaClO ₂ 74.8%, 3 wt.% Of NaClO ₂ when 1 wt% NaClO ₂ treatment to about 50.2% was able to recover the substrate of the high content of delignification rate of 3 parts by weight Treatment with% NaClO ₂ showed a high value of 45.2%. The content of cellulose was 53.4% and 70.6%, respectively, when treated with 1 wt% NaClO ₂ and 3 wt% NaClO ₂ , showing high contents when treated with 3 wt% NaClO ₂ .

The amount of glucose produced after enzymatic hydrolysis was 5.9 g / L and 9.7 g / L, respectively, at 3% by weight NaClO ₂ when treated with 1% by weight NaClO ₂ and 3% by weight NaClO _2. High content in the treatment and 1 wt% NaClO ₂ in the glucose conversion rate And 38.5% NaClO _{2 to} 68.5% and 68.7%, respectively, when treated with 3% NaClO _2. High values were found during treatment.

In NaClO ₂ treatment, as the concentration of NaClO ₂ increases, the amount of recovered substrate decreases, but the cellulose content and glucose content increase, so that the concentration increases when NaClO ₂ treatment is performed in the biomass pretreated with high pressure steam. The more effective it is, the better.

1 wt% NaClO ₂ 3 wt% NaClO ₂ Yield of residue (%) 74.8 50.2 Lignin ^a (%) 33.1 19.3 Talignin rate (%) 6.0 45.2 Cellulose (%) 53.4 70.6 Holocellulose ^b (Holocellulose; cellulose + hemicellulose) (%) 54.0 70.8 Enzyme hydrolysis rate ^c (%) 36.4 50.2 Glucose (%) 5.9 9.7 Glucose Conversion Rate (%) 55.2 68.7

The data in Table 12 above are based on oven dried samples. Lignin ^a represents acid insoluble lignin plus acid soluble lignin. Holocellulose ^b represents cellulose + hemicellulose. Enzymatic hydrolysis condition ^c was hydrolyzed for 96 hours at pH 4.8, 0.1 M sodium citrate buffer, 50 ° C, 150 rpm.

6. Glucose Conversion Analysis of Cellulose Prepared by Multistage Treatment of Alkali and Chlorine Compounds

(1) Multistage Treatment of KOH and NaClO ₂

Firstly, the content of glucose produced by enzymatic hydrolysis of cellulose prepared by multi-stage treatment with 1 wt% KOH aqueous solution and 1 to 3 wt% NaClO ₂ aqueous solution to biomass raw material subjected to high pressure steam pretreatment by the method of Experimental Example 2, and The sugar conversion rate is shown in Table 13.

The yield of the substrate recovered after the multistage treatment of alkali and chlorine was about 50% in 1 wt% KOH + 1 wt% NaClO ₂ and 1 wt% KOH + 3 wt% NaClO ₂ . It was about 4% more effective at 1 KOH + 3 wt% NaClO _{2 than} at 1 wt% KOH + 1 wt% NaClO ₂ . The cellulose content was higher at 77.2% at 1 wt% KOH +3 wt% NaClO ₂ but the glucose production, glucose conversion was higher at 1 wt% KOH + 1 wt% NaClO ₂ .

1 wt% KOH + 1 wt% NaClO ₂ 1 wt% KOH + 3 wt% NaClO ₂ Yield of residue (%) 50.7 50.1 Lignin ^a (%) 15.7 14.3 Talignin rate (%) 55.4 59.4 Cellulose (%) 74.1 77.2 Holocellulose ^b (Holocellulose; cellulose + hemicellulose) (%) 74.4 77.3 Enzyme hydrolysis rate ^c (%) 64.8 65.2 Glucose (%) 12.4 12.2 Glucose Conversion Rate (%) 83.7 79.0

The data in Table 13 above are based on oven dried samples. Lignin ^a represents acid insoluble lignin plus acid soluble lignin. Holocellulose ^b represents cellulose + hemicellulose. Enzymatic hydrolysis condition ^c was hydrolyzed for 96 hours at pH 4.8, 0.1 M sodium citrate buffer, 50 ° C, 150 rpm.

7. Comparison of Glucose Conversion Rate by Chemical Treatment of High Pressure Steam Pretreated Biomass Raw Materials

Glucose conversion was compared when the alkali and chlorine compound aqueous solutions were treated alone and when subjected to multiple stages (see FIG. 4). High pressure steam pretreatment resulted in glucose conversion of 39.1% or higher. When 1% by weight KOH and 1% by weight NaOH were treated alone in biomass pretreated biomass materials, glucose conversion was 67.9% and 67.4%, respectively. Treatment with NaClO ₂ and 3 wt% NaClO ₂ alone showed glucose conversions of 55.2% and 68.7%. Therefore, 1 wt% KOH with high glucose conversion in alkali and 1 wt% NaClO ₂ and 3 wt% NaClO ₂ with high glucose conversion in the chlorine system were treated in parallel to obtain 1 wt% KOH and 1 wt% NaClO ₂ . The multistage treatment of 83.7%, 1 wt% KOH and 3 wt% NaClO ₂ showed 79% glucose conversion. In comparison with the production cost and the conversion rate of glucose produced from these results, it is judged that the combination of weak alkali and weak chlorine treatment is performed on the biomass raw material subjected to the high pressure steam pretreatment.

Claims (6)

Steam blasting the biomass feedstock;
Treating the steam-exploded biomass feedstock with at least one alkali compound selected from the group consisting of sodium hydroxide (NaOH) and potassium hydroxide (KOH);
Treating the biomass raw material treated with the alkali compound with at least one chlorine compound selected from the group consisting of sodium hypochlorite (NaClO) and sodium chlorite (NaClO ₂ ); And
Glucose manufacturing method comprising the step of saccharifying the biomass raw material treated with the chlorine-based compound. The method of claim 1, wherein the vapor decay treatment is performed at 213 to 235 ° C. under a pressure of 20 to 30 kg / cm 2. The method of claim 1, wherein the treating with the alkaline compound comprises treating the aqueous alkali compound solution at a concentration of 1 to 3% by weight. The method of claim 3, wherein the treating with the chlorine compound comprises treating an aqueous solution of chlorine compound at a concentration of 1 to 3 wt%. delete The method of claim 4, wherein the glycosylation is performed by enzyme glycosylation.

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