KR102108097B1 - Method for saccharification of biomass - Google Patents

Abstract

The method of the present invention comprises the steps of sterilizing the biomass at 20 to 90 ° C with sulfite; And by adding an oxidizing agent to the reactant, the step of detoxifying the sulfite; By including, it is possible to suppress or prevent microbial contamination that may occur during the saccharification process, as well as to maximize the glucose yield by enzymatic saccharification as well as fermentation sugar It relates to a method for saccharification of biomass effective to prevent quality degradation.

Description

METHOD FOR SACCHARIFICATION OF BIOMASS

The present invention relates to a method for saccharifying biomass.

In order to cope with the global warming caused by greenhouse gases and the depletion of fossil fuels, research and development have been actively conducted to produce transportation fuels and industrial chemicals from renewable biomass worldwide. In particular, the biochemical industry, which uses edible biomass, such as corn and sugarcane, which can cause potential food problems, is a carbon source for microbial fermentation. A lot of research and development has been conducted to convert the fermentation sugar of biomass (lignocellulosic biomass) to the market, and the market of wood-based fermentation sugar is in the forefront.

Woody biomass is largely cellulose (cellulose, 20 to 50%), hemicellulose (15 to 35%), lignin (10 to 30%) and other ingredients (water soluble starch, free sugar, protein, lipid, pectin, Tannins, alkaloids, organic acids, and various inorganic salts, etc.). Since cellulose is composed of glucose, which is the main nutrient source of fermentation strain, conversion to fermentation sugar will be the core of industrial technology.

When producing glucose from a wood-based biomass, it is common to sequentially perform a pretreatment and a saccharification process because cellulose is not easily converted into glucose only in a form in which the biomass is crushed. Biomass pre-treatment is a process of treating biomass that has been previously crushed or shredded in a physicochemical manner to make each structural component of the biomass easy to fractionate. Saccharification of biomass converts cellulose, which has changed into a more easily hydrolyzed state, into glucose through physicochemical or biochemical methods by dissolving or dissolving some or all of hemicellulose or lignin surrounding cellulose in the pretreatment process. Process.

Enzymatic hydrolysis of biomass is a process of producing glucose by adding an enzyme, cellulase, to the cellulose converted to a more easily hydrolyzed state by a pretreatment process. At this time, in order to promote the hydrolysis action of cellulose, cellulose hydrolase further contains various enzymes such as hemicellulase, hemicellulase, amylase, and pectin hydrolase. In general, enzymatic saccharification is performed at a high temperature of 45 to 55 ° C. This is to promote smooth saccharification reaction through reduction in the viscosity of enzymatic saccharide and prevent microbial contamination that may occur during the enzymatic saccharification process.

However, even under such high temperature enzymatic saccharification, unexpected microbial contamination often occurs. The present inventors discovered that lactic acid was detected in a large amount when the microorganisms were contaminated with saccharides, and as a result of identifying the contaminating microorganisms, they were Bacillus coagulans, a thermophilic strain. The optimal growth temperature of Bacillus coagulance is 50 to 55 ° C, which is almost identical to the reaction temperature of enzyme glycosylation. When the sugars are contaminated by the microorganisms, not only the sugar yield is reduced by the microorganism ingesting and metabolizing the glucose produced during the saccharification process, but also the quality of the fermentation sugar is deteriorated by producing a large amount of lactic acid as a metabolite. As described above, contamination of microorganisms generated in the saccharification process must be prevented because it causes various problems in the production of fermented sugar.

When producing bioethanol using wood-based biomass as a raw material, Inbicon in Denmark generates excessive lactic acid bacteria by re-injecting phenolic decomposition products such as acetic acid, furfural, and vanillin produced in the pretreatment of biomass over a certain amount. It has shown a technique to control not to be used, and the method of using hop acid, virginiamycin, etc. has been suggested for the purpose of suppressing the generation of microorganisms. However, it is obvious that when these substances are added to enzymatic saccharification of biomass, there is a limitation in the use of the fermented sugar produced. Therefore, when enzymatic saccharification is performed to produce a general purpose fermented sugar that can be used for fermentation of various microorganisms while using biomass as a raw material, even if such a microorganism inhibitor is used, it is removed so as not to be contained in the final fermented sugar product. Must be able to. However, this technique has not been reported.

 Sulfite has been widely used in the wine industry since the late 18th century to prevent contamination of microorganisms that may occur during wine production. This sulfite reacts with water to generate sulfur dioxide and this gas sterilizes. A method of producing wine by adding sulfite, maintaining it for one day, and adding yeast before fermenting grapes by this mechanism has been widely used. As such, sulfite has very little residual toxicity, and has been used in the human food industry for a long time. However, in the present inventors' study, when this sulfite is used in the enzyme saccharification process, the residual sulfite inactivates the saccharifying enzyme, thereby lowering the sugar yield. I also found out. In view of this, the present inventors have completed the present invention by putting a lot of effort to develop a technique for applying this sulfite to enzymatic saccharification of woody biomass.

Korean Patent Publication No. 2015-0093483

An object of the present invention is to provide a safe saccharification method of biomass for producing fermented sugar that can be used for cultivation of various microorganisms using wood-based biomass as a raw material.

1. Sterilizing the biomass at 20 to 90 ° C with sulfite; And

Including the step of detoxifying the sulfite by adding an oxidizing agent to the reactant, the method of saccharification of biomass.

2. The method of 1 above, wherein the biomass is an acidic condition, a method for saccharification of biomass.

3. The method of 2 above, wherein the acidic condition is pH 5 or less, a method for saccharification of biomass.

4. In the above 1, further comprising the step of adjusting the pH to 5 or less by adding an acid to the biomass, the method for saccharification of biomass.

5. In the above 1, wherein the sterilization is performed at 30 to 60 ℃, the method of saccharification of biomass.

6. In the above 1, the sulfite is selected from the group consisting of sodium sulfite, sodium metabisulfite, sodium thiosulfate, potassium sulfite, potassium metabisulfite, potassium thiosulfate, calcium sulfite, calcium metabisulfite and calcium thiosulfate. , Biomass saccharification method.

7. The method of 1 above, wherein the sterilization is performed within 1 hour, the method for saccharification of biomass.

8. In the above 1, wherein the sterilization is to sterilize microorganisms containing Bacillus coagulans ( Bacillus coagulans ) contained in the biomass, the method for saccharification of biomass.

9. The method of 1 above, wherein the oxidizing agent is oxygen, hydrogen peroxide or ozone, a method for saccharification of biomass.

10. The saccharification method of biomass according to the above 1, wherein the oxidizing agent is added in an amount of 0.2 to 2 times equivalent of sulfite.

11. The method of 1 above, wherein the detoxification is performed at 20 to 90 ° C., a method for saccharifying biomass.

12. The method of 1, further comprising the step of saccharifying the biomass by adding a glycosylating enzyme after the detoxification.

The method of the present invention can inhibit or prevent microbial contamination that may occur during the saccharification process for the production of fermented sugar of wood-based biomass using sulfite, thereby not only maximizing glucose yield due to enzymatic saccharification but also lowering fermentation sugar quality It is effective in preventing.

FIG. 1 is a graph measuring the production concentration of glucose (FIG. 1-1) and lactic acid production concentration (FIG. 1-2) over time during the saccharification process of fermentation sugar production and when microbial contamination does not occur. (Open symbol: When microbial contamination does not occur, Closed symbol: When microbial contamination occurs).
2 is a graph of the analysis of the saccharification solution by HPLC at 0 and 74 hours of saccharification when contamination of microorganisms occurs. The glycation reaction at 74 hours shows that lactic acid was produced.
FIG. 3 shows a figure in which a microorganism was isolated from a saccharification reactor in which microbial contamination occurred, and 16s rDNA was analyzed and identified for this strain, and as a result, Bacillus coagulans was found.

Hereinafter, the present invention will be described in more detail.

The term “wood-based biomass” used throughout the present specification mainly refers to the entire ground plant, and chemically refers to a biomass having cellulose, hemicellulose, and lignin as structural components. Examples of this wood-based biomass include agricultural byproducts such as corn stalk, straw, barley, rice straw, farm workshop, sunflower stalk, sorghum and singular, energy crops such as reeds, silver grass, and willow hybrids. crops) and woody biomass such as acacia, eucalyptus, birch, and pine.

The term "pre-treatment" of xylem-based biomass, which is a term used throughout this specification, partially or all of hemicellulose or lignin among structural components of biomass by physicochemical or biological methods to facilitate enzymatic saccharification of xylem-based biomass. It means the process of removal. This pretreatment is largely divided into acid catalyst pretreatment including autohydrolysis, liquid hot qater treatment or hydrothermolysis, which mainly hydrolyzes hemicellulose, and alkali reagents such as ammonia, sodium hydroxide, and calcium hydroxide. All treatments for the purpose of hydrolysis of cellulose using enzymes, such as alkali treatment, which mainly dissolves lignin, physical treatment that mechanically crushes to increase the surface area of cellulose, and steam explosion, etc. Includes.

The terms “fibrin hydrolase” or “glycosylase” used throughout this specification include cellulase, endoglucanase, exoglucanase, cellobiohydrolase, and β-glucosidase. Cellulose hydrolase or xylanases, endozylanases, exolylanases, β-xyloxidases, arabinoxylanases, mannase, galactases, pectinases, glucuronidases It is an enzyme preparation containing at least one enzyme selected from the group consisting of hemicellulose hydrolase containing. Examples of such enzyme complexes for enzyme glycosylation include Celluclast® 1.5L or Celluclast® conc BG and NovozymeTM 188 mixture, Cellic CTec2 and Cellic HTec2 mixture, or Cellic CTec3 and Cellic HTec3 mixture, Celluzyme®, Serenity Mixtures of Cereflo® and Ultraflo® (above from Novozymes, Denmark), AccelleraseTM, Laminex® and Spezyme® mixtures (above Genencor Int.) And Rohament® (manufactured by Rohm GmbH).

The term “enzymatic saccharification of biomass”, which is used throughout this specification, produces a sugar or glucose containing sugar by hydrolyzing the woody biomass with a fibrinolytic enzyme, that is, a glycosylation enzyme. It means a process, and is usually a method of dispersing the biomass pretreatment in water and adding cellulase complex enzyme as a fibrinolytic enzyme, followed by stirring while maintaining a constant acidity and temperature.

“Substrate for enzymatic hydrolysis” refers to biomass or its pre-treatment material, which is a target substance for conversion to monosaccharides by hydrolysis with fibrinolytic enzyme, and “saccharification system” is bio It refers to all substances including the saccharification substrate and the saccharification tank inside the saccharification tank of the saccharification device used for the enzyme saccharification of the mass. The “total glucan content” of the saccharifying substrate means the total amount of cellulose contained in the saccharifying substrate, and in some cases, this amount may mean the amount of glucose to be produced by hydrolysis. In addition, “substrate loading” means the total amount of biomass that is introduced into the saccharifier for saccharification of the biomass enzyme, and may be expressed as the ratio of the building material of the biomass pretreatment to the total saccharification weight.

In the present specification, the “fed-batch” method used when injecting a substrate for saccharification into a saccharification group for enzymatic saccharification of biomass refers to a method of additionally injecting a substrate to a saccharification in progress of saccharification. It is a concept that contrasts with the batch method, in which the total amount of saccharification substrate is added at once with the start. The term “conversion rate of cellulose to glucose” refers to the glucose conversion rate of cellulose contained in the substrate for saccharification as a result of enzymatic saccharification of biomass, and the glucose produced by enzymatic hydrolysis of the total amount of cellulose converted to glucose. Calculate as a percentage of the total amount. In addition, all materials in the present invention mean weight or weight ratio unless otherwise specified.

In the present invention, “detoxification” performed before sterilization by adding sulfite to an object to be sterilized with a sterilizing agent and adding saccharification enzyme refers to a process of reducing or eliminating the action of sulfite to reduce the activity of saccharification enzyme, and for the microorganisms of sulfite It is a concept used in a different sense from toxicity.

The method for saccharifying biomass of the present invention comprises sterilizing biomass at 20 to 90 ° C with sulfite; And adding an oxidizing agent to the reactant to detoxify the sulfite.

In the present invention, the sulfite used to sterilize the saccharification system before enzymatic saccharification of the biomass pretreatment includes chemicals capable of killing microorganisms by generating sulfur dioxide harmful to microorganisms, for example, sodium sulfite, meta Sodium metabisulfite, sodium thiosulfate, potassium sulfite, potassium metabisulfite, potassium thiosulfate, calcium sulfite, calcium bisulfite ( calcium metabisulfite) and calcium thiosulfate. These may be used alone or in combination of two or more.

The sulfite can be introduced into the saccharification system in the form of a dried drug or an aqueous solution dissolved in non-ionic water. The concentration showing the sterilizing power for microorganisms is slightly different depending on the type of the compound, and the higher the substrate concentration of the saccharification system should be, so it cannot be greatly limited, but according to the research results, the total saccharification substrate load in the saccharification machine is 25 g / L or more, When 300 g / L or less, sulfites of 2 μM to 40 μM can be used. For example, in the case of using palm farming room hydrothermal treatment as a saccharification substrate, the saccharification system loaded with 25 g / L in the building is treated at a concentration of 5 μM to sterilize with sodium metabisulfite, but the saccharification substrate loaded at 125 g / L In order to sterilize with sodium metabisulfite, it can be treated at a concentration of 16 μM, but is not limited thereto.

As the sulfite, meta-bisulfite, which can generate two sulfur dioxides, can be used in a smaller amount than sodium sulfite, which generates one sulfur dioxide per molecule, so it is preferable.

In order to sterilize microorganisms or their spores that may be contained in biomass, it is advantageous to sterilize under conditions in which sulfur dioxide is generated in large quantities from sulfite. In this aspect, sterilization may be performed at 20 ° C to 90 ° C. If the temperature is less than 20 ° C, the rate of sulfur dioxide production is low, so long-term sterilization is required. If it exceeds 90 ° C, the degree of sulfur dioxide production by temperature increase is negligible, and it is inefficient because a high pressure reactor that withstands vapor pressure must be used. In this aspect, it may be preferably carried out at 30 to 60 ℃.

In addition, in terms of generating a large amount of sulfur dioxide, the biomass under acidic conditions can be sterilized with sulfite. Acidic conditions can be, for example, pH 5 or less. Above pH 5, sulfur dioxide production may be negligible. The lower the pH, the faster the generation of sulfur dioxide, but the amount of acid used is excessive, and in this case, the amount of the neutralizing agent used to neutralize the acid immediately before saccharification is also greatly increased, so in this respect, the pH may be preferably 2 to 5.

If the pH of the biomass is not an acidic condition, the method of the present invention may further include acidifying the biomass by adding an acid. The pH can be acidified to 5 or less, preferably pH 2 to 5. As the acid that can be added, for example, inorganic acids such as sulfuric acid, hydrochloric acid, phosphoric acid, and nitric acid can be used, but are not limited thereto.

In terms of promoting sulfur dioxide production, sterilization can be performed under stirring, and a sulfite aqueous solution can be added to promote sulfur dioxide production within a short time.

Sterilization may be performed, for example, within 1 hour, but is not limited thereto.

The sterilization target may be, for example, all microorganisms including Bacillus coagulans and its spores included in the biomass.

The toxicity of the sulfite naturally decreases over time with stirring, but you have to wait for a long time. For the continuous enzymatic saccharification of biomass, the toxicity of sulfite added for sterilization must be able to be removed at a level that is harmless to the enzyme in a short time.

To this end, the method of the present invention includes the step of detoxifying sulfite by adding an oxidizing agent to the sterile reactant. The oxidizing agent is a substance that oxidizes sulfur dioxide to produce sulfate, and examples thereof include oxygen, hydrogen peroxide, and ozone. These may be used alone or in combination of two or more.

The oxidizing agent can add 0.2 to 2 times the equivalent of sulfite, for example. For example, if 1 g (5.3 mmole) of sodium metabisulfite was used for sterilization of the farm water treatment room hydrothermal pretreatment, 2.4 g to 24 g (0.0053 x 2 x 34 / 0.03) of hydrogen peroxide can be used as a 3% aqueous solution, but is not limited thereto. It does not work.

Detoxification can be carried out, for example, at 20 ° C to 90 ° C. When the temperature is less than 20 ° C, the rate of sulfur dioxide generation is slowed, and the rate of detoxification generated in combination with oxygen may be remarkably slowed. If it exceeds 90 ° C, the degree of increase in detoxification rate due to temperature increase is negligible, and the pressure is high due to pressure. This can be inefficient as a reactor must be used.

Detoxification is for the safety of the enzyme added for the subsequent enzymatic saccharification, and it is recommended that the time is long, but it is desirable to complete it within 24 hours to maintain the continuity of the enzymatic saccharification process, more preferably within 12 hours, Most preferably, it should be completed within 2 hours.

The method of the present invention may further include a step of glycating the biomass by adding a glycosylation enzyme to the sterilized biomass.

In the method of the present invention, the enzymatic saccharification step is a process of converting biomass into cellulose and hemicellulose contained in biomass into monosaccharides such as glucose and xylose. At this time, in order to prevent contamination by external microorganisms in the saccharification process, the saccharification system must be kept closed.

Water can be additionally used for saccharification by adding the cellulase complexase, but it is good to limit the amount to obtain a high concentration of sugar solution after saccharification. Therefore, the ratio of water to solid content during enzymatic saccharification is preferably 1: 3 to 1:10 by weight in terms of drying during solid content.

Cellulase complex enzyme containing hemicellulase is used for the enzyme glycosylation, for example, Cellullast® 1.5L or a mixture of Celluclast® conc BG and NovozymeTM188, a mixture of Cellic CTec2 and Cellic HTec2, or Cellic CTec3 and Mixtures with hemicellulase, mixtures of Celluzyme®, Cereflo® and Ultraflo® (above from Novozymes, Denmark), AccelleraseTM, Laminex® And Spezyme® mixtures (above Genencor Int.), Rohament® (manufactured by Rohm GmbH), and the like.

For hydrolysis of hemicellulose contained in biomass, hemicellulase may be added, and the mixing ratio of cellulase and hemicellulase is preferably about 9: 1 to 10: 0. In addition, the amount of cellulase coenzyme per 1 g during biomass drying is preferably 0.001 g to 0.5 g.

Enzymatic saccharification is recommended to maintain pH 4.8 to 5.2 and temperature 50 ± 5 ° C for the conditions in which the hydrolase exhibits maximum activity, that is, a mixture of Cellic CTec2 and Cellic HTec2, and saccharification is 24 hours unless there is contamination of microorganisms. It is preferable to last for at least 96 hours.

If necessary, the method of the present invention may further include known processes required for biomass saccharification in addition to the above processes.

For example, pretreatment of biomass; Recovery of sugar solution after enzymatic saccharification; Filtration of sugar solution, concentration, removal of impurities; And the like.

The pretreatment process of biomass includes, for example, hydrating a coarse pulverized product or powder of biomass; Pre-treatment of the obtained solid content with hot water; It may include the step of solid-liquid separation of the hot water pretreatment to obtain a solid content.

The hydrating step of the biomass may be performed, for example, by adding water to a coarse wood-based biomass or powder and mixing it, followed by dehydration.

In the process, the saccharification rate and sugar yield are minimized by minimizing the amount of substances capable of inhibiting the activity of cellulose hydrolase, such as inorganic salts, which are contained in a large amount in the herbal biomass when enzymatic saccharification of the pretreatment. ) And at the same time, it is effective to reduce the process cost by reducing the impurity load in the purification process of sugar solution after enzyme saccharification. In addition, this process recovers and recycles useful substances such as starch, free sugars, proteins, lipids, pectins, tannins, alkaloids that can exhibit various physiological activities, organic acids, and inorganic salts contained in extractable substances in biomass, During the pre-treatment process, these substances become useless by reactions such as decomposition, condensation, and modification, or they reduce the likelihood of proteins becoming toxic by a Maillard reaction.

In the above process, the biomass is immersed in water and then extracted or extracted with water at a temperature at which the water solubility of the substance is maximized. Preferably, the biomass is immersed in water, then stirred for 1 to 60 minutes and the aqueous solution can be removed through solid-liquid separation. In this process, most of the extractable components contained in the biomass can be removed, including counter-current extraction, co-current extraction, semi-batch type extraction, and the like. Various extraction methods such as batch type extraction can be used.

The purpose of this process is to extract and remove as much of the extractable material as possible with minimal hot water from the biomass. In the case of batch extraction, the biomass pulverized material is placed in an extractor, hot water is added for extraction, and solid-liquid separation is performed to remove the liquid material. At this time, the removal efficiency of the extractable material starts at a weight ratio of approximately 1: 4 (biomass and As the proportion of water increases). For example, when 1 kg of biomass is added to 20 L of water and heated to 50 ° C., solid-liquid separation is performed to obtain 3 kg of dry matter and 18 L of aqueous solution, 90% of the extractable components are removed, and only the remaining 10% is pretreated. Enter the fair. However, when using the extract in terms of the biomass fraction, it is preferable that the liquid-liquid ratio does not exceed 1:20 in consideration of dehydration cost and water treatment cost.

The removal efficiency of extractables by hot water extraction of biomass increases with increasing temperature during extraction and solid-liquid separation. In addition, the higher the particle size of the biomass, the shorter the immersion time, but considering the energy cost and the efficiency of countercurrent extraction during grinding, it has a size that can be used as a raw material for pretreatment of the biomass, for example, has an average particle size of 0.1 mm to 50 mm. It is advantageous to crush or crush first.

The water content of the solid content after extraction of the extractable material varies depending on the method used and the application equipment, but is preferably 50% to 90%. If necessary, a continuous centrifugal separator, a filter press, a drum filter, or a screw press may be used to obtain the dehydrated solid content as much as possible after the extraction process.

In the method of the present invention, the hydromass pretreatment step of biomass is to maximize the sugar yield while minimizing the generation of microbial inhibitors. It is a hot water pre-treatment performed by adding water under the condition that the yield is maximized.

The above process can be achieved by adding water to the solid content obtained by hydration and pretreatment at 170 to 210 ° C for 1 to 30 minutes. The hot water pretreatment may be performed by a batch type or continuous pretreatment. The amount of water added to the raw material in the hot water pre-treatment, that is, the solid-liquid ratio is not particularly limited as long as it is an amount suitable for carrying out the hydrolysis reaction of the biomass, but in the present invention, the reactants are not washed separately after the pre-treatment, thus producing during the pre-treatment efficiency and pre-treatment. It is preferable that the ratio of raw materials to moisture is a weight ratio of 1: 3 to 1:15 in consideration of carry-over of the microbial growth inhibitory substances to a subsequent process. For example, in the case of batch-type hot water pretreatment of herbal biomass for general purpose fermentation sugar production, water or water and clay minerals are added to the solids recovered by moistening the sunflower stem powder, the solids ratio is adjusted to 8%, and then this is high pressure. It can be placed in a reactor and reacted at 190 ° C. for 5 minutes (refer to Republic of Korea Publication No. 2012-73087 and International Publication No. WO / 2012/087068). At this temperature, the yield of xylose and xylan produced by hydrolysis of hemicellulose is maximized, while the amount of HMF produced by overlysis of perfural and cellulose that is easily decomposed is minimal. On the other hand, since the yield of glucose that can be produced in enzymatic saccharification after pretreatment is lower than that in pre-treatment under more intense conditions, clay minerals during enzymatic saccharification (Republic of Korea Publication No. 2012-73087 and International Publication No. WO / 2012/087068) or polyethylene glycol (US Patent No. 7,972,826) can be used.

In the method of the present invention, the solid-liquid separation step is a step of obtaining a solid content containing a small amount of liquid material by solid-liquid separation from the obtained hot water pretreatment. The solid-liquid separation process may be performed according to all solid-liquid separation processes commonly used in the art, and examples thereof include centrifugation, suction filtration, and pressure filtration. The pre-processed solids obtained by the solid-liquid separation may contain about 2 to 4 times the pre-processed liquid in the building. In the fermentation sugar production method of the present invention, as in many research papers, the pre-processed solids are not washed with hot water, but the subsequent cellulase complex remains Enzyme is added to the saccharification process. It is preferable that the pre-treatment liquid contained in the solid content after solid-liquid separation is adjusted to contain 5% to 30% of the liquid contained in the pre-treatment immediately after pre-treatment. The liquid material contains an appropriate amount of microbial growth inhibitory material to suppress the growth of microorganisms that can be frequently contaminated from the air, such as lactic acid bacteria, at and around 50 ° C, the optimal working temperature of cellulose hydrolase, without any sterilization. It has the advantage of enzymatic saccharification for more than 72 hours.

The pre-treated liquid material recovered by the solid-liquid separation contains a small amount of xylose, a large amount of xylo-oligosaccharides, acetic acid, traces of perfural, and water-soluble lignin decomposition products produced by hydrolysis of hemicellulose, and Most of the impurities are removed by step 1 and can be used as an excellent raw material for reprocessing with xylitol or dietary fiber.

In the method of the present invention, the step of recovering the sugar solution is a step of recovering the sugar solution from the obtained saccharide by repeating solid-liquid separation and extraction. In the recovery step, methods such as continuous centrifugation, filter press, and batch centrifugation may be used. For example, in the batch recovery process, the sugar produced by enzymatic saccharification is repeated 3 to 5 times to recover the supernatant by centrifuging the saccharide and diluting the saccharified residue in water of the same volume and centrifuging to recover the sugar solution. Can recover more than 99%.

In the method of the present invention, the concentration step is a step of filtering, concentrating, and removing impurities from the obtained sugar solution. The final sugar concentration of the sugar solution obtained by the recovery of the sugar solution is diluted in the process of sugar recovery and lowered to about 50% of the initial concentration, resulting in about 60 g / L to 150 g / L. Through the concentration process using can be increased to a sugar concentration of 30% or more. The concentration process may be performed using reverse osmosis, nanofiltration, etc., which are well known in the art. In addition, to reuse the enzyme contained in the sugar solution, it may be recovered using ultrafiltration, or heated to denature to make a precipitate and then removed using solid-liquid separation technology.

Hereinafter, examples will be described in detail to specifically describe the present invention.

Example

Manufacturing example . Palm Classroom Enzymatic saccharification  Preparation of temperament

Water was added to 10 kg of dried fruit bunch of oil palm (10 mm or less) to prepare biomass for hydrothermal treatment with a water content of 65%. This biomass was injected into a continuous high pressure reactor (SuPR2G, Advancebio product, USA) and subjected to a thermo-hydrotreating at 200 ° C for 10 minutes. Water was added to the preheated water treatment to prepare 100 kg of goonjuk, followed by solid-liquid separation with a filter press (Taeyoung Filtration, Korea). The solid obtained in the cake state was crushed with a cutting mill, and a calcium hydroxide suspension was added thereto and mixed to prepare a substrate for saccharification containing a solid content of 24.4% by weight and a ratio of 57.8 g of cellulose to glucose when lyophilized.

Test example  1. Palm Broad-spectrum Enzymatic saccharification

260 g of the enzyme saccharification substrate of Preparation Example 1 was taken and transferred to a fermenter of three fermenters (CNS, Korea), and then nonionic water was added to make the total amount of 2 L. Thereafter, 4.5 mL of Cellic CTec2 and 0.5 mL of Cellic HTec2 were added as cellulose hydrolase, and the fermenter was stirred at 200 rpm while maintaining at 50 ± 0.5 ° C. and pH 5.5 ± 0.1. After enzymatic saccharification, samples of 1 mL are taken at regular time intervals for 72 hours and analyzed by Agilent HPLC (1260 series) equipped with an Agilent Hi-Plex H column and a refractivity index detector to calculate the concentration of glucose and other sugars. Did.

About 23 g / L of glucose was generated 72 hours after the initiation of saccharification in two saccharification reactors, and only about 18 g / L of glucose was produced in one saccharification reactor (Fig. 1), and lactic acid, a microbial metabolite, was also detected. Became (Fig. 2). MRS solid medium (Bacto peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, K ₂ HPO ₄ 2 g / L, MgSO ₄ 7H ₂ O 0.2 g / L, MnCl ₄ H ₂ O 0.05 g / L, Glucose 20 g / L, agar 15 g / L) were plated and cultured at 37 ° C. to confirm the presence of microorganisms. As a result, it was confirmed that microorganisms were present, and as a result of analysis and identification of 16s rDNA, it was found that the thermophilic strain was Bacillus coagulans (FIG. 3). The optimum growth temperature of this Bacillus coagulant was 50 ~ 55 ℃, which was almost identical to the optimum temperature of the glycosylase. As a result, it was confirmed that glucose was converted into lactic acid by metabolism of contaminated Bacillus coagulance, as the concentration of glucose production was decreased in enzymatic saccharification of palm co-fermentation glycosylation substrate.

Test example  2. Artificially Bacillus Core regulation  Contaminated Saccharification  Antiseptic effect of sulfite on

Sodium metabisulfite, NaHSO ₃ , Na ₂ SO ₃ , Na ₂ S ₂ O ₄ , potassium metabisulfite () Potassium metabisulfite) and five other sulfites can be used through experiments. Bacillus coagulants were cultured at 4 ° C and 250 rpm in 4 1 L flasks containing 600 ml MRS medium, and when OD ₆₀₀ reached 3.0 (about 1 × 106 CFU / ml), 24 sterilized 250 ml flasks Then, 100 ml of the cell culture solution was dispensed, and 5 sulfite solutions prepared at 10 g / L each were added to each flask at a concentration of 25 mg / L to 100 mg / L. While culturing in the incubator, the growth amount of the strain was measured during the culture time.

When sulfite was added at a concentration of 100 mg / L, Bacillus coagulance could be completely killed, and when added at a concentration of 50 mg / L, the killing ability was different depending on the type of sulfite, among which sodium metabisulfite ( sodium metabisulfite) had the best killing ability. As a result of this experiment, it was confirmed that all five sulfites have the ability to kill Bacillus coagulance.

Example  One. Bacillus Core regulation  Contaminated Saccharification  Sterilization effect of sulfite on

When using two fermenters to enzymatically saccharify the pre-processed solids as a substrate as in Test Example 1, the Bacillus coagulant of 1 × 10 ⁶ CFU / ml was first contaminated and cultured for 30 hours. When 0.1 g / L of lactic acid was detected in saccharide by growth of Bacillus coagulance, sodium metabisulfite was added as a reagent to 2,000 mg / L in one fermenter. Subsequently, while continuing saccharification, 1 ml of each sample was taken during saccharification to measure the concentration of glucose and lactic acid. As a result, after 72 hours of enzyme glycosylation, about 15 g / L of glucose and 2.5 g / L of lactic acid were generated in the case of a saccharide without sodium metabisulfite. However, in the case of the saccharide added with sodium metabisulfite, the glucose increased to about 20.5 g / L, which was slightly smaller than the glucose concentration of the microorganism-free test group, and the lactic acid did not increase further to 0.1 g / L. In addition, Bacillus coagulability was not detected in the saccharides to which sodium metabisulfite was added.

Example  2. Enzymatic saccharification  Pre-sulfite Saccharified Enzymatic saccharification

As in Test Example 1, 260 g of enzyme saccharification substrates were taken at a time and transferred to a fermenter of three fermenters (CNS, Korea), followed by adding non-ionized water to make a total amount of 2 L. A fermenter was mounted on the fermenter and 1 × 10 ⁶ CFU / ml of Bacillus coagulant active strain was added. While measuring the acidity, an aqueous solution of sulfuric acid (1 N) was added to adjust the pH to 3.5, and the temperature of the fermenter was raised to 60 ° C. and then sodium metabisulfite was added to a concentration of 1,000 mg / L. The mixture was stirred at a rate of 100 times per minute and held for 10 minutes. Then, 27 ml of hydrogen peroxide (3% by weight) was added and stirred for 1 hour to remove the toxicity of sulfite. After the fermenter was adjusted to 50 ± 0.5 ° C and pH 5.5 ± 0.1, 4.5 mL of Cellic CTec2 and 0.5 mL of Cellic HTec2 were added as cellulose hydrolase, and saccharification was performed for 72 hours. During the saccharification, 1 ml of saccharide was taken at 24 hour intervals, and the concentrations of glucose and lactic acid were measured. In addition, this saccharide is a MRS solid medium (Bacto peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, K2HPO4 2 g / L, MgSO ₄ 7H ₂ O 0.2 g / L, MnCl ₄ H ₂ O 0.05 g / L, Glucose 20 g / L, agar 15 g / L) and cultured at 37 ° C. to confirm the presence of microorganisms.

As a result, about 72 g / L of glucose was generated after 72 hours of enzymatic saccharification, and lactic acid was not detected. In addition, colonies were not observed at all in the microbial culture experiment. As a result, it was confirmed that sodium metabisulfite effectively inhibits the generation of microorganisms in enzymatic saccharification of biomass.

Example  3. Saccharification  High-load Saccharification  For sulfite Core regulation  Pollution prevention effect

Similar to Example 2, but the enzyme saccharification substrate was taken at a concentration of about 780 g (260 g × 3), and transferred to a fermenter of three fermenters (CNS, Korea), followed by adding non-ionized water to make a total amount of 2 L. A fermenter was mounted on the fermenter and 1 × 10 5 CFU / ml of Bacillus coagulant active strain was added. While measuring the acidity, an aqueous solution of sulfuric acid (1 N) was added to adjust the pH to 3.5, and the temperature of the fermenter was raised to 60 ° C. and then sodium metabisulfite was added to a concentration of 2,000 mg / L. The mixture was stirred at a rate of 100 times per minute and held for 10 minutes. Thereafter, 54 ml of hydrogen peroxide (3% by weight) was added and stirred for 1 hour to remove the toxicity of sulfite. After the fermenter was adjusted to 50 ± 0.5 ° C and pH 5.5 ± 0.1, 13.5 mL of Cellic CTec2 and 1.5 mL of Cellic HTec2 were added as cellulose hydrolase and saccharification was performed for 72 hours. During the saccharification, 1 ml of saccharide was taken at 24 hour intervals, and the concentrations of glucose and lactic acid were measured. In addition, this saccharide is a MRS solid medium (Bacto peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, K ₂ HPO ₄ 2 g / L, MgSO ₄ 7H ₂ O 0.2 g / L, MnCl ₄ H ₂ O 0.05 g / L, Glucose 20 g / L, agar 15 g / L) was plated and cultured at 37 ° C. to confirm the presence of microorganisms.

As a result, after 72 hours of enzyme glycosylation, glucose was produced at about 40 g / L, and lactic acid was not detected. In addition, colonies were not observed at all in the microbial culture experiment. As a result, it was confirmed that sodium metabisulfite effectively inhibits the generation of microorganisms in enzymatic saccharification of biomass.

Claims (12)

Sterilizing the biomass at 20 ° C to 90 ° C with sulfite;
Detoxifying the sulfite by adding an oxidizing agent to the reactant; And
After the detoxification, the step of saccharifying the biomass at 50 ± 5 ℃ by adding a saccharifying enzyme; includes,
The sterilization is to sterilize microorganisms containing Bacillus coagulans contained in the biomass, a method for saccharifying biomass.
The method of claim 1, wherein the biomass is an acidic condition.
The method of claim 2, wherein the acidic condition is pH 5 or less.
The method for saccharifying biomass according to claim 1, further comprising adjusting the pH to 5 or less by adding an acid to the biomass.
The method according to claim 1, wherein the sterilization is performed at 30 to 60 ℃, the method of saccharification of biomass.
The method according to claim 1, The sulfite is selected from the group consisting of sodium sulfite, sodium metabisulfite, sodium thiosulfate, potassium sulfite, potassium metabisulfite, potassium thiosulfate, calcium sulfite, calcium metabisulfite and calcium thiosulfate. The method of mass saccharification.
The method of claim 1, wherein the sterilization is performed within 1 hour.
delete The method of claim 1, wherein the oxidizing agent is oxygen, hydrogen peroxide or ozone.
The method for saccharifying biomass according to claim 1, wherein the oxidizing agent is added in an amount of 0.2 to 2 times equivalent of sulfite.
The method according to claim 1, The detoxification is carried out at 20 to 90 ℃, the method of saccharification of biomass.
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