JP4064359B2 - Method for separating phosphoric acid and lignocellulose hydrolysis product and method for producing glucose - Google Patents

Description

  The present invention is a method for efficiently separating phosphoric acid and a hydrolyzed product of lignocellulose from a phosphoric acid solution containing a hydrolyzed product of lignocellulose obtained by hydrolyzing lignocellulose with high-concentration phosphoric acid. The present invention relates to a method for producing glucose by hydrolyzing a hydrolysis product of lignocellulose with β-glucosidase.

  Technology related to biomass is being developed worldwide in connection with the reduction of carbon dioxide due to recent global environmental problems. Among them, a method has been proposed in which glucose is obtained from lignocellulose and ethanol or L-lactic acid is produced by fermentation technology. However, the present situation is that efficient industrial production has not yet been carried out (non-patented). Literatures 1-3).

  Among them, the technology that has been developed for many years led by the American National Renewable Energy Laboratory (NREL) is about to be industrialized. These are intended to produce ethanol (bioethanol) for the time being, but use concentrated sulfuric acid or dilute sulfuric acid in the pretreatment and hydrolysis processes. Typical examples include BCI International and Arkenol. There is a way.

  The former method uses concentrated sulfuric acid, but the sulfuric acid is recovered as gypsum by the neutralization method. On the other hand, the latter is a method in which saccharide and sulfuric acid are separated by cation exchange resin using dilute sulfuric acid (Patent Documents 1 to 3).

  Furthermore, as a recent method, there is a method of Controlled Environmental Systems Corporation. In this method, sulfuric acid used is removed by neutralization and anion exchange resin or exclusion chromatography (Patent Document 4).

  On the other hand, the present inventor proposed a method for obtaining glucose by vigorously stirring a cellulosic substance dissolved and / or swollen with a high concentration of phosphoric acid and decomposing the resulting hydrolysis product with an enzyme. However, in this method, a neutralization method and an organic solvent extraction method are used as a method for recovering phosphoric acid (Patent Document 5).

NEDO 2001 Results Report, “Development of Highly Efficient Biomass Energy Conversion Technology / Development of New Ethanol Fermentation Technology Using Cellulosic Biomass / Development of Pretreatment / Saccharification / Ethanol Fermentation Technology” pp. 18-20 NEDO FY 2002 Results Report, “Development of High-efficiency Biomass Energy Conversion Technology / Development of Technology for Producing Ethanol for Fuels by Developing New Ethanol Fermentation Technology Using Cellulose Biomass” (Part 1) NEDO 2002 report, “Development of high-efficiency biomass energy conversion technology / Development of technology for producing ethanol for fuels by development of new ethanol fermentation technology using cellulosic biomass” (Part 2) Japanese National Patent Publication No. 11-506934 US Pat. No. 5,580,389 US Pat. No. 5,820,687 Special table 2001-511418 gazette JP-A-10-11001

In the method disclosed in Patent Document 5, phosphoric acid is recovered using a neutralization method and an organic solvent extraction method, but it is necessary to use an efficient phosphoric acid recovery method in order to further reduce manufacturing costs. Cellulosic substances are treated with high-concentration phosphoric acid, and phosphoric acid is separated from the treatment solution, and then the hydrolysis product is hydrolyzed with enzymes to obtain glucose. Needs to be improved.
Accordingly, an object of the present invention is to efficiently separate phosphoric acid and hydrolysis products from a phosphoric acid solution containing a hydrolysis product obtained by hydrolyzing lignocellulose with phosphoric acid, which has solved the above problems. And a method for producing glucose from lignocellulose capable of mass-producing glucose at low cost.

  As a result of intensive investigations to achieve the above object, the present inventors have found that a phosphoric acid solution containing a hydrolyzed product of lignocellulose obtained by hydrolyzing lignocellulose with a high concentration of phosphoric acid can be efficiently solidified with a separate decanter. Use a column filled with an anion exchange resin that has acid retardation action for the phosphoric acid solution containing lignocellulose hydrolysis products that can be liquid-separated. Thus, it has been found that it can be efficiently separated into an aqueous solution of phosphoric acid and an aqueous solution containing a hydrolysis product of lignocellulose.

In addition, the aqueous solution containing the hydrolyzed lignocellulose product separated by the above column treatment may be used as it is or concentrated by UF (ultrafiltration), RO membrane (reverse osmosis membrane) or NF (nanofiltration). Then, after adjusting the pH of the solution to, for example, about 4 to 6 with an alkaline agent such as sodium hydroxide, in the cellulase (a mixture of three enzymes, endocellulase, exocellulase and β-glucosidase) It has been found that hydrolysis of the lignocellulose hydrolysis product with only β-glucosidase results in hydrolysis in a shorter time than using cellulase (a mixture of the above three enzymes).
On the other hand, the phosphoric acid aqueous solution recovered by the column treatment can be concentrated by using an RO device. By concentrating the concentrated phosphoric acid aqueous solution in a can, it is concentrated by direct can evaporation without being concentrated in the RO device. The concentration cost can be reduced more than the case. The recovered phosphoric acid can be used for hydrolysis of lignocellulose. The present invention has been completed based on these findings.

That is, according to the present invention, a phosphoric acid solution containing a hydrolysis product of lignocellulose obtained by hydrolyzing lignocellulose with high-concentration phosphoric acid is filled with an anion exchange resin having an Acid Retardation action. The hydrolyzed product of lignocellulose is separated in the upward flow, and the separation of phosphoric acid is performed in the downward flow to separate the phosphoric acid and the hydrolyzed product of lignocellulose. A separation method is provided.
Moreover, according to this invention, the manufacturing method of glucose characterized by hydrolyzing the hydrolysis product of the lignocellulose isolate | separated above by (beta) -glucosidase is provided.

  According to the present invention, a solution containing glucose, cellooligosaccharide and other monosaccharides can be produced at low cost from lignocellulose, and glucose crystals can be produced by concentrating and crystallizing the solution after purification. It is. Moreover, it can be used as an inexpensive alternative such as molasses which is generally used as a fermentation raw material such as ethanol and L-lactic acid without purifying the above solution and containing monosaccharides other than glucose.

Next, the present invention will be described in more detail with reference to the best mode for carrying out the invention.
Examples of lignocellulose used as a raw material in the present invention include, for example, old newspapers, magazines, cardboard, office waste paper, wood quality (wood chips, building waste, thinned wood, driftwood, roadside trees or garden tree pruning materials, etc.) Agricultural waste such as bamboo, bagasse, straw, corn cob, linter, cotton, pulp, and waste pulp discharged from paper manufacturers or cellulose-rich components obtained by organosolv treatment such as acetic acid. Of the above lignocelluloses, particularly preferred in the present invention are old newspapers, linters, cotton and pulp.

In the present invention, the lignocellulose is first hydrolyzed using high-concentration phosphoric acid, but the hydrolysis method is not particularly limited. A preferred method is the following method.
Lignocellulose is added to high concentration phosphoric acid to dissolve and / or swell. As the high-concentration phosphoric acid used, 70% by mass or more of phosphoric acid is preferable, and 81-85% by mass or 92-97% by mass of phosphoric acid is particularly suitable for the present invention. Lignocellulose is preferably used at a ratio of about 5 to 20 parts by mass per 100 parts by mass of the high-concentration phosphoric acid.

  Lignocellulose is added to high-concentration phosphoric acid to dissolve and / or swell and then stirred to hydrolyze the lignocellulose. Stirring is not particularly limited as long as hydrolysis of lignocellulose occurs, but for example, it is vigorously stirred for about 1 to 4 hours at a temperature of about 30 to 60 ° C. using a pulper or the like (for example, connected motor 150 It is preferable that the rotation speed of the rotor is 600 to 1000 RPM. By this strong stirring treatment, lignocellulose is hydrolyzed to produce a mixture of amorphous cellulose, cellooligosaccharide, glucose rich monosaccharides other than glucose such as xylose and mannose (hereinafter referred to as “the hydrolysis product of lignocellulose”). Generated).

  In order to separate the lignocellulose hydrolysis product from the high-concentration phosphoric acid solution containing the lignocellulose hydrolysis product, the cake after solid-liquid separation of the phosphoric acid solution (solid content: unhydrolyzed lignocellulose) As a result of various studies on solid-liquid separation methods for minimizing the amount of residual phosphoric acid in the lignocellulose raw material), in other words, improving the recovery efficiency of phosphoric acid, the following method is optimal. I found out. That is, the high-concentration phosphoric acid solution containing the lignocellulose hydrolysis product is poured into the above-mentioned phosphoric acid solution, for example, distilled water having a volume of about 2 to 10 times, and the solid content is sufficiently reprecipitated. By treating the solution containing the solid content as a solid-liquid separation means with a decanter, for example, a horizontal (horizontal) decanter, for example, DS-10V (manufactured by Mitsubishi Chemical Corporation) under the conditions of 2000G to 2500G, a cake (solid It has been found that the amount of residual phosphoric acid in (min) can be minimized. The amount of distilled water is not limited to the above amount, and it is preferable to minimize the amount of residual phosphoric acid in the cake. If the desired amount of residual phosphoric acid cannot be obtained by a single operation, the cake is again dispersed with stirring in distilled water, and the dispersion is again subjected to the above decanter treatment to achieve the desired amount of residual phosphoric acid. Is possible.

  As a result of various examinations on the separation method of the hydrolysis product of phosphoric acid and lignocellulose from the solid-liquid separated solution phase (phosphoric acid solution containing the hydrolysis product of lignocellulose), a negative effect with Acid Retardation (acid retardation) action was obtained. It was found that a column packed with an ion exchange resin can efficiently separate phosphoric acid and lignocellulose hydrolysis products. Examples of the anion exchange resin used here include MA01SS (manufactured by Mitsubishi Chemical), Dowex 11A8 (manufactured by Dow Chemical Japan), AP246 (manufactured by Bayer Japan), and the like. From the viewpoint of separation efficiency, MA01SS is particularly preferable. As the column apparatus, any known system using an ion exchange resin such as a pseudo moving system, a batch system, a fixed bed system, and a fluidized bed system can be used, and the column apparatus is not particularly limited. As a liquid passing method, a phosphoric acid solution (solution phase) containing the above lignocellulose hydrolysis product, preferably a concentrated solution thereof, is passed directly or through a guard column at the bottom of the column packed with the ion exchange resin. After that, the distilled water was allowed to flow in the upward flow, and the phosphoric acid solution containing the hydrolysis product of lignocellulose was recovered from the top of the column while monitoring with a glucose concentration meter and an electric conductivity meter, and then in the downward flow By flowing distilled water, it is possible to discharge and collect the phosphoric acid aqueous solution while monitoring using an electric conductivity meter.

After the aqueous solution containing the lignocellulose hydrolysis product separated and recovered by the column is adjusted to a pH of about 5.5 with an aqueous sodium hydroxide solution, for example, cellulase is added to the lignocellulose hydrolysis product. Glucose can be obtained by hydrolysis. However, not all of this hydrolysis results in glucose, and monosaccharides other than glucose, cellooligosaccharides, and the like are also produced together with glucose. In the present invention, these are collectively referred to as glucose.
Here, as a result of diligent studies to further shorten the reaction time, the hydrolysis product of lignocellulose was added to glucose in a short time by adding only β-glucosidase rather than cellulase (mixture of the above three enzymes). I found that it could be decomposed. In addition, the aqueous solution containing the recovered lignocellulose hydrolysis product may be used by any means rather than using it as it is, particularly preferably by concentrating it using an NF (nanofiltration) membrane. From the point of view, it is preferable. Moreover, although the usage-amount of (beta) -glucosidase is not specifically limited, Usually, it is about 1-10 mass parts per 100 mass parts of hydrolyzed products of lignocellulose.

  When hydrolyzing the lignocellulose hydrolysis product with β-glucosidase, it is possible to carry out the reaction while collecting and reusing β-glucosidase by incorporating a UF membrane into the reaction apparatus to be used. On the other hand, it is also possible to separate the glucose produced by incorporating an NF (nanofiltration) membrane from the remaining unhydrolyzed product and recycle β-glucosidase to continue the reaction.

  Examples of the UF device include, but are not limited to, an agitation type ultra holder UHP-62K manufactured by Advantec, a Pellicon mini folder manufactured by Nippon Millipore, ProFlux M12, and the like. Examples of the UF membrane used in the UF apparatus include Q0100 (fraction 10,000) manufactured by Advantec, Biomax-8 (fraction 10,000) manufactured by Nihon Millipore, and the like. It is not limited.

After hydrolyzing the lignocellulose hydrolysis product with β-glucosidase, the resulting solution containing glucose is usually concentrated and used, but the concentration cost is reduced by using an NF membrane for concentration. It is possible to make it.
Examples of the pressure vessel to which the NF membrane is attached include, but are not limited to, Nitto Denko Membrane Master C40-B, Nippon Millipore Remolino, and the like. Examples of the NF film include DL and DK manufactured by Desalination Systems, but are not limited thereto.

  The concentrated solution is usually decolorized. However, for decolorization, the use of activated carbon is not preferred because it adsorbs saccharides (especially cellooligosaccharides). For example, basic ion exchange resin, particularly VP OC 1074 manufactured by Bayer AG. , MP 500, IRA404J manufactured by Organo, etc. are effective, but are not limited thereto.

  Furthermore, in order to roughly separate glucose from the hydrolysis product of β-glucosidase, for example, use of Desalination Systems G-50 (GM) as a UF membrane, and NF-45 made by Film Tech as an NF membrane, etc. However, it is not limited to these.

  Further, if necessary, in order to increase the purity of glucose (original glucose only) contained in the crude fractionation solution, the crude fractionation solution is separated from glucose using an existing separation column having a simulated moving bed. It can also be separated into other sugars. Examples of the ion exchange resin packed in the separation column include UBK555 manufactured by Mitsubishi Chemical Corporation, VP OC 1800 manufactured by Bayer Corporation, and the like, but are not limited thereto.

  On the other hand, in the present invention, the aqueous phosphoric acid solution separated and recovered from the column filled with the anion exchange resin needs to be concentrated in order to reuse the phosphoric acid. Usually, an evaporation operation is required for concentration, but this process is energy intensive and increases the concentration cost. Therefore, as a result of various studies, it is possible to reduce the total concentration cost by first concentrating the diluted phosphoric acid aqueous solution with a reverse osmosis (RO) membrane apparatus and then further concentrating with an evaporator.

  The concentration process by the reverse osmosis membrane is not particularly limited, and a known batch type (dead end filtration), circulation type (cross flow filtration), or a method of permeating once in a cross flow without circulation, a constant concentration It is possible to carry out any of the methods (diafiltration) of circulating in a cross flow while maintaining the same.

Examples of the pressure-resistant container used in the batch type include, but are not limited to, a membrane master C40-B type manufactured by Nitto Denko Corporation, and Remolino manufactured by Nippon Millipore Corporation.
Moreover, as an apparatus used by a circulation type, RO / UF module, flat membrane testing machine TS-RU-10 type etc. by Toray Industries, Inc. are mentioned, for example. Examples of the reverse osmosis composite membrane used in the circulation apparatus include NTR-70SWC manufactured by Nitto Denko Corporation, SU-810 manufactured by Toray Industries, Inc., SW-4040 manufactured by Film Tech Co., etc. If it is an aromatic polyamide film | membrane which has a rate, it will not be limited to these.

The phosphoric acid aqueous solution concentrated using the RO membrane is concentrated to 70 mass% or more as a phosphoric acid concentration by an evaporator, and mixed with polyphosphoric acid, 89 mass% phosphoric acid or 85 mass% phosphoric acid as appropriate to hydrolyze lignocellulose. Reused.
On the other hand, the permeated liquid of RO membrane can be reused as re-precipitated water of high-concentration phosphoric acid solution of lignocellulose hydrolysis product after passing through activated carbon or ion exchange resin column to remove impurities if necessary. .

  The present invention will be described more specifically with reference to the following examples. “%” In the text is based on mass unless otherwise specified. “Glucose” in the text refers to the original glucose (D-glucose).

[Example 1]
180 g of used newspaper shredded by MS Shredder 122MA manufactured by Meiko Shoji Co., Ltd. is added to a 5 L (liter) beaker containing 1,800 ml of 85% phosphoric acid, heated to 60 ° C. and stirred vigorously for 2 to 4 hours. The used newspaper was dissolved and / or swollen and hydrolyzed. The obtained slurry containing the hydrolysis product was poured into 5 times volume of distilled water with vigorous stirring, and the resulting diluted solution was 2,000 G-2 with a separation decanter DS-10V manufactured by Mitsubishi Chemical Corporation. The solid and liquid were separated by a centrifugal force of 500G. The phosphoric acid residual rate in the cake was 0.7% or less after one operation.

  The hydrolyzed phosphoric acid solution obtained by this solid-liquid separation operation was concentrated with a rotary evaporator, and 160 ml of this concentrated liquid was packed with an anion exchange resin MA01SS manufactured by Mitsubishi Chemical Corporation having an Acid Retardation action (inner diameter 44. 0cm, 46.5cm in height), distilled water as eluent and operated in the upward flow under the conditions of SV: 0.888, LV: 0.414m / hr, The effluent (hydrolysis product stream) was monitored with a conductivity meter and a glucose concentration meter. As shown in FIG. 1, an increase and a decrease in conductivity were first observed, and then an increase and a decrease in glucose concentration were observed. The hydrolysis product fraction was collected. After the hydrolysis product fraction was almost discharged from the column, distilled water was allowed to flow from the uppermost stage of the column to convert it from an upward flow to a downward flow, and an aqueous phosphoric acid solution was recovered. While the phosphoric acid recovery fraction was monitored with a conductivity meter, a high concentration (high conductivity) portion of the phosphoric acid aqueous solution was recovered. On the other hand, the low concentration (low conductivity) portion was reusable instead of distilled water used for dilution in the solid-liquid separation process.

  The aqueous solution containing the hydrolysis product separated by the column is adjusted to pH 5.1 with an aqueous sodium hydroxide solution, 300 ml of the solution is placed in a 500 ml Erlenmeyer flask, and the solution is stirred and kept at 50 ° C. in a thermostatic bath. In addition, 1 ml of Novozymes Japan Novozyme 188 was added as β-glucosidase, and the glucose concentration produced over time was determined as Gluco. Measured with Jr. In addition, hydrolysis was performed in the same manner as described above instead of cellulase (Multifect GC manufactured by Genencor International Japan Limited). As shown in FIG. 2, when β-glucosidase was used, the reaction reached a plateau in 0.5 to 1 hour, but when cellulase was used, it took 20 hours or more.

After completion of the reaction, enzymes such as enzymes from a solution containing the hydrolysis products of the above enzymes by a UF apparatus in which Prostack PSHN AG021 (manufactured by Millipore Japan, Inc.) (pore size 0.45 μm) is incorporated into ProFlux M12 (manufactured by Millipore Japan) Was removed. In order to obtain glucose from this permeation solution, dialysis was performed using an NF membrane (DL, DK) manufactured by Desalination Systems described in Table 1, and the permeation inhibition state of glucose was examined. As shown in Table 1, these NF membranes prevent glucose permeation. The permeation inhibition rate of glucose was 95.4% in DL.
Moreover, although the permeation-preventing property of glucose was confirmed also about UF membrane (G20 (GK)) by Desalination Systems, the permeation-inhibiting property of glucose similar to said NF membrane made by the said company was shown.

  After decolorizing the solution containing glucose and the like after dialysis with a basic ion exchange resin (VP OC 1074 manufactured by Bayer Co.), contaminating ions were removed with a mixed bed column of cation exchange resin and anion exchange resin, Monosaccharides other than glucose were removed and purified by column chromatography as necessary.

  On the other hand, several types of phosphoric acid solutions with different fractionation ranges separated and recovered by the column were used as a circulation device using RO / UF module / flat membrane tester TS-RU-10 manufactured by Toray Industries, and manufactured by Toray Industries as RO module. The solution was concentrated using SU-810 (abbreviated as SU) and SW-4040 (abbreviated as SW) manufactured by Film Tech. As shown in Table 2, the phosphoric acid solution could be concentrated 4 to 6 times.

上記のＲＯ装置で燐酸溶液を濃縮した後、ササクラ社製ＲＨＣ−５で蒸発濃縮して、７０％以上の燐酸溶液を回収して再利用した。

After concentrating the phosphoric acid solution with the above-mentioned RO apparatus, it was evaporated and concentrated with RHC-5 manufactured by Sasakura, and 70% or more of the phosphoric acid solution was recovered and reused.

[Example 2]
100% L of 85% phosphoric acid was put into an HV pulper (volume 0.3 m ³ ) manufactured by ASEC Co., and 10 kg of used newspaper was dissolved while stirring vigorously. A portion of the resulting slurry was placed in a 5 L beaker and vigorously stirred for 2 to 4 hours while maintaining the temperature at 60 ° C. to hydrolyze used newspaper. The slurry containing the hydrolysis product could be treated in the same manner as in Example 1.

The solution containing glucose and monosaccharides other than glucose separated by the above method can be used, for example, as a substitute for molasses such as ethanol fermentation and lactic acid fermentation. By purifying the solution with an ion exchange column, a glucose product is obtained. It is also possible.
In addition, the RO membrane used in the phosphoric acid concentration step in this manufacturing method can also be applied to phosphoric acid concentration in other fields such as plating.

It is a figure which shows the glucose concentration and electrical conductivity in each discharge | emission fraction of the aqueous solution of the hydrolysis product of a lignocellulose isolate | separated using the anion exchange resin MA01SS (made by Mitsubishi Chemical Corporation) in Example 1. FIG. FIG. 3 is a graph showing changes over time when the hydrolyzed products of lignocellulose separated in column in Example 1 were hydrolyzed with β-glucosidase (188: trade name Novozyme188) and cellulase (GC: trade name Multifect GC), respectively. is there.

Claims (4)

A phosphoric acid solution containing a hydrolysis product of lignocellulose obtained by hydrolyzing lignocellulose with high-concentration phosphoric acid is used to form a lignocellulose using a column packed with an anion exchange resin having an acid retardation action. A separation method characterized in that the hydrolysis product of cellulose is separated in an upward flow, and the separation of phosphoric acid is carried out in a downward flow to separate the hydrolysis product of phosphoric acid and lignocellulose. A method for producing glucose, comprising hydrolyzing the hydrolyzed lignocellulose product separated in claim 1 with β-glucosidase. The method for producing glucose according to claim 2 , wherein the aqueous solution of phosphoric acid separated in claim 1 is concentrated with an RO membrane (reverse osmosis membrane), and further evaporated and reused as at least part of phosphoric acid. An aqueous solution of hydrolysis products separated lignocellulose in claim 1, NF concentrated (nanofiltration), the hydrolysis products by the addition of this β- glucosidase to hydrolyze claim 2 The manufacturing method of glucose of description.

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