JP2011078327A - Method for separating fermentation inhibitor from sugar solution - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for separating a fermentation inhibitor from a sugar solution, by which the fermentation inhibitor can efficiently be separated from the sugar solution containing the fermentation inhibitor, such as a sugar solution obtained by hydrolyzing a polysaccharide-based biomass such as lignocellulose. <P>SOLUTION: The method for separating the fermentation inhibitor from the solution containing the sugar and the fermentation inhibitor to obtain the sugar solution is characterized by allowing a polystyrene-based resin to adsorb or hold the fermentation inhibitor to separate the sugar solution. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for separating a fermentation inhibitor from a sugar solution containing a fermentation inhibitor and a method for producing a useful substance such as ethanol and xylitol using a sugar solution obtained by using the method.

In Japan, 70% of the country is forested and rich in polysaccharide biomass such as lignocellulose. In recent years, trial production of bioethanol has been started as a national policy, and if bioethanol production is performed using waste wood, thinned wood, etc., it will be very advantageous in terms of cost. In order to produce bioethanol from polysaccharide-based biomass, it is necessary to hydrolyze the biomass to form monosaccharides or disaccharides that can be used for fermentation microorganisms such as yeast. In the process, fermentation inhibitors such as furfurals and phenols are always generated. In addition, xylitol is a substance useful as a sweetener and the like, and fermentation production from polysaccharide biomass has also been attempted, but there is also a problem of a decrease in yield due to generation of a fermentation inhibitor.
Conventionally, these fermentation-inhibiting substances have been removed with ion exchange resins or distillation, and recently activated carbon (for example, Patent Document 1), but none of them has advantages and disadvantages and is not economical. For example, ion exchange resins are selective, and only about 50% of hydroxymethylfurfural (HMF) can be removed. Distillation requires a large amount of heat and is not cost effective. Activated carbon requires a high temperature treatment of 700 ° C. for its production and has a problem that its production and regeneration are very expensive.
Moreover, although this inventor has disclosed the production | generation method of the raw material for ethanol fermentation which removed the coloring substance from molasses or waste molasses using hydrophobic chromatographic resin (patent document 2), it is from polysaccharide biomass, such as lignocellulose. Therefore, there has been a demand for a method that can remove fermentation inhibitors such as HMF more efficiently.

JP 2005-270056 JP JP 2009-95282 JP

  An object of the present invention is to provide a method capable of efficiently separating a fermentation inhibitor from a sugar solution containing a fermentation inhibitor such as a sugar solution obtained by hydrolyzing polysaccharide biomass such as lignocellulose.

  The present inventor has intensively studied to solve the above problems. As a result, by performing column chromatography using a polystyrene-based resin, the fermentation inhibitor can be efficiently removed from the sugar solution containing the fermentation inhibitor, and by using the sugar solution thus obtained, It discovered that fermentation production efficiency, such as ethanol and a xylitol, improved, and completed this invention.

That is, the present invention provides the following.
(1) A method for obtaining a sugar solution by separating a fermentation inhibitor from a solution containing sugar and a fermentation inhibitor, wherein the fermentation inhibitor is adsorbed or retained on a polystyrene resin and separated. .
(2) The solution containing the sugar and the fermentation inhibitor is a polysaccharide biomass hydrolyzate (1
) Method.
(3) The method according to (2), wherein the polysaccharide biomass is lignocellulose.
(4) The polystyrene resin is Amberlite (registered trademark) XAD4 resin, (1)
The method in any one of-(3).
(5) The method according to any one of (1) to (4), wherein the fermentation-inhibiting substance is one or more substances selected from the group consisting of furfural, HMF, and vanillin.
(6) Further, the fermentation inhibiting substance adsorbed or retained on the resin is eluted from the resin and recovered.
The method in any one of (1)-(5) including the process to include.
(7) The method according to (6), wherein the fermentation inhibitor is eluted using an alcohol having 1 to 3 carbon atoms.
(8) The method according to (7), wherein the alcohol is isopropanol or methanol.
(9) The method according to (7) or (8), wherein after the fermentation inhibitor is eluted using the alcohol, the resin is regenerated and reused.
(10) The elution is performed by passing the alcohol from the upper side to the lower side of the column installed in the vertical direction, and the regeneration of the resin is performed by passing the alcohol through the column from the lower side to the upper side of the column. The method according to (9), which is carried out by passing a washing solution.
(11) A step of obtaining a sugar solution by the method according to any one of (1) to (10), and culturing a microorganism that converts sugar to a useful substance using the obtained sugar solution, thereby fermenting the useful substance. A method for producing a useful substance, which includes a production step.
(12) The method according to (11), wherein the useful substance is ethanol or xylitol.

(13) A method for purifying a substance from a solution containing one or more kinds of substances selected from the group consisting of furfural, HMF and vanillin, wherein the substance is purified using a polystyrene-based resin.
(14) The polystyrene-based resin is Amberlite (registered trademark) XAD4 resin, (1
The method according to 3).

According to the present invention, HMF that has been most difficult to remove and that has the highest fermentation inhibition ability can be almost completely removed from the hydrolysis solution of polysaccharide biomass. Thereby, alcohol fermentation can be performed with very high efficiency.
In addition, the fermentation inhibitor adsorbed or retained on the column can be eluted with a small amount of alcohol, and the resin can be easily regenerated with the column packed. The recovered fermentation inhibiting substances such as vanillin, furfural and HMF can also be used as raw materials for fragrances, synthetic rubbers, furan resins, nylons, biofuels and the like in various industries.

Chromatogram when fermentation inhibitor is eluted with 70% methanol using 25 ml of Amberlite XAD4 resin. Chromatogram when fermentation inhibitor was eluted with 70% isopropanol using 25 ml of Amberlite XAD4 resin. Chromatogram when fermentation inhibitor is eluted with 70% methanol using 100 ml of Amberlite XAD4 resin.

The method of the present invention is a method for obtaining a sugar solution by separating a fermentation inhibitor from a solution containing sugar and a fermentation inhibitor, and separating the fermentation inhibitor by adsorbing or holding it on a polystyrene resin by interaction. It is characterized by that.

  Examples of the sugar include monosaccharides such as galactose, glucose, fructose, mannose, and xylose, disaccharides such as sucrose, maltose, and lactose, and combinations thereof. In this, the saccharide | sugar produced by hydrolyzing cellulosic biomass is more preferable.

  Fermentation-inhibiting substance means a substance that inhibits the growth of microorganisms or reduces substance production efficiency in the fermentation process in which useful substances are produced by culturing microorganisms in a medium containing a carbon source (sugar). . Among them, fermentation inhibitory substances produced in the process of subjecting polysaccharide biomass such as lignocellulose to acid treatment or supercritical water treatment are preferable, and specific examples include furfural, HMF, and vanillin.

The solution containing the sugar and the fermentation inhibitor may be a liquid (preferably an aqueous solution or a buffer) obtained by dissolving the sugar and the fermentation inhibitor, but hydrolyzate polysaccharide biomass such as lignocellulose. It is preferably a solution containing a sugar obtained by the above and a fermentation inhibitor such as furfural, HMF, vanillin.
Hydrolysis of polysaccharide-based biomass such as lignocellulose can be performed by a known method such as hydrolysis with water in a subcritical state or supercritical state, hydrolysis with acid, and enzymatic saccharification.

  The polystyrene resin used in the method of the present invention may be a resin having a polystyrene skeleton, and may be a copolymer of styrene and another monomer such as a styrene-divinylbenzene copolymer. For example, specifically, Amberlite (registered trademark) XAD4, XAD1180N, XAD1600, XAD2000, Amberlite (registered trademark) FPX66, FPX68, and Amberlite (registered trademark) EX4 are exemplified. Of these, Amberlite XAD4 is more preferable. This resin is a copolymer having a huge network structure and has a small average pore size and a translucent appearance. Therefore, the surface area of the resin is larger than that having a large average pore diameter.

Separation is preferably performed by column chromatography.
Column chromatography using a polystyrene resin can be performed according to the procedure of column chromatography using a normal hydrophobic adsorption resin.
For example, first, a column packed with a polystyrene resin is equilibrated with water or a buffer solution. Next, a solution containing a sugar and a fermentation inhibitor is passed through the column and passed through the column. Here, sugar passes through the column, whereas fermentation inhibitors such as furfural and vanillin are adsorbed to the column resin, while HMF is held there for a certain period of time, so the difference between this adsorption and holding time is utilized. Sugar and fermentation inhibiting substances can be separated.
That is, purified sugar can be obtained by collecting the flow-through fraction. The presence of sugar can be monitored by the Brix degree or absorbance at 195 nm (A195).
If the solution containing sugar and fermentation inhibitor is a hydrolyzate of polysaccharide biomass, it may be loaded on the column as it is, but if it contains insoluble matter, centrifuge to prevent clogging of the resin. It is preferable to load the column after removing the insoluble matter by the membrane separation method.

For the purpose of purifying sugar, fermentation inhibitor such as vanillin adsorbed on the resin does not need to be recovered, but HMF elutes as a very broad peak fraction after a certain time after passing through the column. Come. Therefore, it must be eluted from the column together with the adsorbed or retained fermentation inhibitor to prepare for the next sugar separation operation. In addition, fermentation inhibitory substances such as vanillin, furfural, and HMF are useful as fragrances, synthetic rubbers, furan resins, nylons, raw materials for producing biofuels, etc., and are therefore preferably eluted and recovered from the column.
The solvent used for the elution of the fermentation inhibitor is not particularly limited as long as it is a solvent that can elute the fermentation inhibitor from the polystyrene resin. In particular, in the case of HMF, since the adsorptive power to the resin is weak, it can be eluted from the column by passing water. On the other hand, in the case of furfural and vanillin completely adsorbed with the resin, it is preferable to elute with an alcohol having 1 to 3 carbon atoms in order to obtain as few fractions as possible. Here, examples of the alcohol having 1 to 3 carbon atoms include methanol, ethanol, propanol, and isopropanol, and isopropanol is more preferable. The alcohol having 1 to 3 carbon atoms used for elution may be a hydrous alcohol. In that case, the alcohol concentration is preferably 50% or more, and more preferably 70% or more.
The elution of the fermentation inhibitor can be monitored, for example, by absorbance at 280 nm (A280).

  In the case where a fermentation inhibitor is collected using an alcohol having 1 to 3 carbon atoms, when a plurality of fermentation inhibitors are present, it is preferable to obtain each fermentation inhibitor by distillation using a difference in boiling points. For example, the boiling points of furfural, HMF, and vanillin are 162 ° C, 115 ° C, and 285 ° C, respectively.

  Moreover, the C1-C3 alcohol used for the elution of a fermentation inhibitory substance can also be collect | recovered and reused by methods, such as distillation.

In addition, the column after recovering the alcohol having 1 to 3 carbon atoms can be regenerated and reused using a column cleaning solution such as water.
When collecting alcohol with 1 to 3 carbon atoms remaining in the column with a column cleaning solution such as water, the water has a higher specific gravity when water is passed over the remaining alcohol, that is, from the top of the column. May occur, and it may be difficult to efficiently recover the remaining alcohol. Therefore, it is preferable to install the column in the vertical direction and to flow a cleaning solution such as water in the direction opposite to the case of the sample solution and alcohol, that is, from the bottom to the top of the column.

Since the sugar solution obtained by the method of the present invention contains almost no fermentation-inhibiting substance, it can be suitably used for the fermentation production of useful substances such as alcohols such as ethanol, sugar alcohols such as xylitol, carboxylic acids, and amino acids. .
That is, the present invention is characterized in that a sugar solution from which a fermentation inhibitor is removed by the above method is obtained, and a microorganism that converts sugar into a useful substance is cultured using the obtained sugar solution to obtain a useful substance. To provide raw materials for the production of useful substances.

Hereinafter, the fermentation production of ethanol will be described.
Yeast is added to the sugar solution that is the raw material for ethanol fermentation, and ethanol fermentation is performed under anaerobic conditions. Fermentation is promoted by static culture at 30 ° C. Usually, ethanol accumulates in the medium by reaction for 1 to 72 hours, and ethanol is obtained by recovering this by distillation or the like. As the ethanol-fermenting microorganism, any known ethanol-producing microorganism can be used, and specific examples include yeast ( Saccharomyces cerevisiae ) and zymomonas mobilis . These microorganisms may be wild strains, mutant strains or genetically modified strains.

In the case of using Saccharomyces cerevisiae , those stored by slanting or freezing may be used, but commercially available baker's yeast may be used. When yeast in a state preserved with slant or the like is used, it is desirable to increase the amount and activity of the yeast by performing pre-culture using a liquid medium before use in fermentation.
Although the production of ethanol using a microorganism that converts sugar into ethanol has been described above, other useful substances can be produced according to known methods using microorganisms that produce known useful substances. For example, xylitol useful as a sweetener can also be efficiently produced using a sugar solution from which a fermentation inhibitor is removed obtained by the method of the present invention.
It is known that lignocellulose contains cellulose and hemicellulose, and that the abundance ratio of the latter is high in plant-based biomass. When plant-based biomass containing a large amount of hemicellulose is decomposed by dilute sulfuric acid decomposition or the like, xylose is generated in the same manner as glucose. Therefore, xylitol can be produced by fermentation using a solution containing xylose obtained by the method of the present invention. Examples of the method for fermentative production of xylitol include the methods described in JP-A-11-192095 and the like.

  The present invention is a method for purifying a substance from a solution containing one or more kinds of substances selected from the group consisting of furfural, HMF and vanillin, wherein the substance is purified using a polystyrene resin. A method is also provided. The solution containing one or more substances selected from the group consisting of furfural, HMF, and vanillin is not limited to the above-mentioned sugar and a solution containing these substances, and may be a solution containing these substances and other substances. . The purification method is as described in the item of the separation method.

  The following examples illustrate the present invention more specifically. However, the present invention is not limited to the following examples.

[Example 1]
An aqueous solution containing the sugar and fermentation inhibitory substances shown in Table 1 was prepared. This composition simulates the composition of a lignocellulose hydrolysis solution with supercritical water.

As a polystyrene chromatography resin, 25 ml of Amberlite XAD4 (organo) was packed in a column tube (diameter 1.4 cm × 16 cm) and equilibrated with ion-exchanged water.
From the upper side, 354 ml of the aqueous solution having a pH of 4.7 was passed through the obtained column (flow rate: 11.5 μl / second), and then ion-exchanged water was further flowed. The liquid passing through the column was fractionated into 2.1 ml fractions, and A280 and Brix degree were monitored for each fraction. Then, after the Brix degree became 0.1 or less (from the time when the elution amount was 354 ml), the liquid flow was switched to 70% methanol. All column flow operations were performed at room temperature.

  The results are shown in FIG. In the elution volume of 0 to 336 ml (through fraction), the Brix degree was 0.1 and sugar was detected, while the A280 value was small, indicating that HMF, furfural and vanillin were almost absent. It was. The sugar recovery rate in the flow-through fraction was calculated to be 90% or more.

In contrast, in the 70% methanol elution fraction (360 to 460 ml), the A280 value was large, indicating that HMF, furfural and vanillin were eluted with 70% methanol. The recovery rate of these fermentation inhibitors in the 70% methanol elution fraction was calculated to be 99% or more. In addition, although the Brix degree becomes large after 360 ml, this is because 70% methanol has a Brix degree of 5 to 6, and sugar is not contained in this fraction.

From the above, it was found that by using a polystyrene-based resin, sugars and fermentation inhibiting substances can be separated, and further, the fermentation inhibiting substances adsorbed on the resin can be eluted with 70% methanol. The half width of the peak fraction of the fermentation inhibitor eluted with 70% methanol was 50 ml.
After flowing 70% methanol, ion-exchanged water was flowed from the bottom to the top to regenerate the column.

[Example 2]
300 ml of the aqueous solution (pH: 4.8) shown in Table 2 was passed through the polystyrene resin column (flow rate: 12.2 μl / second), and then ion-exchanged water was allowed to flow. The flow-through was divided into 4.4 ml fractions, and A280 and A195 (which are much more sensitive to sugar concentration than Brix degree) were monitored for each fraction. And after A195 became 0.01 or less (from the time of elution amount 352 ml), the liquid flow was switched to 70% isopropanol. All column flow operations were performed at room temperature.

  The results are shown in FIG. In the column-flowing solution (through fraction) with an elution volume of 0 to 330 ml, the A195 value was 0.01 or more and sugar was detected (A195 not shown), while the A280 value was small, and HMF, furfural and vanillin Was found to be hardly included. The sugar recovery rate in the flow-through fraction was calculated to be about 97%.

  On the other hand, in the isopropanol elution fraction (374-414 ml), the value of A280 was large, indicating that HMF, furfural and vanillin were eluted with 70% isopropanol. The recovery rate of these fermentation inhibitors was calculated to be about 98%.

As described above, it was found that by using a polystyrene-based resin, sugars and fermentation-inhibiting substances can be separated, and further, the fermentation-inhibiting substances adsorbed on the resin can be eluted with 70% isopropanol. The half-width of the peak fraction of fermentation inhibitor eluted with 70% isopropanol is 20
It was found that all fermentation inhibitors were eluted in a smaller amount when 70% isopropanol was used than when 70% methanol was used.
Even after flowing 70% isopropanol, the column was regenerated by flowing ion-exchanged water from the bottom to the top of the column.

[Example 3]
The above polystyrene resin is packed into a large column (100 ml for XAD4 resin, column size is 2.2 cm x 40 cm in diameter), and 745 mL of the aqueous solution (pH is 4.9) in Table 2 is passed through (flow rate 11.3 μl / sec)
Then, ion exchange water was poured. The passing solution was divided into 4.5 ml fractions, and A280 and Brix degree were monitored for each fraction. Then, after the Brix degree became 0 (from the time when the elution amount was 892 ml), the liquid flow was switched to 70% isopropanol. All column flow operations were performed at room temperature.

  The results are shown in FIG. In the pass-through fraction, sugar was detected using the Brix degree as an index (the Brix degree is not shown), while the A280 value was small, indicating that HMF, furfural and vanillin were hardly contained.

On the other hand, in the 70% isopropanol elution fraction (896 to 1011 ml), the value of A280 was large, and it was found that HMF, furfural and vanillin were eluted with 70% isopropanol. The recovery rate of these fermentation inhibitors was calculated to be about 94%.
After flowing 70% isopropanol, ion exchange water was flowed from the bottom to the top of the column to regenerate the column.

  From the above, it was found that the experiment can be scaled up sufficiently.

  In addition, when the same experiment as Example 1 was performed using Amberlite XAD7HP (organo company) which is an acrylic resin, the retention time of fermentation inhibitors, particularly HMF, was short (at an elution amount of 50 to 55 ml). It was found difficult to obtain a sufficient amount of a sugar solution that does not contain a fermentation inhibitor.

According to the present invention, a fermentation inhibitor can be removed almost completely at room temperature with an adsorbing resin from a hydrolyzed solution of polysaccharide biomass that can be used for ethanol fermentation or the like without impairing the yield of saccharides. As a result, the fermentation inhibitor contained in the hydrolysis solution can be removed to a concentration that does not affect the fermentation, so that a large amount of bioethanol can be produced from abundant polysaccharide biomass in Japan. It is thought that it can make a significant contribution to the achievement of the target for CO2 reduction.
Fermentation-inhibiting substances such as vanillin, furfural and HMF adsorbed or retained on the resin can be recovered using alcohol. These substances are raw materials such as fragrances, synthetic rubbers, furan resins, nylon and biofuels. Useful as.

Claims (14)

A method for obtaining a sugar solution by separating a fermentation inhibitor from a solution containing a sugar and a fermentation inhibitor, wherein the fermentation inhibitor is adsorbed or held on a polystyrene-based resin for separation. The method according to claim 1, wherein the solution containing the sugar and the fermentation inhibitor is a polysaccharide biomass hydrolyzate. The method according to claim 2, wherein the polysaccharide-based biomass is lignocellulose. The method according to any one of claims 1 to 3, wherein the polystyrene-based resin is Amberlite (registered trademark) XAD4 resin. The method according to any one of claims 1 to 4, wherein the fermentation inhibitor is one or more substances selected from the group consisting of furfural, hydroxymethylfurfural and vanillin. Furthermore, the method as described in any one of Claims 1-5 including the process of eluting and collect | recovering the fermentation inhibition substance adsorbed | sucked or hold | maintained at resin from resin. The method according to claim 6, wherein the fermentation inhibitor is eluted using an alcohol having 1 to 3 carbon atoms. The method according to claim 7, wherein the alcohol is isopropanol or methanol. The method according to claim 7 or 8, wherein after the fermentation inhibitor is eluted using the alcohol, the resin is regenerated and reused. The elution is performed by passing the alcohol from the upper side to the lower side of the column installed in the vertical direction, and the regeneration of the resin is performed by passing the column washing liquid from the lower side to the upper side after passing the alcohol. The method according to claim 9, wherein the method is performed. A step of obtaining a sugar solution by the method according to any one of claims 1 to 10, and a step of performing fermentation production of a useful substance by culturing a microorganism that converts sugar to a useful substance using the obtained sugar solution. A method for producing a useful substance. The method according to claim 11, wherein the useful substance is ethanol or xylitol. A method for purifying a substance from a solution containing at least one substance selected from the group consisting of furfural, hydroxymethylfurfural and vanillin, wherein the substance is purified using a polystyrene resin. The method according to claim 13, wherein the polystyrene-based resin is Amberlite (registered trademark) XAD4 resin.

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