JP2002355086A - Method for producing acetic acid bacterial cellulose using xylose as raw material - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an acetic acid bacterial cellulose having a large scope of uses by fermenting and assimilating xylose in lignocellulose waste materials such as woody wastes or agricultural industrial wastes. SOLUTION: This method for producing cellulose comprises culturing a microorganism belonging to the genus Acetobacter and having the ability to produce the cellulose in a culture medium in which xylulose in produced by isomerization of the xylose which is a glucide source and producing the cellulose in the culture medium. The method for producing acetic acid bacterial cellulose comprises carrying out the isomerization of the xylose by aseptically adding xylose isomerase into the culture medium and providing a means for suppressing the lowering of pH value of the culture medium with an acidic metabolite of the glucide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing cellulose using xylose derived from woody waste or agricultural waste as a raw material, and more particularly, to the isomerization reaction of xylose to xylulose. Acetic acid bacteria Acetobacter xylinus
(Acetobacter xylinus) The present invention relates to a method for producing acetic acid bacterial cellulose in which xylulose is selectively fermented and assimilated.

[0002]

BACKGROUND OF THE INVENTION Today, as the serious impact on natural environment on a global scale due to the large-scale consumption of fossil resources, which has been facilitated so far, has been recognized, the recyclability and other characteristics of the raw materials of plant resources have been renewed. It is highly evaluated and has high expectations for various technologies that can be used effectively. For example, if sugars in lignocellulosic wastes such as woody wastes and agricultural wastes, such as thinned woods, construction wastes, and sawn timbers that have been discarded, can be used as effective resources, not only effects from an economic standpoint but also It greatly contributes to the preservation of the natural environment.

By the way, polysaccharides such as woody wastes and agricultural wastes are composed of six-carbon sugars (glucose, mannose, galactose) and five-carbon sugars (xylose, arabinose). Among them, xylan, which is a polysaccharide of glucose and cellulose, which is a polysaccharide of xylose, occupies a major constitution. Therefore, monosaccharides obtained by hydrolysis of woody wastes and agricultural wastes are mostly glucose and xylose, and among these monosaccharides, glucose has conventionally been involved in the fermentation reaction of microorganisms. Utilize liquid fuel (ethanol), cell protein (Single Ce)
ll Protein) or other chemical industrial raw materials.

[0004] However, fermentation and conversion of 5-carbon sugars to useful substances are extremely low, if not impossible, depending on the cultivation conditions, and cannot be used for industrial applications. At present, like glucose,
Regarding xylose, it has been a major challenge to realize a technique for performing fermentation and conversion to useful substances with high efficiency by utilizing the fermentation reaction of microorganisms.

[0005]

DISCLOSURE OF THE INVENTION The present invention relates to a microorganism having the ability to produce cellulose belonging to the genus Acetobacter in a medium in which xylose is produced by isomerization of xylose, which is a carbohydrate source. Culturing, providing a method for producing acetic acid bacterial cellulose by producing cellulose in a medium,
An object of the present invention is to solve the above-mentioned conventional problems.

[0006] In the above structure, the means for isomerizing xylose to xylulose may have a structure in which xylose isomerase is aseptically added to the medium.

Further, in the above structure, the microorganism having the ability to produce cellulose belonging to the genus Acetobacter may be:
Acetobacter xylin
us) IFO-15606 strain and / or IFO-13693 strain, and the acetic acid bacterium Acetobacter xylinus
(Acetobacter xylinus)
In some cases, the system may be provided with means for suppressing the decrease in the pH value.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, hestrin and schlum medium (glucose 2%, peptone 0.5%, yeast extract)
0.5%, citric acid 0.115%, disodium hydrogen phosphate 0.27
% Dissolved in pure water), replacing glucose, which is a carbohydrate raw material, with xylose, and adding IFO-15606 of Acetobacter xylinus to this medium.
The strain or IFO-13693 strain is inoculated, and xylose isomerase is aseptically added to the medium as a means for isomerizing xylose of xylose.

[0009] By the addition of the xylose isomerase, isomerization of xylose to xylulose proceeds by an enzymatic reaction in the medium, and this isomerization continues until the ratio of the two reaches equilibrium. The inoculated acetic acid bacteria Acetobacter xylinus selectively ferment and assimilate the isomerized xylulose to produce cellulose.

When xylulose is reduced by fermentation and assimilation, until the ratio of xylose and xylulose equilibrates,
Further, the isomerization reaction with xylose isomerase continues, xylulose is compensated, and the ratio between the two reaches equilibrium. Thereafter, this cycle is sequentially repeated.

The acetic acid bacterium Acetobacter xylinus (Aceto)
As the fermentation and assimilation of xylulose by bacter xylinus progresses, the pH value of the medium due to the acidic metabolites of carbohydrates decreases, causing a stagnant phenomenon of cellulose production. For this reason, it is necessary to take measures to suppress a decrease in the pH value. Means for suppressing the decrease in pH value include lowering the concentration of citric acid in the medium, increasing the concentration of disodium hydrogen phosphate, and adding an alkali reagent.

Next, a specific example of the embodiment according to the present invention will be described. First, using a culture tube having a diameter of 4 cm and a volume of 100 ml, an acetic acid bacterium, Acetobacter xylinus, was inoculated into 50 ml of a medium containing hestrin and schlum containing xylose instead of glucose. After static culture, xylose was not fermented or assimilated and there was no or very little production of cellulose acetate.

On the other hand, when xylose isomerase is added aseptically, the xylose and xylulose in the culture medium are enzymatically reacted until the ratio between xylose and xylulose reaches equilibrium.
Isomerized gradually to xylulose, and the inoculated acetic acid bacteria selectively ferment and assimilate the isomerized xylulose, as shown in Table 1 in FIG. Produced in meaningful yield. That is, the acetic acid bacteria Acetobacter xylinus IF0-15606 strain or IFO-
In 13693 strains, acetic acid bacterial cellulose was efficiently produced with a yield to sugar of about 10%.

The amount of xylulose was determined by resorcinol-
The measurement was carried out by a hydrochloric acid method (Kulka's method) at 615 nm of visible light. This xylulose quantification method is not affected even if xylose coexists.

In this embodiment, xylose in the medium is isomerized to xylulose by aseptic addition of xylose isomerase, but this isomerization reaction of xylose is added. According to the amount of xylose isomerase, it progresses gradually until the ratio of xylose to xylulose in the medium reaches at least equilibrium.
The results were confirmed by the experimental results shown in FIG.

Xylose isomerase is aseptically added to a hestin and schlum medium containing xylose instead of glucose as a carbohydrate source, inoculated with acetic acid bacterium Acetobacter xylinus, and allowed to stand at 30 degrees Celsius for one month. As a result of the incubation, it was confirmed that the pH value of the medium dropped to 4.0 or less. Therefore, in the present embodiment, an operation of suppressing a decrease in pH due to an acidic metabolite of the saccharide of the acetic acid bacterium Acetobacter xylinus is performed. This operation is performed by lowering the concentration of citric acid in the medium, increasing the concentration of disodium hydrogen phosphate, or adding an alkaline reagent. FIG. 3 shows the experimental results of this operation. According to this experiment, the IF0-15606 strain of Acetobacter acetobacillus xylinus was controlled by controlling the pH value so that the pH value of the medium did not drop below 4.8. Per 100ml of medium
It is found to produce more than 0.3 g of acetic acid cellulose.

[0017]

EXAMPLE An acetic acid bacterium, Acetobacter xylinus IFO-15606, was cultured under the following conditions, and an acetic acid bacterium cellulose production experiment was performed. (A) Hestrin and schlum medium (glucose 2%, peptone 0.5
%, Yeast extract 0.5%, citric acid 0.115%, hydrogen phosphate 2
0.27% sodium dissolved in pure water), containing xylose (3%) instead of glucose (2%) as a carbohydrate source, and (ii) a medium containing acidic metabolites of carbohydrates of Acetobacter xylinus. The concentration of the disodium hydrogen phosphate is increased from 0.27% to 0.5-1.5%
Then, xylose isomerase (210 units per 50 ml of medium) was aseptically added thereto, inoculated with acetic acid bacterium Acetobacter xylinus, and allowed to stand still at 30 ° C. for one month.

FIG. 4 is a table showing the results of the experiment.
According to this, it was found that the IFO-15606 strain of acetic acid bacterium Acetobacter xylinus produced more than 0.3 g of acetic acid cellulose per 100 ml of medium, unless the pH value of the final medium dropped below 4.8. The sugar yield has also increased to 30%.

[0019]

According to the present invention, the production of acetic acid cellulose by using xylose as a carbohydrate raw material can be realized by the constitution and operation described above. Xylose, which is a saccharide raw material in the present invention, is obtained by steaming / explosion treatment of lignocellulose waste such as woody waste or agricultural waste, supercritical water treatment, acid hydrolysis, enzyme hydrolysis, or kraft pulp mill. Can be discharged in large quantities by the xylanase treatment of unbleached pulp in the above, and can be easily supplied at low cost. Acetobacter cellulose is pure cellulose that does not contain any lignin or hemicellulose, has excellent physical properties of high crystallinity and high elasticity, functional food materials, high-quality acoustic diaphragms, medical materials, The range of application such as filtration membranes for filtration is extremely large. As described above, according to the present invention, it is possible to realize the production of a high-value material called acetic acid bacterial cellulose from woody waste or agricultural waste, which has been extremely limited in the past, realizing a new industrial field. It is very large to contribute to.

[Brief description of the drawings]

FIG. 1 is a table showing experimental results of cellulose yield and sugar yield by various acetic acid strains in a xylose-only medium and a xylose / xylulose medium.

FIG. 2 is a graph showing the correlation between the concentration of xylulose and the amount of xylose isomerase added.

FIG. 3 is a graph showing the relationship between the final pH value of a medium and the yield of cellulose acetate.

FIG. 4 is a table showing the relationship between conditions for suppressing the pH value of a medium and the yield of cellulose acetate.

Continued on the front page F-term (reference) 4B064 AF12 CA02 CA21 CB30 CC03 CC07 CD02 CD09 DA01 DA10 DA20

Claims (3)

[Claims] 1. A microorganism having the ability to produce cellulose belonging to the genus Acetobacter is cultured in a medium in which xylose is produced by isomerization of xylose as a carbohydrate source. A method for producing cellulose acetate from xylose as a raw material, wherein the cellulose is produced in the cellulose. 2. The production of acetic acid bacterial cellulose from xylose according to claim 1, wherein the isomerization of xylose is performed by aseptically adding xylose isomerase to the medium. Method. 3. A microorganism belonging to the genus Acetobacter which has the ability to produce cellulose, is selected from the IFO-15606 and / or IFO-13693 strains of Acetobacter xylinus, and the Acetobacter acetobacter. -Acetobacter cellulose from xylose as claimed in any one of claims 1 to 2, further comprising means for suppressing a decrease in the pH value of the medium due to an acidic metabolite of saccharide of Acetobacter xylinus. Manufacturing method.