JPH05284990A - Production of bacterium cellulose by spinner culture - Google Patents

Abstract

PURPOSE:To efficiently produce bacterium cellulose by using a spinner culture tank having a specific coefficient of shear rate, culturing a bacterium capable of producing cellulose under a stirring condition of a specific oxygen transfer rate to increase production rate and maximum accumulative concentration of bacterium cellulose. CONSTITUTION:A stirring shaft rotatable out of a spinner culture bath is installed at the central part of the spinner culture tank, the shaft is equipped with a bottom paddle, having a lower part in close vicinity to the bottom of the spinner culture tank, arranged at the bottom the tank and an arm paddle and a strip extending in the direction of a shaft are attached to a part higher than the bottom of paddle of the stirring shaft to constitute a lattice blade. Plural baffle plates set along the axial direction from the bottom to the top are attached at intervals to a side wall face of the spinner culture tank to give the spinner culture tank having >=15 coefficient of shear rate. A bacterium capable of producing cellulose is cultured by using the spinner culture tank under a stirring condition of >=0.5X10<6>mol/ml/minute to efficiently produce bacterium cellulose at increased production rate and in the increased maximum accumulative concentration.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing microbial cellulose.

Microbial cellulose can be used in various industrial materials, clothing materials, medical materials, functional materials, food materials and the like.

[0003]

2. Description of the Related Art Microbial cellulose, in a produced state, has extremely fine fibers (called microfibrils, fibrils, ribbons, etc.) made of cellulose having extremely high crystallinity and uniaxial orientation. It creates a tangled network-like structure. According to electron microscope observation, the width of this fiber is about several tens to several hundreds nm. Since a large amount of liquid is contained in the interstices of this network-like structure, the appearance of the microbial cellulose produced is gel-like. The microbial cellulose content is about 1% based on the weight of the gel material. Further, the solid content contained in this gel-like substance includes not only microbial cellulose but also microbial cells and the like.
It is disclosed in JP-A-61-113601 that a suspension (disaggregated product) of short fibers can be obtained by disaggregating such a gel-like substance with a commercially available mixer or the like.

Up to now, no report has been known regarding a method for producing microbial cellulose, which has been carried out focusing on the shear rate coefficient of a stirred culture tank.

[0005]

The object of the present invention is to increase the productivity of microbial cellulose particularly in culture systems other than static culture such as shaking culture and stirring culture.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventors have cultivated a cellulose-producing microorganism using a stirring culture tank having a specific shear rate coefficient. As a result, they have found that microbial cellulose can be produced more efficiently than before, and have completed the present invention.

That is, the present invention uses a stirred culture tank having a shear rate coefficient of 15 or more and an oxygen transfer rate of 0.5 × 10 ^-6.
The present invention relates to a method for producing microbial cellulose by culturing a cellulose-producing microorganism under a stirring condition of mol / ml / min or more.

The shear rate coefficient of the stirred culture tank of the present invention is 15
Desirably 19 or more is more efficient than conventional
It is preferable because microbial cellulose can be produced. Stay here
The shear rate coefficient depends on the shape of the stirring blade.
It is a value. This shear rate coefficient was calculated in 1957 by Met.
According to the following formula introduced by zner and Otto
(AIChE Journal,Three
(1), 3-10). That is, the average shear in the stirred culture tank
Speed γ_av(M / sec), stirring rotation speed is n (rps)
Then, the shear rate coefficient K = γ_av/ N. general
In the case of turbine blades, it is about 10-13.

The value of the shear rate coefficient K used in the present invention was calculated by using power data obtained from an experiment using a maltose solution which is a Newtonian fluid and a carboxymethylcellulose solution which is a non-Newtonian fluid. To explain the outline of the calculation method, first, the apparent viscosity of the carboxymethylcellulose solution was calculated from the flow curve of the carboxymethylcellulose solution using the assumed value K ′ of the shear rate coefficient. Further, the apparent Reynolds number in the stirred culture tank was calculated from the apparent viscosity value.
Next, a double logarithmic plot (power curve) with the apparent Reynolds number on the horizontal axis and the power number on the vertical axis was prepared. The value of K ′ at which this power curve is the same as that of the maltose solution was defined as the shear rate coefficient K.

As such a stirring device, a stirring shaft which can be rotated from the outside of the tank is provided at the center of the inside of the stirring culture tank, and the lower end of the stirring shaft is placed close to the bottom of the stirring culture tank and placed at the bottom of the tank. The bottom paddle of the stirring shaft is attached, and the arm paddle and the strip extending in the axial direction are attached to the portion above the bottom paddle of the stirring shaft to form a lattice blade and the side wall surface of the stirring culture tank from the bottom to the top. The agitated culture tank with multiple baffles along the axial direction installed at intervals is raised,
Specifically, there is one disclosed in detail in JP-A-61-200842. The most important feature of this stirring culture tank is the shape of its stirring blade. The stirring blade that has been often used in conventional fermenters is often a turbine type, but the stirring blade described in the above publication has a special shape and is smaller than the conventional turbine type. The mixing efficiency of the liquid becomes large even with the stirring power and the stirring rotation speed. In particular, this stirring blade is said to be suitable when the culture solution has a high viscosity.

Further, as the stirring culture condition used in the present invention, it is desirable that the oxygen transfer rate is 0.5 × 10 ⁻⁶ mol / min / min or more. More preferably 1.0 x
It may be 10 ⁻⁶ mol / min / min or more. The oxygen transfer rate is the amount of oxygen transfer from the gas phase to the liquid phase per unit time and culture solution unit volume, calculated from the difference between the oxygen partial pressures contained in the supply gas and the exhaust gas. The oxygen partial pressure can be measured with a normal oxygen electrode.

The microbial cellulose in the present invention includes cellulose and a heteropolysaccharide having cellulose as a main chain and glucans such as β and α. In the case of a heteropolysaccharide, constituent components other than microbial cellulose are
Hexoses such as mannose, fructose, galactose, xylose, arabinose, rhamnose, and uronic acid,
Examples include pentose sugar and organic acids. These polysaccharides may be a single substance, or two or more types of polysaccharides may be mixed. The microbial cellulose is not particularly limited as long as it is as described above.

Specifically, as a microorganism that produces microbial cellulose, Acetobacter pasteurianus (Aceto
bacter pasteurianus) ATCC23769, FERM
P-12884, or A. aceti, A. xylinum, A. ransen
s), Sarcina ventricul
i), Bacterium xyloide
s), Pseudomonas spp, Agrobacterium spp, Rhizobium spp, algae, molds and the like can be used.

For the production and accumulation of microbial cellulose,
A general method for culturing ordinary bacteria using the above-mentioned microorganism may be followed. That is, it may be added to a normal nutrient medium containing a carbon source, a nitrogen source, inorganic salts, and other organic trace nutrients such as amino acids and vitamins, if necessary. The temperature may be controlled at 20 ° C to 40 ° C to carry out the culture.

To give a concrete example, the medium is fructose 50.0 g / l, corn steep liquor 5
0 ml / l, ammonium sulfate 3.0 g / l, phosphoric acid 1
Potassium 1.0 g / l, magnesium sulfate heptahydrate 1.0
g / l, phytic acid 100 mg / l, ammonium iron citrate 15 mg / l, calcium chloride 15 mg / l, ammonium molybdate 1 mg / l, zinc sulfate heptahydrate 2
mg / l, manganese sulfate tetrahydrate 1 mg / l, copper sulfate pentahydrate 0.02 mg / l, nicotinic acid 0.5 mg / l, pyridoxine hydrochloride 0.5 mg / l, thiamine hydrochloride 0.5
mg / l, calcium pantothenate 0.2 mg / l, riboflavin 0.2 mg / l, folic acid 0.02 mg / l, biotin 0.02 mg / l, yeast extract 100 mg / l,
A malt extract having a composition of 100 mg / l (pH 5.0) may be used. Further, a particularly preferable culture temperature is 25 to 30 ° C.

The gel-like substance containing the microbial cellulose thus produced contains not only liquid components but also bacterial cells and medium components. Therefore, dilute alkali, dilute acid, organic solvent, hot water, surfactant, etc. may be used alone or It is purified by washing in combination. The gel-like substance is
It is possible to purify by washing with an organic solvent, hot water, a surfactant or the like alone or in combination.

[0017]

EXAMPLES The present invention will be specifically described below with reference to examples.

Example 1 As a microbial cellulose production medium, fructose 40.0 g / l, corn steep liquor 50
ml / l, ammonium sulfate 3.0 g / l, potassium phosphate 1 g / l, magnesium sulfate heptahydrate 1.0 g
/ L, phytic acid 100 mg / l, ammonium iron citrate 15 mg / l, calcium chloride 15 mg / l, ammonium molybdate 1 mg / l, zinc sulfate heptahydrate 2 m
g / l, manganese sulfate tetrahydrate 1 mg / l, copper sulfate pentahydrate 0.02 mg / l, nicotinic acid 0.5 mg / l, pyridoxine hydrochloride 0.5 mg / l, thiamine hydrochloride 0.5 m
g / l, calcium pantothenate 0.2 mg / l, riboflavin 0.2 mg / l, folic acid 0.02 mg / l, biotin 0.02 mg / l, yeast extract 100 mg / l, malto extract 100 mg / l (pH 5.0 ) Composition was used. 400 ml of a medium having this composition was poured into a 1 L volume of a stirring culture tank (shear rate coefficient: 19.5) that was independently produced as shown in FIG. 1 for main culture.

First, as a seed culture, 100 ml of the above medium was placed in a 500 ml baffled flask, and Acetobacter pasteurianus FERM P-1.
After being inoculated with 2884, the one that was cultured at 200 rpm for 3 days at 30 ° C. was used. This was once crushed with a blender and then inoculated into the main culture. The inoculation concentration was 10%.

The main culture temperature is 30 ° C. and the stirring speed is 4
The rpm was set to 00 rpm and the ventilation amount was set to 1 VVM. While measuring the fructose concentration during the culture, when it fell below 1%,
Fructose was additionally added. In addition, medium components other than fructose were added at the same concentrations as at the start of culture at 48 hours and 72 hours of culture.

As a comparative control, a stirring culture tank equipped with turbine blades as shown in FIG. 2 was used. The difference from the stirring culture tank of FIG. 1 was that the shape of the stirring blade was different. The shear rate coefficient of this stirred culture tank was 12. The culture using the stirring culture tank of FIG. 1 was designated as culture A, and the culture using the stirring culture tank of FIG. 2 was designated as culture B. In addition, in the culture using the stirred culture tank of FIG. 2, the oxygen transfer rate (mol / ml / min)
In order to make the same as the case where the stirring culture tank of FIG. 1 was used, the culture was also carried out for the stirring speed of 600 rpm (culture C). The oxygen transfer rate in this case is 1.5 ×
It was 10 ⁻⁶ mol / ml / min.

120 hours after the start of the culture, the culture was terminated. After completion of the culture, all solids in the culture solution were collected by centrifugation. The recovered product was immersed in a 2% sodium hydroxide solution at room temperature for 24 hours to remove bacterial cells and media components other than microbial cellulose. This purification operation was repeated three times until the collected product became almost white to obtain a purified microbial cellulose. The purified microbial cellulose was thoroughly washed with water to remove it, then dried and weighed. The measurement results are shown in Table 1. The culture process is shown in FIG.

[0022]

[Table 1]

As in the present invention, when the stirring culture was carried out in the stirring culture tank having a high shear rate coefficient, the microbial cellulose could be produced more efficiently than when the ordinary fermentation tank was used. Further, in the case of a normal fermenter, as the accumulation amount of microbial cellulose increases, the viscosity of the culture solution increases, stirring and mixing was not sufficiently performed, in the fermenter of the present invention, in visual observation Also, the culture solution inside the fermenter was uniformly stirred. Therefore, the final accumulation concentration of microbial cellulose also increased.

[0024]

Example 2 Culture was performed in the same medium as in Example 1. However, as the culturing device, the culturing was performed using a 3.0-liter Max Blend fermenter (MBF type) manufactured by Sumitomo Heavy Industries, Ltd. (Culture D). Note that the amount of overhanging medium is 2
It was set to L. As a comparative control, culture was performed in a fermenter equipped with two stages of turbine-type stirring blades having the same diameter as the stirring blades (culture E). Acetobacter, Pasteurianus ATCC53263 was used as the strain. Seed culture preparation conditions,
The aeration conditions, culture temperature, etc. were the same as in Example 1. In both cultures D and E, the stirring speed was set to 400 rpm. The shear rate coefficient K was 20 in the culture D and 13 in the culture E.

After culturing for 5 days, accumulation of microbial cellulose shown in Table 2 was recognized at the end.

[0026]

[Table 2]

[0027]

According to the method of the present invention, the maximum cumulative concentration of microbial cellulose can be increased in agitation culture, which is effective for industrial use.

[Brief description of drawings]

FIG. 1 shows a stirred culture tank (shear rate coefficient is 19.5).

FIG. 2 shows a stirring culture tank (shear rate coefficient is 12) equipped with turbine blades.

FIG. 3 shows the progress of culturing when using each agitation culture tank.

Claims (2)

[Claims] 1. A stirring culture vessel having a shear rate coefficient of 15 or more and an oxygen transfer rate of 0.5 × 10 ⁻⁶ mol / ml.
A method for producing microbial cellulose, which comprises producing microbial cellulose by culturing the cellulose-producing microorganism under a stirring condition of not less than 1 minute. 2. A stirring shaft that can be rotated from the outside of the tank is provided at the center of the inside of the stirring culture tank, and a bottom paddle that is arranged at the bottom of the stirring culture tank so that the lower end is close to the bottom surface of the stirring culture tank is attached to the shaft. Then, an arm paddle and a strip extending in the axial direction are attached to a portion of the stirring shaft above the bottom paddle to form a lattice blade, and a plurality of sidewalls of the stirring culture tank are arranged along the axial direction from the lower part to the upper part. The production method according to claim 1, wherein a stirring culture tank in which baffle plates of a book are installed at intervals is used.