JPH05284989A - Production of bacterium cellulose - Google Patents

Abstract

PURPOSE:To efficiently produce bacterium cellulose by culturing a bacterium capable of producing cellulose while controlling partial pressure of dissolved oxygen to >= critical partial pressure of dissolved oxygen and concentration of saccharide in a specific range to improve accumulative concentration of cellulose of bacterium. CONSTITUTION:A bacterium [e.g. Acetobacter pasteurianus (FERM P-12,884) capable of producing cellulose is inoculated into a liquid medium containing fructose, corn steep liquor, ammonium sulfate, monopotassium phosphate, magnesium sulfate, etc., subjected to spinner culture at 30 deg.C for 20 hours while introducing an oxygen-containing gas controlling partial pressure of dissolved oxygen in the medium in a range of critical partial pressure of dissolved oxygen to 0.063atm and concentration of saccharide in 0.5-20g/l to form and accumulate bacterium cellulose as a gelatinous substance. Then the bacterium cellulose is separated and cleaned with a diluted acid, an organic solvent, hot water, a surfactant, etc., alone or in combination to efficiently product bacterium cellulose.

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 Japanese Unexamined Patent Publication (Kokai) No. 62-175190 discloses a technique of increasing the productivity of microbial cellulose in aeration and agitation culture by using a specific strain having high microbial cellulose productivity. ..
In this publication, the oxygen concentration is 30% air saturated (corresponding to 0.063 atm when converted to the dissolved oxygen partial pressure).
There is only a description that it will be retained in. Fermentation production of cellulose at low dissolved oxygen partial pressure was unknown.

Further, Japanese Patent Application Laid-Open No. 63-202394,
JP-A-1-273599 discloses a technique for producing microbial cellulose while maintaining the concentration of carbon source in the medium at 0 to 0.5 g / l. In this publication,
In the first stage, the concentration of the carbon source was maintained at 10 to 20 g / l, so that only the growth of microorganisms was performed, and then the concentration of the carbon source was maintained at 0 to 0.5 g / l in the later stage. Producing cellulose. The maximum accumulated amount of cellulose in this case is about 5 g / l. However, there is no information on the fermentative production of microbial cellulose under the condition of high carbon source concentration.

[0005]

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

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that by maintaining the dissolved oxygen partial pressure at or above the critical dissolved oxygen partial pressure in stirring culture, microorganisms The inventors have found that cellulose can be efficiently produced and that microbial cellulose can be efficiently produced by limiting the sugar concentration in the medium in agitation culture, and completed the present invention. That is, the present invention relates to a method for producing microbial cellulose, which comprises performing culture while stirring while maintaining the dissolved oxygen partial pressure at or above the critical dissolved oxygen partial pressure. The present invention also relates to a method for producing microbial cellulose, which comprises performing culture with stirring while controlling the sugar concentration in the medium to 0.5 to 20 g / l.

The agitation culture referred to in the present invention refers to shaking culture, vibration culture, aeration and agitation culture, airlift type culture in which agitation is performed by aerating an oxygen-containing gas.

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 may be any as long as it is as described above.

The microorganisms that produce microbial cellulose as described above are not particularly limited, but one example thereof is Acetobacter pasteurian.
us) ATCC23769, FERM P-12884,
Alternatively, A. aceti and A. xyli
num), A. ransens, Sarcina ventriculi, Bacterium xyloides, Pseudomonas bacteria, Agrobacterium bacteria, Rhizobium bacteria, etc. 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 10 ° C to 40 ° C to carry out the culture.

[0011] To give a concrete example, as the medium,
Fructose 50.0 g / l, corn steep liquor 50 ml / l, ammonium sulfate 3.0 g / l, potassium monophosphate 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 5
Water salt 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 0.02mg / l, yeast extract 100mg /
l, malto extract 100 mg / l (pH 5.
The composition of 0) may be used. Moreover, a particularly suitable culture temperature is 25 ° C to 30 ° C.

In the present invention, the dissolved amount of oxygen in the liquid is expressed by the dissolved oxygen partial pressure. Although the dissolved amount of oxygen in a liquid is often expressed by a dissolved oxygen concentration (for example, ppm or mol / l), in the present invention, a liquid in equilibrium with 1 atm of air (that is, an oxygen concentration of 21%). The oxygen concentration in the solution was defined as a dissolved oxygen partial pressure of 0.21 atm. That is, the dissolved amount of oxygen in the liquid in equilibrium in which 1 atm of air was aerated without inoculation of the bacteria was 0.21 atm in terms of the dissolved oxygen partial pressure, and the cells were inoculated and proliferated. When oxygen is consumed and oxygen dissolved in the culture medium disappears, the partial pressure of dissolved oxygen is 0 atm. In practice, a commercially available oxygen electrode may be used to measure the dissolved oxygen partial pressure.

The critical dissolved oxygen partial pressure referred to in the present invention is defined below. In the case of aerobic bacteria, generally, the respiratory rate of the bacteria increases with an increase in the dissolved oxygen partial pressure, but when the dissolved oxygen partial pressure exceeds a certain value, no further increase in the respiratory rate is observed. The dissolved oxygen partial pressure at this time is called the critical dissolved oxygen partial pressure.

In the present invention, the critical dissolved oxygen partial pressure is 0.00
Since it is around 2 atm, the dissolved oxygen partial pressure is 0.00
It is necessary to maintain at least 2 atm and preferably at least 0.005 atm. Generally, in fermentation, as the concentration of the microorganisms in culture increases, the oxygen consumed by respiration increases, and the dissolved oxygen decreases. Therefore, as a method for maintaining the dissolved oxygen at the above concentration, the amount of aeration is increased, the stirring speed is increased, the gas-liquid interface area between the aeration gas and the culture solution is increased, and oxygen gas is positively mixed with the aeration gas. ,
Operations such as pressurizing the culture system may be performed.

Regarding sugar as a carbon source, the yield of microbial cellulose may be increased by controlling the concentration in the culture solution to a certain level during the production of microbial cellulose.
Accumulated concentration may increase. A certain level is 0.5-
In the range of 20 g / l, it is preferably 5 to 15 g / l. As the sugar to be used, one or more kinds of fructose, glucose, sucrose, galactose, mannose, sugar alcohol and the like can be used.

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.

[0018]

Example 1 As a microbial cellulose production medium, fructose 50.0 g / l, corn steep liquor 50 m
l / l, ammonium sulfate 3.0 g / l, potassium phosphate 1 g / l, magnesium sulfate heptahydrate 1.0 g /
1, 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 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 of this composition was put into a 1 L volume of a stirring culture tank as shown in FIG. 1 to carry out main culture.

As seed culture, 100 ml of the above medium was placed in a 500-ml baffled flask, and Acetobacter pasturianus FERM P-12884 was added.
After culturing at 30 ° C. for 3 days at 200 rpm after inoculation, After crushing this with a blender once,
The main culture was inoculated. The inoculation concentration was 10%.

The culture temperature of the main culture was 30 ° C., the stirring speed at the start of the culture was 400 rpm, and the aeration rate was 0.3 VVM. The culture solution was sampled during the culturing, the fructose concentration was measured, and when the fructose concentration fell below 1%, fructose was additionally added so as to be 50 g / l. In addition, medium components other than fructose were added at the 48th and 96th hours of culturing so as to have the same concentration as at the start of the culturing.

The dissolved oxygen partial pressure decreased as the culture time passed. Dissolved oxygen partial pressure 0.01a
From the point of time when tm was reached, the aeration rate was increased stepwise to control the dissolved oxygen partial pressure to be 0.01 to 0.02 atm. From the time when the aeration amount reached 1 VVM, the dissolved oxygen partial pressure was controlled by gradually increasing the stirring speed.
From the time when the aeration amount was 1 VVM and the stirring speed was 1400 rpm, oxygen was positively added to the aeration gas to gradually increase the dissolved oxygen partial pressure in the aeration gas to control the dissolved oxygen partial pressure. Culturing was performed at 120 hours under these conditions. The microbial cellulose accumulation concentration in this culture is 1
It was 5.7 g / l.

[0022]

Example 2 The critical dissolved oxygen partial pressure was measured. Example 1
In the same culture as described above, the aeration was temporarily stopped at 20 hours of culture, and the temporal change in the dissolved oxygen partial pressure by the dissolved oxygen electrode was measured while only stirring was performed. The partial pressure of dissolved oxygen decreased linearly with time. However, 0.005 atm to 0.0 in terms of dissolved oxygen partial pressure
From around 02 atm, the decreasing speed slowed down and the linearity was lost. From this result, the critical dissolved oxygen partial pressure is 0.002a.
It was determined to be around tm. An example of this measurement chart is shown in FIG.

[0023]

Comparative Example 1 As a comparative example of Example 1, the culture was carried out for 120 hours under the initial conditions, that is, in the state where the stirring speed was 400 rpm and the air was aerated (aeration 0.3 VVM). The microbial cellulose accumulation concentration in this culture was measured. Compared with the case of Example 1, almost no cellulose was produced from around 20 hours of culture when the dissolved oxygen partial pressure became almost 0 atm, and the microbial cellulose accumulated concentration was 6.2 g / l.

[0024]

Example 3 Culture was performed under the same conditions as in Example 1. However, only the control of the fructose concentration was carried out as follows, unlike in Example 1. The culture solution was sampled during the culture, and while measuring the fructose concentration using liquid chromatography, 100% (w / v) fructose aqueous solution was continuously added so that the fructose concentration became 15 g / l. The culture was carried out for 120 hours and the microbial cellulose accumulation concentration was measured. The accumulated concentration of microbial cellulose was 21.0 g / l.

[0025]

Example 4 Culture was carried out in the same manner as in the method of Example 3. However, the dissolved oxygen partial pressure was controlled to 0.2 to 1%. Culture 1
It went to the 20th hour. The accumulated concentration of microbial cellulose was 14.1 g / l.

[0026]

Comparative Example 2 According to the method of Example 3, the fructose concentration was controlled to 0.2 g / l or less, and the cells were cultured. The accumulated concentration of microbial cellulose was 7.8 g / l.

[0027]

Example 5 Culture was performed in the same manner as in the method of Example 3. However, the fructose concentration was controlled so as to be 1 g / l, and the culture was performed. Accumulation concentration of microbial cellulose is 18.0g
/ L. The results are shown in Table 1 together with the results of Examples 1, 3, 4 and Comparative Examples 1, 2.

[0028]

[Table 1]

[0029]

Example 6 Culture was carried out in the same manner as in the method of Example 5. However, sucrose, glucose and mannitol were used respectively. The results are shown in Table 2.

[0030]

[Table 2] In both cases, the dissolved oxygen concentration was 1-2%, and the sugar concentration was 1 g / l.
Controlled.

[0031]

EFFECTS OF THE INVENTION According to the present invention, microbial cellulose can be produced more efficiently, especially in stirring culture.

[Brief description of drawings]

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

FIG. 2 shows a measurement chart of critical dissolved oxygen concentration.

Claims (3)

[Claims] 1. A method for producing microbial cellulose, which comprises stirring culture while controlling the dissolved oxygen partial pressure to be not less than the critical dissolved oxygen partial pressure to less than 0.063 atm. 2. The production method according to claim 1, wherein the culture is performed while controlling the sugar concentration in the medium to 0.5 to 20 g / l. 3. A method for producing microbial cellulose, which comprises stirring culture while controlling the sugar concentration in the medium to 0.5 to 20 g / l.