JPS60251889A - Preparation of poly (beta-hydroxybutyric acid) - Google Patents

Abstract

PURPOSE:To collect poly (beta-hydroxybutyric acid) (PHB) from a culture in high yield, by cultivating Azotobactor vinelandi or its variant in medium having a weight ratio of C in a carbon source to N in a nitrogen source in a given range. CONSTITUTION:A strain (e.g., IFO 3741) belonging to the genus Azotobactor or its varient is cultivated in a medium having a weight ratio of C in a carbon source to N in a nitrogen source of 6-50. The cultivation is usually carried out at <=50 deg.C at 2-11pH, and, in order to promote growth of the bacteria, the cultivation is preferably carried out aerobically by shaking culture, aerated spinner culture, etc. After the cultivation is over, a mold is separated by centrifugal separation, etc., the mold is washed with water, acetone, etc., and PHB is extracted with a solvent such as chloroform, etc. The extracted solution is then blended with a mixed solution of methanol/water (4/1), etc. PHB is coagulated, and dried to give solid PHB. By this method, since multiplication ratio of PHB- producing mold is high, and PHB content in the mold is not reduced, PHB is obtained in high yield.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the production of poly(β-hydroxybutyric acid) by microorganisms.
The present invention relates to a method for producing PHB (hereinafter referred to as PHB).

PHB is a thermoplastic polyester represented by the following formula, and exhibits biodegradability, bioabsorption, high crystallinity, piezoelectricity, optical activity, etc., and is resistant to acids, alkalis, light, air, and most organic solvents. Has resistance.

Since PHB can be processed into threads, fibers, membranes, etc., it can be used as an alternative to polypropylene, as well as in sutures, surgical bottles, etc.
It is useful as a material for coating materials, piezoelectric films, etc.

It is known that PHB can be produced using microorganisms using carbohydrates such as glucose and fructose as raw materials. The microorganism that produces PHB is Alcaligenes.
Alcaligenes faceca
lis), Alcaligenes Fist Rulandi (Al,
ruhlandi), Alcaligenes larus (A1
．． 1arus), Alcaligenes aquamarinus (A1. aquamarinus), 7/l/Calligenes II Eutrophus (Al, eutrophus)
s), Bacillus megaterium
megaterium), Pseudomonas facilis, Azotobacter
rchroococum), Azotobacter vinellandii (/L vi, ne], andi), etc. are known.

When these microorganisms are cultured using a medium that uses carbohydrates as a carbon source, PHB accumulates within the microbial cells, and after the microbial cells are destroyed by, for example, ultrasonication, PHB is extracted using a PHB-soluble solvent. do.

However, microorganisms of the genus Azotobacter produce P.
It is said to be preferable because HB can be extracted (Japanese Patent Application Laid-open No. 5
5-118394). In particular, strains of Azotobacter vinelandii (hereinafter abbreviated as vinelandii species) are
PHB is large and convenient to separate from the culture medium, and the molecular weight of the PHB produced is approximately 2 million, which is higher than PHB obtained from other microorganisms.
It has features such as excellent HB performance. However, this microorganism has the drawbacks common to nitrogen-fixing bacteria, such as consuming a lot of energy for nitrogen fixation and therefore having a low growth rate and cell yield. If a strain of Vignerandii is cultured in a nitrogen-containing medium, it is possible that energy will not be consumed for nitrogen fixation and the growth rate will increase. However, when a nitrogen source is added to the medium, the growth rate increases, but PHB in the bacterial cells
The productivity of PHB does not improve because the content decreases (
Journal of Bacteriology Volume 91 No. 1 60
4-610, 1966).

The present inventors used a strain of Vignerandi sp.
As a result of research on the production method, we found culture conditions that allow the growth rate of microorganisms to be high and that do not reduce the PHB content in the cells.

The present invention is based on this knowledge, and involves culturing Azotobacter vinellandii or a mutant strain thereof in a medium in which the weight ratio of carbon in the carbon source to nitrogen in the nitrogen source is 6 to 50:1.
This is a method for producing poly(β-hydroxybutyric acid), which is characterized by collecting poly(β-hydroxybutyric acid) from a culture.

Bacterial strains of A. and Binnerandi species used in the present invention include, for example, IFO3741, IFO12018, and IF00.
Examples include 16581. Of course, it is also possible to use these mutant strains.

As the carbon source, sugars such as glucose and fructose are used. The concentration of carbon source in the medium is 10-50
ill is preferred. As the nitrogen source, ammonium salts such as ammonium nitrate, ammonium chloride, and ammonium sulfate are used. A phosphorus source, inorganic salts, vitamins, etc. may be added to the medium. As the phosphorus source, phosphoric acid compounds such as dipotassium phosphate, dibasic phosphate, and the like are preferred.

In the present invention, the weight ratio of carbon in the carbon source to nitrogen in the nitrogen source in the culture medium (C7N ratio) is 6 to 5°:1, preferably 10 to 40:1, and this is determined as follows. calculate. The weight of carbon in 100g of glucose is 409, and the weight of nitrogen in 50g of ammonium nitrate is 17.5.9, so gluconis 100.9 and ammonium arsenate 50.9.
The C/N ratio of the medium containing lit is 2.3. Based on this calculation method, the C/N ratio in the medium is appropriately adjusted.

When the C/N ratio in the medium is lower than 6, the PHB content in the bacterial cells decreases, and when the C/N ratio is higher than 50, the growth rate of microorganisms decreases and the yield of PHB decreases.

When carrying out the present invention, the c/N ratio is between 6 and 50.
A strain of Binnerandi species or a mutant strain thereof is cultured in a medium. Cultivation is usually carried out at a temperature of 50°C or lower and at pI (2-1
This is done within a range of 1. In order to promote the growth of microorganisms, it is usually preferable to culture the microorganisms aerobically by shaking, aerating, or the like.

Two-stage culture can also be performed.

After culturing, the bacterial cells are separated, for example, by centrifugation, and then washed with water, acetone, methanol, etc.
Extract PHB. For this extraction, a solvent in which PHB is soluble,
For example, chloroform, methylene chloride, ethylene carbonate, etc. are used. Add methanol/water (4/1) to the extract.
), add n-hexane, etc. to solidify PHB,
Upon drying, a solid PHB is obtained.

According to the method of the present invention, the growth rate of PHB-producing bacteria is high and the PHB content in the bacterial cells does not decrease, so that PHB can be obtained at a high yield.

Example 1 Azotobacter vinelandii Il'i'0135'8
A culture solution containing 0.6 g of No. 1 was inoculated into a liquid medium (C/N ratio = 34.3, pH 7.7) 17 having the composition shown in Table 1.

Table 1 Glucose 30 j; l/13 Ammonium nitrate 1 Dipotassium phosphate 5 Magnesium sulfate 0.4 Calcium chloride 0.11 // Ferrous sulfate 0.012 u Sodium molybdate 0.0025 7' Sodium chloride 0 .4 After culturing with aeration in a mini jar fermentor for 48 hours, the cells were separated by centrifugation, washed with water and acetone, and extracted with chloroform. Next, it was coagulated with n-hexane and then dried to obtain 7.8 g of PHB. The PHB content in the bacterial cells was 65%.

Examples 2 to 8 PHB was obtained by culturing in the same manner as in Example 1 using a medium having a C/N ratio shown in Table 2 at °C. The results are shown in Table 2.

Table 2 Comparative Example Glucose 30 i/-Q and 7 ammonium arsenate 7! ? /Using a medium containing Numa (C/N ratio 4.9),
Culture was carried out in the same manner as in Example 1 to obtain PHB.

As a result, the amount of bacterial cells was 12.5g, the PHB content was 20%, and the PHB content was 20%.
The yield of HB was 2.5 g.

Applicant: Mitsubishi Rayon Co., Ltd. Agent: Patent Attorney Masao Kobayashi

Claims (1)

[Claims] The weight ratio of carbon in the carbon source to nitrogen in the nitrogen source is 6 to 50:
1. A method for producing poly(β-hydroxybutyric acid), which comprises culturing Azotobacter vinellandii or a mutant strain thereof in the medium according to item 1, and collecting poly(β-hydroxybutyric acid) from the culture.