JPH07170989A - Production of poly-beta-hydroxybutyric acid using microorganism - Google Patents

Abstract

PURPOSE:To efficiently produce poly-beta-hydroxybutyric acid from microorganism by using an inorganic substance as a sole carbon source for photosynthesis without necessitating an organic carbon source or a reducing substance such as H2. CONSTITUTION:This process for the microbial production of poly-beta- hydroxybutyric acid comprises the cultivation of blue-green algae under light- irradiation using an inorganic carbon source as the carbon source, the cultivation of the product under a dark anaerobic condition to obtain an organic acid and/or an alcohol, the cultivation of a microorganism capable of accumulating poly-beta-hydroxybutyric acid using the organic acid, etc., as a carbon source to effect the accumulation of poly-beta-hydroxybutyric acid in the microbial cell and the separation of the accumulated poly-beta-hydroxybutyric acid from the cell.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is a microorganism capable of obtaining an organic acid and / or alcohol from a culture solution of cyanobacteria and accumulating poly-β-hydroxybutyric acid using the organic acid and / or alcohol as a carbon source. To culture poly-β-
The present invention relates to a method for producing hydroxybutyric acid.

[0002]

BACKGROUND OF THE INVENTION Poly-β-hydroxybutyric acid (hereinafter referred to as PHB) is a biopolymer produced and accumulated by microorganisms as a carbon source or an energy source in the cells, and is a thermoplastic resin degradable by microorganisms. As a substance, it is expected to be applied in various fields such as pharmaceuticals, agricultural chemicals, medical materials, and industrial materials.

Various methods for accumulating PHB in microbial cells have been proposed so far (see, for example, Japanese Patent Laid-Open No. Sho 5).
9-220192, JP-A 64-27483 and JP-A 1-222788). However, in any of these cases, an organic carbon source is required as an assimilating carbon source.

A method for producing PHB by utilizing a special microorganism without the need for an organic carbon source has also been proposed. For example, Journal of Fermentation and Bioenginee
In ringVol.69, No.3, 170-174 (1990), Alcaligenes eutrophus (Alcaligenes eutrophus)
s) is used to produce PHB using carbon dioxide as the carbon source. However, with this method, P
In addition to carbon dioxide, H ₂ which is a reducing substance is essential for HB production.

In addition, Journal of Bacteriology, Vol.172,
No.5,2791-2792 (1990), the blue-green algae Spirulina
ulina) is capable of accumulating PHB by photosynthesis without requiring an organic carbon source or a reducing substance such as H ₂ . However, with this method, the PHB content is extremely low and practical application cannot be expected.

The problem of the present invention is that
It is an object to provide a method for efficiently producing PHB by culturing a microorganism without using an organic carbon source or a reducing substance such as H ₂ .

[0007]

Means for Solving the Problems As a result of intensive research to solve the above problems, the present inventors have cultivated cyanobacteria under light irradiation using an inorganic carbon source such as carbon dioxide and metal bicarbonate. ,
The organic acid and / or alcohol is obtained from the culture solution by culturing the culture under dark anaerobic conditions, and the organic acid and / or alcohol is used as a carbon source to culture a microorganism capable of accumulating PHB. As a result, they have found that PHB can be efficiently accumulated in the cells, and completed the present invention.

That is, according to the present invention, cyanobacteria are cultured under light irradiation using an inorganic carbon source as a carbon source, and then the culture is cultivated under dark anaerobic conditions to obtain organic acid from the culture. And / or alcohol is obtained, and the obtained organic acid and / or alcohol is used as a carbon source to cultivate a microorganism capable of accumulating poly-β-hydroxybutyric acid. Provided is a method for producing poly-β-hydroxybutyric acid using a microorganism, which accumulates hydroxybutyric acid and collects poly-β-hydroxybutyric acid from bacterial cells.

First, the process of producing an organic acid and / or alcohol by culturing cyanobacteria will be described.

Ia) In culturing cyanobacteria under light irradiation using an inorganic carbon source, any cyanobacteria that releases organic acid and / or alcohol into the culture solution under dark anaerobic conditions can be used. It may be one. Examples of cyanobacteria that are preferably used include Spirulina platensis Foundation Earth and Human Environment Forum (hereinafter referred to as GEF) deposit NIES-39, Spirulina platensis GEF deposit
NIES-46, Oscillatoria l
imnetica) GEF deposit NIES-36, Microcystis aeruginosa GEF deposit NI
ES-44, Microcys aeruginosa
tis aeruginosa) GEF deposit NIES-87, Nostoc commune GEF deposit NIES-24 and Synechococcus elongatus (Synechococcus elonga)
tus) Micromachine Lab. deposited FERMP-12393 and the like.

Since the above-mentioned cyanobacteria are autotrophic microorganisms, when they are cultured under light irradiation, NaHCO ₃ , K ₂ H
PO ₄ , NaNO ₃ , K ₂ SO ₄ , NaCl, MgSO
₄ , it is not necessary to add organic additives such as glucose, peptone, yeast extract, etc. to the medium as in the case of culturing a heterotrophic microorganism, which grows well in an aqueous medium containing only inorganic salts such as CaCl ₂ .

As the carbon source, only an inorganic carbon source is used. Typical examples of the inorganic carbon source are carbon dioxide, bicarbonate and the like.

As the culture solution, depending on the strain to be used,
For example, SOT medium (Table 1), MA medium (Table 2), MD
M medium (Table 3), BG-11 medium (Table 4) and the like can be appropriately selected and used.

The culture of cyanobacteria is a so-called bright place culture carried out under light irradiation. The illuminance of this photopic culture is, for example, 10
0 to 10000 looks, preferably 500 to 3000
It looks.

At the stage of culturing under light irradiation, it is also preferable to carry out aeration stirring culture. Culture time, culture temperature, p
The culture conditions such as H are appropriately set so that the production amount of organic acid and / or alcohol is maximized depending on the strain used. Generally, the culturing time is 3 hours or more, the culturing temperature is 20 to 60 ° C., preferably 25 to 55 ° C.
H is preferably 6 to 11, and more preferably 7 to 9.

Ib) Next, culturing of cyanobacteria under dark anaerobic conditions is carried out, for example, by the following operation. That is, as described above, the cyanobacterial cells cultured under light irradiation are separated from the culture solution, and the obtained cyanobacterial cells are inoculated into another culture solution,
This is cultivated in the dark under anaerobic conditions.

Means for separating the cyanobacterial cells from the culture solution include filtration, centrifugation and the like. Another culture solution inoculated with the separated cyanobacteria is Tris-hydrochloride buffer, hydrochloride buffer, phosphate buffer or the like. The pH of these buffers is 6-1
1, preferably 7 to 10. The anaerobic state is ensured by replacing the air in the culture vessel with an inert gas such as nitrogen gas or helium gas. It is also preferable to carry out the culture with stirring using a shaking culture device or the like. The culturing conditions such as culturing time and culturing temperature are appropriately set so as to maximize the production of organic acids and alcohols depending on the strain used. Generally, the culture time is 5 to 20 hours, and the culture temperature is 20.
The temperature is -60 ° C, preferably 25-55 ° C, and the culture is performed in the dark with the light blocked.

Thus, by culturing cyanobacteria, lower organic acids such as formic acid, acetic acid and lactic acid, alcohols such as ethanol, or both organic acids and alcohols are produced in the culture solution. The organic acid and / or alcohol may be collected from the culture medium, but may be directly used in the form of a solution in the culture medium for the next step.

Identification of the organic acid and alcohol and measurement of the concentration in the culture solution are carried out by a method using high performance liquid chromatography, gas chromatography or the like.
In order to collect and purify the organic acid and / or alcohol from the culture broth, a technique generally used for collecting and purifying organic compounds can be adopted.

Next, the step of culturing a microorganism capable of accumulating PHB by using an organic acid and / or alcohol as a carbon source will be described.

IIa) In this culturing step, various inorganic components capable of proliferating microorganisms capable of accumulating PHB are added to a culture solution containing an organic acid and / or alcohol, and the microorganism accumulates PHB. Culture under the culture conditions.

The lower limit of the concentration of the organic acid and / or alcohol in the culture medium may be a concentration at which the microorganism used can accumulate PHB, and the upper limit may be a concentration at which the culture of the microorganism is not inhibited.

Examples of the inorganic component to be added include calcium salt, magnesium salt, potassium salt, sodium salt, manganese salt, zinc salt, iron salt, copper salt, molybdenum salt,
A cobalt salt, a nickel salt, a chromium salt, a boron compound or the like is appropriately selected. The microorganism that can accumulate PHB is PHB.
There is no particular limitation as long as it is a microorganism capable of accumulating in cells, but in practice, Alcaligenes eutrophus (Alca
ligenes eutrophus) ATCC17699, Alcaligenes faecalis ATCC8750, Rhodospirillum rubrum ATCC2590
3, Rhodobacter sphaeroides
eroides) ATCC17023 and the like are used. The culturing conditions such as culturing time, culturing temperature, pH and the like are appropriately set so that the amount of PHB accumulated by the microorganism becomes maximum depending on the microorganism used. Generally, the culture time is 3 to 96 hours, preferably 8 to 48 hours, and the culture temperature is 20 to 40 ° C, preferably 2
5 to 35 ° C., pH 6 to 11, preferably 7-1
It is 0. Under such conditions, the culture is carried out aerobically or anaerobically depending on the strain. Further, in order to increase the efficiency by using the photosynthetic microorganism, it is also preferable to carry out a light culture. The illuminance in the light culture is, for example, 100 to 10000 lux, preferably 500 to.
It is 3000 looks. In some cases, the culture may be performed with stirring using a shaking incubator or the like.

IIb) PH thus accumulated in the cells
In order to collect B from the bacterial cells, the following known processing operations are collected, for example. That is, bacterial cells are separated from the culture solution by means of individual liquid separation such as filtration and centrifugation, and PHB is extracted from the obtained bacterial cells or, if necessary, from the bacterial cells destroyed by ultrasonication. The extract is purified by a known treatment method if necessary. As the extraction solvent, for example,
Halogenated hydrocarbons such as chloroform and 1,2-dichloroethane are preferred.

The identification of PHB and the measurement of the concentration in the culture medium are
After the PHB is hydrolyzed into a monomer as required, it is carried out by a technique using, for example, high performance liquid chromatography, gas chromatography or the like.

[0026]

EXAMPLES Next, the present invention will be specifically described with reference to some examples of the present invention.

Example 1 I) Spirulina platensi
s) 100 mg of wet cells of NIES-39 deposited in GEF was inoculated into a culture solution of SOT medium (Table 1) and cultured at a temperature of 30 ° C for 10 days under irradiation with light of 1200 lux. At that time, air was supplied to the culture medium to carry out aeration stirring culture.

Next, the culture solution was filtered using a filter paper to obtain about 3 g of wet bacterial cells. 50 mL of the obtained wet cells
30 mM phosphate buffer, pH 8, in Erlenmeyer flask
mL was inoculated. After closing the Erlenmeyer flask with a rubber stopper on the mouth, nitrogen gas was supplied using an injection needle to replace the inside of the flask with nitrogen gas, thereby ensuring an anaerobic state. Then, the culture was performed at 30 ° C. for 10 hours with light being blocked while stirring using a shaking incubator. As a result, formic acid 2.4 mM, acetic acid 9.7 mM, ethanol 6.2 mM,
A culture solution containing 0.5 mM lactic acid was obtained. The concentration was measured by the gas chromatography method.

II) To 30 mL of this culture solution, 1 mg of wet cells of Rhodospirillum rubrum ATCC25903 was inoculated into a culture solution of pH 7 obtained by adding the inorganic components shown in Table 5, and 1300 Lux at a temperature of 30 ° C. The cells were cultured for 24 hours under the irradiation of light. PHB due to cells after culturing
The production amount was 4.2 mg.

Collection and quantification of PHB were carried out as follows. That is, put the freeze-dried cells into a 10 mL test tube with a screw cap, and add 2 mL of chloroform.
Then, 2 mL of a 3 wt% sulfuric acid-methanol solution was added, the mouth was capped, and the reaction was carried out at 100 ° C. for 3.5 hours. By this reaction, after PHB was hydrolyzed to the monomer, 1 mL of water was added, and the mixture was vigorously shaken for 10 minutes, the lower chloroform layer separated into two layers was taken out, and this chloroform layer was subjected to gas chromatography to obtain the peak of methyl hydroxybutyrate. The amount of PHB was calculated and calculated from the area.

Example 2 Rhodospirillum rubrum
The same operation as in Example 1 was carried out except that Rhodobacter sphaeroides ATCC17023 was used instead of ATCC25903. As a result, the amount of PHB produced by the cells after culturing was 1.2 mg.

Example 3 Spirulina platensis G
Nostoc commune GEF deposit NIES-24 was used instead of EF deposit NIES-39, and SOT
The procedure was exactly the same as in Example 1 except that the MDM medium (Table 3) was used instead of the medium. As a result, a culture solution containing 0.2 mM formic acid, 2.5 mM acetic acid, and 1.5 mM lactic acid was obtained. The PHB production from Rhodospirillum rubrum ATCC25903 was 0.9 mg.

Example 4 Spirulina platensis G
EF Deposit NIES-39 instead of Synechococcus elongatus FERM P-1
The same procedure as in Example 1 was performed except that 2393 was used and BG-11 medium (Table 4) was used instead of the SOT medium. As a result, formic acid 3.4 mM, acetic acid 5.6 mM,
A culture solution containing ethanol 2.8 mM and lactic acid 4.6 mM was obtained. Also, Rhodospi
rillum rubrum) PHB production from ATCC25903 is 1.
It was 9 mg.

[0034]

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[0035]

INDUSTRIAL APPLICABILITY According to the present invention, PHB can be efficiently used from microorganisms without using an organic carbon source or a reducing substance such as H _{2 using} only an inorganic substance as a carbon source for photosynthesis.
Can be produced.

Front Page Continuation (72) Inventor Yasuo Asada 1-1102 Kasuga, Tsukuba City, Ibaraki Prefecture (72) Inventor Yutaka Tokiwa 46-12 Sakuragaoka, Tsuchiura City, Ibaraki Prefecture

Claims (1)

[Claims] 1. An organic acid and / or alcohol is obtained by culturing cyanobacteria under light irradiation using an inorganic carbon source as a carbon source, and further culturing the culture under dark anaerobic conditions, By culturing a microorganism capable of accumulating poly-β-hydroxybutyric acid using the obtained organic acid and / or alcohol as a carbon source, poly-β-hydroxybutyric acid is accumulated in the microbial cells, A method for producing poly-β-hydroxybutyric acid using a microorganism, which comprises collecting poly-β-hydroxybutyric acid.