JPH07155192A - Method for producing poly-3-hydroxybutyric acid - Google Patents

Abstract

(57) [Summary] [Structure] Methanol-assimilating bacteria capable of producing poly-3-hydroxybutyric acid are cultured in a culture medium such that methanol is used as a carbon source and the culture pH is a limiting factor for growth. -Hydroxybutyric acid is accumulated in the cells and collected. [Effect] In the production of poly-3-hydroxybutyric acid according to the present invention, poly-3-hydroxybutyric acid can be produced in high yield by a simple operation of only lowering the culture pH.
Further, since the raw material is methanol, the raw material cost is low, and there is no fear of contamination by various bacteria. Therefore, poly-
It becomes possible to produce 3-hydroxybutyric acid on a large scale, economically and stably.

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 poly-3-hydroxybutyric acid (hereinafter referred to as PHB).

[0002]

2. Description of the Related Art PHB is a thermoplastic polymer that is produced and accumulated in the cells of many microorganisms as an energy storage substance and exhibits excellent biodegradability and biocompatibility. It has attracted attention as a plastic, and it has been expected for many years to be applied to various fields such as medical materials such as surgical threads and materials for fixing bone fractures, and sustained release systems for gradually releasing drugs and pesticides. Especially in recent years
Since synthetic plastics have become a serious social problem from the viewpoints of environmental pollution and resource environment, PHB has attracted attention as a biopolymer that does not depend on petroleum, and some production methods of PHB have been reported so far (Japanese Patent Publication No. Sho). 63-3243
8, Japanese Patent Publication No. 5-997, Japanese Patent Publication No. 02-20238, Japanese Patent Publication No. 03-65154). In these publications, PHB is obtained by continuously culturing cells of the genus Alcaligenes using growth as a carbon source (hereinafter also referred to as a substrate) under growth-restricting conditions by a method such as limiting nitrogen or phosphorus. Manufacturing method (Japanese Patent Publication No. 02-20238),
A method for producing PHB by batch-culturing cells of the genus Azotobacter and Protomonas under growth limiting conditions as in the case of cells of Alcaligenes (Japanese Patent Publication No.
7, Japanese Patent Publication No. 03-65154) and the like, but these methods are insufficient for industrial production due to high production cost.

That is, in the above-mentioned invention, the main nutrient for growing the bacterial cells, that is, the carbon source is expensive, the production cost of PHB cannot be kept low, and the accumulation of PHB by continuous culture is insufficient. However, there are drawbacks such as a complicated manufacturing process such as two-step culture.

The cost of the raw material (ie substrate) is PHB
It is an important factor in the overall cost of production. Although there is a description of a method for producing PHB using glucose, sucrose or the like as a raw material in Japanese Examined Patent Publication No. 02-20238 and Japanese Examined Patent Publication No. 63-32438, these methods cannot avoid an increase in PHB production cost. As for the culture method using inexpensive methanol as a substrate, for example, according to the description of JP-A-56-117793, a microorganism of Methylobacterium organophyllum sp. Culture continuously without limitation. At this time, PHB is not accumulated intracellularly. Then, it is continuously transferred to a second culture tank and cultured in the second culture tank with nitrogen or phosphorus as a rate-determining factor for growth. At this time, PHB is accumulated intracellularly for the first time. The method of performing two-stage culture using two culture tanks in series has the drawbacks that the process is complicated and a satisfactory PHB content cannot be obtained.

[0005] Further, Japanese Patent Publication No. 02-20238 describes that Methylobacterium organophyllum NCIB 11483 strain was continuously cultured with a methanol substrate under nitrogen limitation, but it was the best achieved under these conditions. P
The HB content is as low as about 11%.

[0006]

The object of the present invention is to solve the above-mentioned problems in the prior art, and to use PHB in a simple method by using an inexpensive bacterium capable of assimilating methanol.
An object of the present invention is to provide a production method capable of producing a large amount of rice at low cost.

[0007]

The present inventors have used a methanol-assimilating bacterium capable of producing PHB to
As a result of intensive studies on a method for inexpensively and simply producing PHB, the pH of the culture solution for culturing these bacteria was
Is maintained at a pH significantly lower than the optimum pH for the growth of the bacterium, thereby limiting the growth and culturing,
The present inventors have found that PHB can be accumulated in large amounts in parallel with cell growth, and arrived at the present invention. That is, the present invention is
A methanol-assimilating bacterium having the ability to produce HB,
Culturing with methanol as a carbon source, synthesizing and accumulating PHB in the cells, and obtaining PHB from the cells, in the method for producing PHB, low pH culture in which the culture pH is a limiting factor for growth. Is a method for producing poly-3-hydroxybutyric acid.

In the present invention, PHB is -OCH.
(CH ₃₎ a polyester material composed of CH ₂ CO- comprising repeating units. The bacterium used in the present invention may be a bacterium having an ability to produce PHB and having an ability to assimilate methanol, and examples thereof include genus Methylobacterium, genus Xanthobacter, genus Hyphomicrobium, and Paracoccus. Examples thereof include bacteria belonging to the genera Methylobacillus and Ankylovacta, but practically preferred are bacteria belonging to the genus Methylobacterium.

As the medium used for the culture of the present invention, an ordinary medium containing a carbon source, a nitrogen source, and inorganic salts is used. Methanol is used as the carbon source. The methanol may be pure, and crude methanol or waste containing methanol may be used. As a nitrogen source,
There is no particular limitation as long as it is a substance that can be assimilated by the bacterium to be used, and examples thereof include ammonia, urea, nitric acid, or organic nitrogen-containing substances such as yeast extract and malt extract. As the inorganic salts, phosphates, potassium salts, sodium salts, sulfates and metal salts such as magnesium, iron, calcium, zinc, manganese, cobalt, copper and molybdenum are used. It should be noted that the use of a carbon compound which can be assimilated by the bacterium to be used or its inclusion as a carbon source together with methanol is not prevented.

The culture conditions vary depending on the strain used, but generally the temperature is 25 to 40 ° C., preferably 30.
~ 38 ° C. The optimum temperature for obtaining good PHB productivity often differs depending on the strain. The culture is carried out aerobically under such conditions, but for that purpose, air or oxygen is aerated, and stirring is carried out if necessary in order to effectively dissolve oxygen in the culture solution. Generally, the dissolved oxygen concentration in the culture solution is preferably 0.3 ppm or more.

The culture tank may be of any type as long as it is an aeration and stirring tank, and for example, a mechanical stirring tank, an air lift type culture tank and a bubble column type culture tank can be used. As a method for supplying the medium, a carbon source, a nitrogen source, various inorganic salts, various additives and the like are supplied all at once or individually, continuously or intermittently. For example, methanol may be supplied to the culture tank as a mixture with other medium components, or may be supplied to the culture tank separately from the other medium components. The pH control of the culture solution is usually performed using ammonia gas or ammonia water. When nitrogen is sufficiently supplied as a medium component for cell growth, a non-nitrogen base such as caustic soda or caustic potash can be used to control the pH.

[0012] For the accumulation of PHB, a method of limiting medium components other than carbon source is usually used. It is preferred to limit the components such as nitrogen, phosphorus, sulfur, potash, and trace elements such as manganese, zinc, copper. In contrast to this, the culture according to the present invention is performed without any limitation on the medium components. Culturing can be carried out batchwise, semi-continuously or continuously. That is, in batch culture, a method is used in which the amount of a medium component required for the growth of bacterial cells is batched or fed stepwise as the bacterial cells grow. In continuous culture, a method of continuously supplying a required amount of medium components in a fixed amount is used. In any culture method, the medium components are supplied without any limitation. The supply of each mineral component without deficiency can be confirmed by continuously measuring each mineral ion present in the culture solution by ion chromatography according to a conventional method.

The present invention can be batch, semi-continuous or continuous. It is possible to perform each culture method alone or in combination with each culture method (for example, two-stage culture using two culture tanks). When performing batch culture or fed-batch culture according to the present invention, the culture at a low pH according to the present invention may be started from the beginning of the culture, or the low pH according to the present invention may be obtained after the cell concentration has risen to a certain value. You may start culture | cultivation with. When the culture at low pH according to the present invention is carried out from the beginning of the culture, the PHB is synthetically accumulated in parallel with the growth of the bacterial cells. After culturing in a pH range normally used to raise the bacterial cell concentration to a certain value and then switching to the low pH culture according to the present invention, synthetic accumulation of PHB starts after the low pH culture is started. As described above, it is surprising and unknown that PHB is synthetically accumulated only by lowering the culture pH without limiting the supply of oxygen to the fermenter or limiting the nutrients required for growth. .

The setting of pH in the present invention depends on the strain. The strain p grows best, that is, it has a higher specific growth rate than the pH (hereinafter referred to as optimum pH)
The PHB content tends to increase as H decreases. As a result of examining the relationship between the culture pH, the specific growth rate, and the PHB content, a remarkable increase in the PHB content was observed when the specific growth rate was controlled to 30% or less of the maximum specific growth rate. The decrease in specific growth rate in the low pH range is remarkable, and setting of an excessively low pH is not desirable.

In the culture of the present invention, the amount of methanol supplied is controlled so that the concentration of residual methanol in the culture solution becomes constant. Industrially, there is a method in which the residual methanol concentration in the culture solution is measured with time by an analyzer such as gas chromatography and the amount of methanol supplied is automatically adjusted by the signal. The residual methanol concentration in the culture is usually 1
0 to 3000 ppm, preferably 200 to 2000
It is maintained at about ppm. The residual methanol concentration can also be known by measuring methanol in the culture waste gas with a hydrocarbon meter or an analyzer such as gas chromatography. In this state, only pH limits the growth of the bacteria.

When the present invention is carried out by continuous culture, it is carried out as follows. As a method for maintaining a steady state in a continuous culture system, from the standpoint of saving the substrate, culturing is performed while limiting the supply rate of the substrate, so-called substrate-limited culturing is generally used. Unlike substrate rate-controlled culture, it is a pH-controlled culture method in which culture is performed while maintaining a steady state by limiting the specific growth rate under low pH conditions, that is, the only factor that limits growth is pH. After switching to the continuous culture of the present invention,
The pH is adjusted to control the growth rate, and at the same time, the supply amount of the medium or methanol is controlled in the same manner as in batch culture so that the residual methanol concentration in the culture solution becomes constant. In this way, a constant steady state is obtained under constant aeration conditions, in which only pH is limiting the growth of the fungus.

Prior to the continuous culture under the pH limitation of the present invention, the bacteria are actively grown and precultured until the concentration of the bacteria in the culture solution reaches a predetermined value. As a preculture, for example, batch culture performed while sufficiently supplying the substrate and other medium components and oxygen, or batch culture performed at a low pH alone, or substrate and other medium components subsequent to these batch cultures Continuous culture, which is carried out at an optimum pH while supplying sufficient amount. The pre-culture is performed by a usual method, and the culture temperature, the substrate and the medium components, the substrate concentration in the medium or the culture medium, and the like are as described above.

Subsequent to the pre-culture, the above-mentioned continuous culture in which the pH is limited is carried out. Bacterial cell concentration in the culture solution at the initial stage of continuous culture in the present invention (dry cell basis, the same applies hereinafter)
Is not particularly limited, but it is usually desirable to transfer to the main continuous culture after reaching a concentration that is about the same as or slightly lower than the bacterial cell concentration in the steady state of the present invention. In addition, the cell concentration in the culture solution during continuous culture in the present invention is usually 10 to 10
It is 0 g / l.

In order to change the growth rate of the bacterium during continuous culture, it can be arbitrarily changed by changing the set pH. That is, in order to increase the growth rate, the conditions may be selected so that the set pH is higher, and conversely, to decrease the growth rate, the conditions may be selected such that the set pH is lower. The PHB content in the cells increased as the set pH was lowered and the average residence time in the culture tank became longer. A dramatic increase in PHB content was observed when the average residence time was set to 15 hours or more. To be

From the thus obtained culture broth, cells are separated and recovered by usual solid-liquid separation such as filtration or centrifugation, and if necessary, washed with water to obtain cells.
Obtained by extracting a halogenated hydrocarbon such as chloroform or 1,2-dichloroethane as an extractant from the bacterial cells thus obtained, or from the bacterial cells further destroyed by sonication if desired. PHB is separated from the obtained PHB extract by a means known per se such as coagulation precipitation by mixing this with a poor solvent. If necessary, it can be further purified to obtain high-purity PHB.

[0021]

EXAMPLES The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples. Example 1 Protomonas exto
rquens) K (Microtechnology Research Institute, No. 8395) was used. According to recent literature, this bacterium belongs to the genus Methylobacterium (IJBo
usfield and PNGreen; Int.J.Syst.Bacteriol., 35 , 209
(1985), T. Urakami et al .; Int. J. Syst. Bacteriol., 4
3 , 504-513 (1993)).

A batch culture medium (medium A) having the following composition was prepared per 1 L of industrial water. Composition of medium for batch culture (Medium A) methanol _{2 g KH 2 PO 4 3 g} (NH 4) 2 SO 4 1 g MgSO 4 · 7H 2 O 1 g Yeast extract _{0.2g FeC 6 H 5 O 7 ·} xH 2 O 60 mg ZnSO ₄ .7H ₂ O 20 mg MnCl ₂ .4H ₂ O 10 mg CaCl ₂ .2H ₂ O 40 mg CuSO ₄ .5H ₂ O 1 mg KI 1 mg (NH ₄ ) ₆ Mo ₇ O ₂₄ .4H ₂ O 1 mg CoCl ₂ .6H ₂ O 1 mg H ₃ BO ₃ 1 mg NaCl 50 mg

1.5 L of the medium A was placed in a 3 L culture tank, sterilized by heating at 120 ° C. for 20 minutes, cooled and adjusted to pH 5.5 with aqueous ammonia, and 200 ml of a seed matrix prepared separately was added thereto. The cells were inoculated and batch culture was carried out at 32 ° C. with aeration of air. The pH during batch culture was automatically controlled to 5.5 with 10% ammonia water. The methanol concentration in the culture broth was measured by gas chromatography continuously, and
Methanol was automatically supplied in the range of 00 to 1500 ppm. The rotation speed of the stirrer is 1000 rp
m, and the ventilation amount was 1 vvm. After some lag time,
Proliferated logarithmically. Cultivation was continued to a cell concentration of 36 g / l, but the specific growth rate during this period was 0.049 h ^-1 (generation time 14.1 hours), and the maximum specific growth rate at the optimum pH (6.6) was 0.23 h. It was suppressed to 21% of ^-1 .
The culture results are shown in Table 1. Table 1 shows the culture time and cell concentration,
The relationship of PHB content is shown. The PHB content was constant at about 40% throughout the culture period. Various mineral ions in the culture solution were measured with ion chromatography over time, and none of the components was insufficient.

[0024]

[Table 1]

The analysis of PHB was carried out as follows. The cells were collected by a centrifuge, washed twice with pure water, and dried with hot air (100 ° C.) to obtain dried cells. About 80 mg of dried cells was placed in a test tube with a screw cap, and 1 ml of chloroform and a methanol-sulfuric acid solution containing an internal standard (internal standard: benzoic acid 200 mg / 100 ml, sulfuric acid 3.5).
1% by volume) and heat treated at 120 ° C for 90 minutes,
The polymer contained in the cells was decomposed and methyl esterified. After the reaction was completed, 1 ml of pure water was added, and the mixture was vigorously stirred and then centrifuged to obtain an organic solvent layer. The PHB component content was calculated by analyzing the organic solvent layer by gas chromatography. Gas chromatographic analysis conditions Device: Shimadzu GC-7AG Column: Reoplex 400 chromosor
b G AW-DMCS 10% (60-80 mesh) Column temperature: 160 ° C Injection port temperature: 250 ° C

Comparative Example 1 Bacteria were cultured in the same manner as in Example 1 except that the pH during the culture was changed to 6.6. The culture results are shown in Table 2. Shortly after inoculation, it grew logarithmically. The culture was continued until the cell concentration was 30 g / l, but the specific growth rate in the main culture was 0.2
It was 3 h ^-1 (generation time 3.0 hours). The PHB content was 4% or less throughout the culture period.

[0027]

[Table 2]

Example 2 Same as Example 1 except that the pH during culture was changed to 6.0.
Then, the fungus was cultured. Shortly after inoculation, it grew logarithmically
It was The specific growth rate at pH 6.0 is 0.206h ^-1(world
It was 3.4 hours). Cell concentration of 15g / l
At some point, the set pH was changed to 5.2. low pH
As a result, the specific growth rate gradually decreases and eventually
0.004 to 0.005h^-1(Generation time 140-170
Time). Culture time, cell concentration, PHB content
The results are shown in Table 3. PHB content is pH 6.0
Is as low as 3-4%, but gradually rises after changing to pH 5.2
And finally rose to about 55%. Each mine in the culture solution
Ral ions were measured with ion chromatography over time.
However, none of the ingredients were deficient.

[0029]

[Table 3] Table 3 ────────────────────────────── Incubation time Cell concentration PHB content pH (hr) (g / L) (%) ────────────────────────────── 20 2.3 2.8 6.0 23 4.2 3.0 6.0 27 27 9.8 4.1 6.0 29 29 15.0 3.3 6.0 41 20.6 12.1 5.2 5.2 56 28.2 35.5 5.2 74 35.6 47.7 5.2 97 40.4 55.3 5.2 ───────────────────────────────

Example 3 Protomonas exto
rquens) K (Microtechnology Research Institute, No. 8395) was used. Three
1.5 L of the medium A was placed in an L-volume culture tank, sterilized by heating at 120 ° C. for 20 minutes, cooled, and then cooled to pH 6. with ammonia water.
The seed culture was adjusted to 5, and 200 ml of separately prepared seed mother was inoculated into this, and batch culture was performed at 33 ° C. while aerating air.
The pH during batch culture was automatically controlled to 6.5 with 25% aqueous ammonia. The concentration of methanol in the culture broth was continuously measured by gas chromatography and measured at 500-1500p.
Methanol was automatically supplied so as to be in the range of pm. Stirrer rotation speed 1450 rpm, aeration amount 1 vvm
And When the bacterial cell concentration reached about 10 g / l, the set pH was changed to 5.5, and at the same time, continuous supply of the continuously prepared medium (medium B) and continuous discharge of the culture solution were started, Transferred to continuous culture. The culture pH was automatically controlled to 5.3 using 10% aqueous ammonia. The composition of the continuous medium was as follows, and was sterilized by heating at 120 ° C. for 20 minutes and cooled before use. However, methanol was injected after sterilizing filtration with a microfilter.

Medium composition for continuous culture (medium B) Methanol 60 g KH ₂ PO ₄ 3 g MgSO ₄ .7H ₂ O 1 g (NH ₄ ) ₂ SO ₄ 1 g FeC ₆ H ₅ O ₇ .xH per 1 L of industrial water ₂ O 60 mg ZnSO ₄ .7H ₂ O 20 mg MnCl ₂ .4H ₂ O 10 mg CaCl ₂ .2H ₂ O 40 mg CuSO ₄ .5H ₂ O 1 mg KI 1 mg (NH ₄ ) ₆ Mo ₇ O ₂₄ .4H ₂ O 1 mg CoCl ₂ .6H ₂ O 1 mg H ₃ BO ₃ 1 mg NaCl 50 mg Defoamer (Silicone KM-75) 1 g

The methanol concentration in the culture broth should be as low as possible in order to reduce the loss of methanol,
It was controlled to 500 ppm. After shifting to continuous culture, the growth rate gradually decreased, and the average residence time was about 40 hours, and the steady state was reached. The concentration of each mineral ion in the culture solution was continuously measured by ion chromatography, but none of the components was insufficient. The culture was continued under these conditions for 10 days, but the culture results were stable. In the culture broth at this time, 21 g of cells containing about 52% of PHB was present per liter. The cell yield was 0.35 g and the PHB yield was 0.18 g per 1 g of methanol. After that, the average residence time was changed by changing the culture pH, and the relationship between the average residence time and the PHB content was investigated. The results are shown in Table 4.
It was shown to. The PHB content in the bacterial cells increased as the set pH was lowered and the average retention time in the culture tank became longer. A dramatic increase in PHB content was observed when the average retention time was set to 15 hours or more. It was

[0033]

[Table 4] Table 4 ───────────────────────── pH average residence time PHB content (hr) (%) ────── ─────────────────── 6.6 4.3 2.5 5.0 6.0 4.3 5.7 15.1 29.8 5.5 20 .6 40.2 5.3 40.2 52.0 ──────────────────────────

[0034]

INDUSTRIAL APPLICABILITY According to the present invention, PHB can be produced on a large scale, economically and stably. Since the PHB production according to the present invention is carried out by a simple operation that only lowers the culture pH, it is easy to manage the operation after setting the optimum conditions, and compared with the conventional process of limiting the mineral components of the culture medium. , Dramatic labor saving is achieved. Since the raw material (substrate) is methanol, the raw material cost is very low, but the better point that the substrate is methanol is that it is a very limited substrate, so there is no contamination of bacteria by general bacteria and It is to be able to easily continue stable culture for a certain period.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Toraichi Tahara Niigata City, Niigata City, Tayuhama, Niiwari, 182 Niigata Research Center Niigata Research Center

Claims (1)

[Claims] 1. A methanol-assimilating bacterium having an ability to produce poly-3-hydroxybutyric acid is cultured by using methanol as a carbon source to synthesize and accumulate poly-3-hydroxybutyric acid in the cells, From bacterial cells to poly-3-
A method for producing poly-3-hydroxybutyric acid, which is characterized in that in the method for producing poly-3-hydroxybutyric acid for obtaining hydroxybutyric acid, low pH culture is performed such that the culture pH is a growth limiting factor.