KR101618852B1 - Method for mass production of succinic acid with carbon dioxide aeration at early stage of cultivation - Google Patents

Abstract

The present invention relates to a method for producing succinic acid through initial carbon dioxide aeration, and more particularly, to a method for producing succinic acid by culturing actinobacillus succinogenes having succinic acid production ability, To a method for producing the same.
The present invention is based on the premise of production of a high concentration of succinic acid by carbon dioxide, which is an expensive medium component, and the shortest time aeration is carried out to reduce the production cost. The magnesium carbonate, which is one of the expensive medium components, · Heavy).

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for mass production of succinic acid through carbon dioxide aeration at the initial stage of culture,

The present invention relates to a method for producing succinic acid through initial carbon dioxide aeration, and more particularly, to a method for producing succinic acid by culturing actinobacillus succinogenes having succinic acid production ability, To a method for producing the same.

Succinic acid is a C4-based organic acid that can be applied to various fields in medicine, foodstuffs, and petrochemical processes, and its value is very high because it forms an international market of more than 20 trillion won annually. In the US Department of Energy, it has been selected as one of the top ten most important substances in the future.

Succinic acid is called straight-chain dicarboxylic acid (HOOC-CH ₂ CH ₂ -COOH), and in 1550 there is evidence that R. agricola was obtained from amber, a fossilized resin, also called succinic acid. Succinic acid is a colorless, columnar or plate-like crystal with a molecular weight of 118.09, a melting point of 185 ° C, a boiling point of 235 ° C and a specific gravity of 1.564.

Succinic acid is one of the major organic acids that constitute the TCA circuit and is a kind of carboxylic acid. In the TCA circuit, succinic acid is produced by dehydrogenation and decarboxylation of alpha-keto glutaric acid to form succinyl Co-A and then converted to succinic acid. Succinic acid is converted to succinic acid by fumaric acid by succinic acid dehydrogenase do.

Succinic acid is produced by chemical synthesis and microbial fermentation. Only a small amount of succinic acid, which is used for specific purposes such as pharmaceuticals, food additives and preservatives, is produced by microbial fermentation. On the other hand, most of the industrially used succinic acid is synthesized from n-butane and acetylene derived from crude oil or liquefied natural gas by major chemical companies in the US, Europe, Japan and China.

However, the succinic acid synthesis process using a chemical method has a problem that a large amount of toxic solid waste, waste solution and waste gas (including carbon monoxide) are emitted during the manufacturing process, and there is a problem of using a fossil raw material, . In addition, there is a problem that the production cost continuously increases due to the continuous rise in oil prices.

Therefore, it is urgent to research and develop a production method of succinic acid using microbial fermentation as a substitute method. Due to recent advances in fermentation technology, it has reached a level that can replace conventional processes that have been produced through hydrogen substitution, and a lot of research is underway in the world.

Korean Patent No. 10-1402659 (entitled "Actino Bacillus succinogenes and a method for producing succinic acid using the same"), which is excellent in the productivity, yield and process stability of succinic acid and is applicable to mass production processes, Actinobacillus succinogenes KCTC 12233BP and a method for producing succinic acid using the same are disclosed. Korean Patent Laid-Open No. 10-2012-00115878 (titled "succinic acid high productivity medium") was developed through a statistical method in a succinic acid-high productivity medium. It was developed using glucose as a carbon source and an expensive yeast extract and a low- And a succinic acid-producing culture medium capable of mass-producing succinic acid on an industrial scale at a low cost by combining an immersion liquid at an optimum ratio.

It is an object of the present invention to develop and provide a method for producing succinic acid at a high concentration by improving the injection method of carbon dioxide in the production of succinic acid.

The present invention relates to a method for producing succinic acid by cultivating actinobacillus succinogenes having succinic acid production ability, wherein the cultivation is carried out by aerating the carbon dioxide gas only for 3 to 6 hours in the initial stage of culture, And a method for producing succinic acid is provided.

Actinobacillus < RTI ID = 0.0 > ( Actinobacillus < / RTI > succinogenes ) supplied carbon dioxide essential for anaerobic induction and succinic acid production by aerating the carbon dioxide for 48 hours. However, carbon dioxide is very expensive and it is disadvantageous in that it is not economically feasible to apply industrialization continuously. However, when the carbon dioxide is not augmented, the production of succinic acid decreases. Therefore, it is necessary to develop a strategy of producing succinic acid at a high concentration while aerating a minimum amount of carbon dioxide. However, in the present invention, a method of producing succinic acid at a high concentration without using a large amount of carbon dioxide was developed by aerating the carbon dioxide gas only for 3 to 6 hours in the initial stage of culture.

In the method for producing succinic acid according to the present invention, it is preferable that the carbon dioxide is injected at 0.4 to 0.8 vvm (aeration volume / medium volume / minute) during the culture.

On the other hand, in the method for producing succinic acid according to the present invention, it is preferable to use a culture medium in which magnesium carbonate (MgCO ₃ ) is added. This is because magnesium carbonate can be an excellent source of carbonate ions necessary for culture. At this time, the medium may further include glucose, yeast extract, solid matter of corn steep liquor, and sodium bicarbonate (NaHCO ₃ ). At this time, the medium preferably contains 100 to 110 g / L of glucose, 10 to 15 g / L of yeast extract, 15 to 20 g / L of corn steep liquor solid, 8 to 12 g / L of sodium hydrogencarbonate (NaHCO ₃ ) And 90 to 110 g / L of magnesium. More preferably, it is more preferable that the solid content of the corn steep liquor is from 17 to 20 g / L, from 10 to 110 g / L of glucose, from 12 to 15 g / L of yeast extract, from 9 to 11 g / L of sodium hydrogencarbonate (NaHCO ₃ ) 105 g / L.

On the other hand, in the medium composition of the present invention, magnesium carbonate can be used, for example, magnesium carbonate heavy. The magnesium carbonate heavier has a formula of (MgCO ₃ ) _4. (MgOH) ₂ .5H ₂ O, which is lower in cost than magnesium carbonate, so that the productivity of the present invention production process can be improved through its use.

On the other hand, in the succinic acid production method of the present invention, the culture may be a batch culture. The batch culture is easier to use than the other cultivation methods and industrially, so that the present invention can produce succinic acid at a high concentration even by batch culture which is a simple culture method.

The present invention is based on the production of a high concentration of succinic acid. The carbon dioxide, which is an expensive medium, is aesthetized for the shortest time, resulting in an innovative reduction in production cost. Magnesium carbonate, which is one of the expensive medium components, · Heavy). Therefore, the present invention has a considerable competitiveness in industrial application and is expected to further enhance the possibility of domestic industrialization of succinic acid as a platform chemical.

1 shows the steps of converting carbon dioxide into carbonate ion when dissolved in water and the equilibrium constant of the conversion reaction.
FIG. 2 is a graph showing the degree of change of succinic acid production amount with aeration time when a carbon dioxide gas is aerated in various times in a 5 L fermentation tank. FIG.
3 is a graph showing the amount of residual glucose in a 5 L fermenter according to aeration time when carbon dioxide gas is aerated at various times.
4 is a graph showing the degree of change of succinic acid production amount in the fermentation tank with aeration time when a carbon dioxide gas is aerated in various times in a 50 L fermentation tank.
FIG. 5 is a graph showing the residual glucose amount with time in a 50 L fermentation tank when carbon dioxide gas is aerated at various times. FIG.

The production of succinic acid using Actino Bacillus succinogenes is very important for the effective supply of carbon dioxide and carbon dioxide, which is the so-called total carbonate concept (see FIG. 1). 1 shows the steps of converting carbon dioxide into carbonate ion when dissolved in water and the equilibrium constant of the conversion reaction.

The most expensive medium for the study of succinic acid production in the laboratory scale production process is carbon dioxide gas, which is designed in conjunction with a plant that generates carbon dioxide gas on an industrial scale (such as linkage with an alcohol production plant) .

However, the above-mentioned linkage of the plant causes various limitations such as the condition and the place. Therefore, it is essential to supply CO 2 gas efficiently to produce Succinic acid, which is cheap in sales price, at a low production cost. Here, productivity of succinic acid is maintained, and the operation method of supplying CO 2 gas to the minimum can be applied more industrially And to develop such a method.

In the present invention, absolutely were to develop a succinic acid production process, considering the "economic" is required, for example in the main of the production medium bicarbonate ion of the present invention supply source of the MgCO ₃ mass compounds of low MgCO ₃ for industrial application Heavy (MgCO ₃ heavy: (MgCO ₃ ) 4 (MgOH) ₂ .5H ₂ O). Although 100 g / L of industrial low-cost compound MgCO ₃ · heavy is supplied to the medium, 100 g / L MgCO ₃ actually exists in about 70%.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

[ Example  One: Actino Bacillus SuShinogenes  ( Actinobacillus succinogenes ) ≪ / RTI >

 1. Experimental Method

(1) Actinobacillus Actinobacillus Succinogenes ) and storage

As succinic acid producing strains, Actinobacillus ( Actinobacillus ), purchased from the American Type Culture Collection (ATCC) succinogenes ) A mutant strain, UK13 strain (KCTC 12233BP) derived from ATCC 55618 was used (Korean Patent Registration No. 10-1402659).

For solid culture, TSA (tryptic soy agar) medium (15 g of pancreatic digest of casein, 5 g of papaic digest of soybean, 5 g of NaCl, 15 g of agar and 1 L of distilled water) was used.

Production growth culture for increasing the microbial cells prior to culturing, TSB (tryptic soy broth) medium (pancreatic digest of casein 17 g, papaic digest of soybean 3 g, dextrose 2.5 g, NaCl 5 g, K 2 HPO 4 (potassium phosphate dibasic 2.5 g, distilled water 1 L) was used. This resulted in highly active microbes.

For storage of the strain, the cells obtained from the liquid culture were stored at 4 ° C. or 20% glycerol stock (stock), stored at -80 ° C., taken out as needed, and inoculated into a solid passage culture medium, And cultured.

(2) Cultivation conditions of Actinobacillus succinogenes for production of succinic acid

Single colonies grown in the above solid medium were aseptically collected and inoculated into liquid medium at 1% (v / v).

In both growth and production cultures, 1% of the seed culture was inoculated with respect to the culture volume, and 100 g / L of glucose, 10 g / L of yeast extract, 15 g / L of corn steep liquor, 10 g / L of sodium hydrogencarbonate, Production medium containing 100 g / L heavy was used.

The growth of the first cells was performed in a shaking incubator at 38 ° C and 200 rpm for 1 to 2 days, and the cells were cultured in a 50 ml glass tube in a 5 ml culture volume. The primary growth culture was incubated for 12 to 15 hours, and then the secondary growth culture was inoculated into the fermenter after culturing for 6 to 12 hours in a 250 ml flask with a culture volume of 30 ml.

After the growth culture, the production culture was carried out in a 5 L stirred tank reactor at a culture volume of 3 L to cultivate succinic acid.

  (3) Analysis of yield of succinic acid

For the quantitative analysis of succinic acid in the culture medium of Actinobacillus succinogenes , samples were taken in 1.5 ml microtube, diluted by serial dilution, and then filtered twice using 0.45 ㎛ filter paper And analyzed by HPLC under the following conditions.

- Analysis temperature: 25 ℃

- flow rate: 0.8 ml / min

- Mobile phase: 0.01NH ₂ SO ₄

- Analysis time: 20 minutes

- Sample injection amount: 10 μl

Column: Organic acid column (Bio-Rad, Aminex HPX 87H, 125-0140)

- Detector: UV detector

- Detection wavelength: 210 nm

  (4) per-analysis

The supernatant was centrifuged at 12,000 rpm for 10 minutes and the supernatant was centrifuged three times for 10 minutes at 12,000 rpm to obtain supernatant. Then, the supernatant was collected by filtration using a 0.45 μm HPLC filter paper, Respectively. The sugar analysis conditions using HPLC were as follows.

- Analysis temperature: 40 ℃

- Flow rate: 1.2 ml / min

- mobile phase: acetonitrile: water = 75: 25 (v / v)

- Analysis time: 15 minutes

- Sample injection amount: 20 μl

- Column: Amine column (250 mm x 46 mm, RS tech)

- Detector: RI detector

  (5) Determination of the amount of bacterium of Actinobasilus suisinogenes

First, the culture medium cultured in the fermenter was recovered, centrifuged at 12000 rpm for 10 minutes, and washed 3 times with distilled water or saline water. Then, after drying at 100 ° C for 10 to 12 hours, the weight was measured. Alternatively, the recovered culture was homogeneously mixed and the absorbance was measured at 660 nm using a spectrophotometer.

The amount of cells can be measured by turbidity. In the case of the culture containing magnesium carbonate in the production medium, the magnesium carbonate is completely dissolved by diluting 50 times with 1N hydrochloric acid to measure the absorbance.

 2. Experimental results

Actinobacillus Actinobacillus succinogenes ), the control group ('0 hour' aeration group) in which no carbon dioxide gas was supplied, 1.5, 3, 4.5, and 6% from the start of culture to confirm the change of succinic acid production according to the various aeration conditions of carbon dioxide (Fig. 1). The results are shown in Table 1 and Table 2, respectively.

Culture conditions Culture medium Liquid medium containing 100 g / L of glucose, 10 g / L of yeast extract, 15 g / L of corn steep liquor, 10 g / L of sodium hydrogencarbonate and magnesium carbonate · Heavy 100 g / Strain Actino Bacillus SUSHINOGENES UK13 Incubation period 48 hours Stirring speed 300 rpm Hydrogen ion concentration Adjust to 6.8 Carbon dioxide aeration time 0 1.5 3 4.5 6 48

After the carbon dioxide gas was aerated for a time corresponding to the various conditions described above, external air was prevented from flowing into the fermentation tank to prevent contamination. In addition, the 0 hour aeration group as the control group and the 3 hour aeration group as the most economical compared with the production amount were specifically cultured twice each.

The experimental results are shown in Table 2, Fig. 2 and Fig. FIG. 2 is a graph showing the degree of change of succinic acid production amount with aeration time when a carbon dioxide gas is aerated in various times in a 5 L fermentation tank. FIG. 3 is a graph showing the amount of residual glucose in a 5 L fermenter according to aeration time when carbon dioxide gas is aerated at various times.

Carbon dioxide aeration time 0 hours ⁴⁾ 1.5 hours 3 hours ⁴⁾ 4.5 hours 6 hours 48 hours Final cell mass (g / L) 11.90 11.90 11.18 11.17 11.1 11.18 Production of succinic acid (g / L) 53.07 59.69 85.51 82.64 85.73 89.98 Remaining glucose amount (g / L) 21.93 9.74 0.07 0 0 0 Productivity (g / L / hr) 1.19 1.24 1.78 1.7 1.82 1.85 Y _{P / S} (g SA / g glu) ¹⁾ 0.60 0.60 0.78 0.76 0.8 0.82 Y _{X / S} (g cell / g glu) ²⁾ 0.12 0.12 0.10 0.10 0.1 0.1 Y _{P / X} (g SA / g cell) ³⁾ 4.82 5.02 7.65 7.40 7.87 7.95

1) Y _{P / S} : Production yield of glucose non-succinic acid (SA) used

2) Y _{X / S} : Growth yield of non-cell mass of glucose used

3) Y _{P / X} : amount of bacterium Unsaturated (SA) production yield

4) The average value of the culture results of the No. 2 culture

As a result of the experiment, as shown in Table 2 and FIG. 2, it was found that the culture (0 hour aeration) in which carbon dioxide gas was not aerated was 53.07 g / L on average and the production of succinic acid was small. In the case of culture aeration only for 1.5 hours from the start of culture, it was observed that the production of succinic acid was increased to a small level, and the increase was not great.

However, the production of succinic acid was significantly increased in the case of cultivation in which the carbon dioxide gas was aerated only for 3 hours from the start of culture. It was confirmed that succinic acid was produced at an average of 85.51 g / L in the culture in which carbon dioxide gas was aerated for 3 hours, and productivity of 1.78 g / L / hr and succinic acid yield of 0.78 g / g of glucose (Table 2, Fig. 2). Which was substantially the same in terms of the amount of succinic acid produced as compared with the supply of carbon dioxide for 48 hours.

On the other hand, the cultures fed for 4.5 hours and 6 hours showed 82.64 g / L and 85.73 g / L succinic acid production, respectively, as compared with those cultured with carbon dioxide gas for 3 hours .

On the other hand, the above results could be solved by the fact that the bicarbonate ion is supplied smoothly together with the carbon dioxide gas in the early stage of cultivation, and the succinic acid metabolic process acts more strongly. MgCO ₃ · Heavy contained in the production medium combination supplied bicarbonate ions and supplied gaseous carbon dioxide to form an environment rich in total carbonate in the inoculated cells (see FIG. 1). Therefore, it was predicted that the daughter cells of the above-mentioned cells had more metabolic processes using more carbon dioxide.

As a result, it was found that, by simply aeration of carbon dioxide gas for a short time in the early stage, the supply environment of abundant carbonic acid ions is improved compared with the culture without aeration of carbon dioxide gas. Thus, the production amount of succinic acid is about 1.3 times It is possible to interpret it as.

On the other hand, the active metabolic activity of the cells upon carbon dioxide aeration was further confirmed by observing the residual glucose concentration shown in Fig. The culture without carbon dioxide gas aeration showed that glucose could not be used at all, and it was confirmed that carbon dioxide gas was used for a further 1.5 hours aeration. However, it was observed that glucose was used in cultures in which carbon dioxide gas was aerated for 3 hours or more.

[ Example  2: Through expansion of culture Actino Bacillus SuShinogenes  ( Actinobacillus  succinogenes ) Succinic acid production]

In order to confirm that the results of the above Example 1 show the same effect even under the expanded culture condition, the culture without carbon dioxide aeration, the culture in which carbon dioxide is aerated at 0.6 vvm for 6 hours, and the carbon dioxide at 0.6 vvm Were performed in a 50 L fermentor.

Cultivation was carried out in the same manner as in Example 1 except that the fermentation tank was cultivated and a working volume of 5 L was cultivated. The experimental results are shown in Table 3 below.

Culture conditions 0 hour 6 hours 48 hours Final cell mass (g / L) 11.9 11.14 11.19 Final SA (g / L) 62.26 79.16 74.94 Residual glu. conc. (g / L) 30.73 0 0 Productivity (g / L / hr) 1.30 1.65 1.56 Y _{P / S} (g SA / g glu) ¹⁾ 0.80 0.72 0.69 Y _{X / S} (g cell / g glu) ²⁾ 0.15 0.10 0.10 Y _{P / X} (g SA / g cell) ³⁾ 5.23 7.11 6.70

1) Y _{P / S} : Production yield of glucose non-succinic acid used

2) Y _{X / S} : Growth yield of non-cell mass of glucose used

3) Y _{P / X} : yield of bismuth non-succinic acid production

As a result, it was found that the 5-L fermenter as a whole exhibited the same phenomenon. In the culture without aeration of carbon dioxide, 62.26 g / L of succinic acid was produced, and the culture for 6 hours aeration was 79.16 g / It was confirmed that the culture produced 74.94 g / L of succinic acid. As a result, it was confirmed that the culture obtained by aerating the carbon dioxide gas for 6 hours produced about 1.3 times as much succinic acid than the culture not aerated with the carbon dioxide gas (Table 4 and FIG. 4). FIG. 4 is a graph showing the degree of change of succinic acid production amount in a fermenter according to aeration time when a carbon dioxide gas is aerated in a 50 L fermenter at various times. FIG.

On the other hand, the change in the residual glucose concentration and the culture without aerating the carbon dioxide gas showed that glucose was not completely used (Table 4 and Fig. 5). FIG. 5 is a graph showing the amount of residual glucose according to aeration time when a carbon dioxide gas is aerated in a 50 L fermenter at various times. FIG.

According to the results of the experiment of the present invention, it was concluded that there is a metabolic process of biosynthesizing succinic acid through the absorption of bicarbonate ions in the cells during the adaptation period of the cells, and a synergistic effect is caused by aeration of the carbon dioxide gas. As a result, it was predicted that the same amount of succinic acid could be produced even at an industrial fermentation scale of 10 times increase,

Claims (7)

A method for producing succinic acid by culturing Actinobacillus succinogenes having succinic acid production ability,
The cultivation was carried out in the same manner as in Example 1 except that 100 to 110 g / L of glucose, 10 to 15 g / L of yeast extract, 15 to 20 g / L of corn steep liquor solid, 8 to 12 g / L of sodium hydrogencarbonate (NaHCO ₃ ) magnesium carbonate · heavy) of 90 to 110 g / L, carbon dioxide gas was aerated at an infusion rate of 0.4 to 0.8 vvm (aeration volume / medium volume / minute) only for 3 to 6 hours in the initial stage of culture, Lt; RTI ID = 0.0 > a < / RTI >
delete delete delete delete delete The method according to claim 1,
The above-
Lt; RTI ID = 0.0 > a < / RTI > batch culture.

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