KR100842869B1 - Culture method by providing nitrogen and carbon source automatically and apparatus therefor - Google Patents

Abstract

A fed-batch culturing method is provided to supply carbon source as well as nitrogen source to adjust a pH of a culture solution, thereby obtaining a product with high yield and making a culture process convenient. A fed-batch culturing method for supplying nitrogen source and carbon source automatically in accordance with pH change comprises the steps of: (a) measuring a pH of a culture solution; (b) after determining an amount of the nitrogen source to be added from the measured pH value, supplying the determined amount of the nitrogen source to the culture solution; and (c) after determining an amount of the carbon source to be added from the added amount of the nitrogen source, supplying the determined amount of the carbon source to the culture solution, wherein the nitrogen source is ammonia water or ammonia gas and the carbon source is glucose. Further, the step (b) is performed by a feedback control method.

Description

Feeding cultivation method and apparatus for automatically supplying nitrogen source and carbon source {Culture Method by Providing Nitrogen and Carbon Source Automatically and Apparatus Therefor}

Figure 1 shows one embodiment of the fed-batch culture apparatus according to the present invention.

Figure 2 is a graph showing the results of measuring the consumption of ammonia water and glucose and the production of threonine while culturing according to the conventional (preliminary) culture method.

3 is a graph showing the consumption ratio of ammonia water and glucose as a result of culturing according to a conventional (preliminary) culturing method.

Figure 4 is a graph showing the results of measuring the production of threonine while culturing according to the culture method of the present invention.

5 is a graph showing the results of measuring the production of threonine while culturing according to another culture method of the present invention.

<Description of the code for each main part of the drawing>

100: fed-batch culture apparatus 110: control unit

120: culture tank 130: pH meter

140: nitrogen source chamber 150: nitrogen source supply pump

160: carbon source chamber 170: carbon source supply pump

The present invention relates to a fed-batch cultivation method and apparatus for automatically supplying a nitrogen source and a carbon source, and more particularly, a fed-batch cultivation method for measuring the pH of a culture medium and automatically supplying a nitrogen source and a carbon source according to the value thereof, and Relates to a device.

"Federated cultivation" means a culture in which one or more nutrients are fed into the culture continuously immediately after the start of fermentation or after the culture has reached some stage, or when the supplied nutrient is depleted from the culture. In fed-batch cultures, for example, pH adjustment is generally used to maintain the desired growth conditions, and the essential nutrients are fed to the culture to prevent the depletion of one or more essential nutrients. Microbial cells will continue to grow at a growth rate determined by the nutrient supply rate. In general, a single nutrient, a carbon source, is the limiting factor for growth. Other kinds of nutrient limitations may also be used, for example, by nitrogen sources, by oxygen, by sulfur and by phosphorus.

In general, in the production process of a product containing nitrogen and carbon as the main constituents, a carbon source or a nitrogen source is used as a biosynthetic raw material of the target product, so that the concentration of the target product can be kept to a maximum without depleting the carbon source or nitrogen source in the culture medium. In order to maintain the system, a carbon source or a nitrogen source must be supplied continuously. To this end, the culture medium is periodically taken in the production process of the product, and the concentration of carbon source or nitrogen source in the culture solution is measured, and sugar, ammonia water, ammonia gas or ammonium ions are additionally added.

Therefore, in most biological processes, the supply strategy of these additional carbon or nitrogen sources has a large impact on productivity, but automation is difficult because the additional amounts and rates of carbon or nitrogen sources are determined after periodic sampling and analysis. There is a risk of this being lowered.

In order to solve this problem, a culture method for automatically supplying components required for various cultures including a carbon source or a nitrogen source has been developed, and related techniques are described in the following documents.

Korean Patent Application No. 1983-0000063 relates to a method for automating the glutamic acid fermentation process by exhaust gas analysis. The method of cultivating oil price by measuring the concentration of penicillin and the depletion state of fermented sugar by continuously measuring carbon dioxide concentration in glutamic acid fermentation exhaust Disclosed is a method for automating a glutamic acid fermentation process characterized by automating a penicillin (or surfactant) administration process and an additional sugar addition process, which are essential for an industrial glutamic acid fermentation process.

Korean Patent No. 10-0392411 relates to a method for increasing yeast concentration in culture by automatically adding glucose and ammonium when reusing liquor waste liquor as a culture substrate of yeast.In the case of glucose, the dissolved oxygen concentration of the culture is automatically During measurement, when dissolved oxygen concentration increases above a certain level, a certain amount of glucose is added, and in the case of ammonium, ammonium hydroxide is used as a pH adjuster after the addition of glucose is started to be automatically supplied by the pH controller.

However, the automation method of Korean Patent Application No. 1983-0000063 has a disadvantage that it can be applied only when the final product is glutamic acid. In addition, the automated method of the Republic of Korea Patent No. 10-0392411 should measure the dissolved oxygen concentration to determine the addition amount and rate of glucose, and at the same time to measure the pH in the culture medium to determine the addition amount and rate of ammonium Ammonium has a problem in that both indicators (dissolved oxygen concentration and pH) in the culture should be measured.

The present inventors have confirmed that the addition of a carbon source when adding a nitrogen source to adjust the pH of the culture solution to increase the yield of the product and provide convenience to the experimenter and completed the present invention.

The present invention as one aspect, (1) measuring the pH of the culture; (2) determining an additional amount of a nitrogen source from the measured pH value and supplying it to the culture solution; (3) It provides a fed-batch culture method for automatically supplying the nitrogen source and the carbon source according to the pH change, including the step of determining the additional amount of the carbon source from the additional amount of the nitrogen source and supplying the culture medium.

In another aspect, the control unit; Culture tank; A pH meter for measuring the pH of the culture solution; A nitrogen source chamber for storing a nitrogen source; A nitrogen source pump or valve for supplying a nitrogen source from the nitrogen source chamber to the culture tank; A carbon source chamber for storing a carbon source; Provided is a fed-batch culture apparatus in which a nitrogen source and a carbon source are automatically supplied according to a pH change including a carbon source pump or a valve for supplying a carbon source to a culture tank from a carbon source chamber.

The present invention (1) measuring the pH of the culture; (2) determining an additional amount of a nitrogen source from the measured pH value and supplying it to the culture solution; (3) relates to a fed-batch cultivation method for automatically supplying a nitrogen source and a carbon source according to a pH change including determining an additional amount of a carbon source from the additional amount of the nitrogen source and supplying it to the culture solution.

The fed-batch culture method of the present invention is particularly applicable to culturing microorganisms to produce any compound, preferably amino acids, more preferably threonine, including carbon and nitrogen as products.

The fed-batch cultivation method of the present invention can adjust the nitrogen source, in particular pH, to measure the pH of the culture medium in the culture and when the measured pH value is outside the specified pH value in order to achieve an optimum production yield. It is characterized by supplying a carbon source while supplying a nitrogen source.

Thus, the "nitrogen source" is preferably ammonia water or ammonia gas capable of adjusting pH.

In addition, the term "carbon source" includes all carbon sources available by microorganisms, and depends on various conditions such as the type of microorganism, the product, and the environment, and for example, glucose, mannose, fructose, galactose, sucrose, and maltose. Mannitol, arabinose, xylose, dextrin, starch and the like.

According to the fed-batch culture method of the present invention, the pH of the culture solution is measured using a pH meter or the like during culturing, and when the measured pH value deviates from the specified pH value to achieve an optimum production yield, the pH is adjusted. Add nitrogen source. In the present invention, it is preferable to use a feedback control method when adding a nitrogen source to adjust the pH.

Then, the additional amount of the carbon source is calculated from the additional amount of the nitrogen source in order to add the carbon source simultaneously with the nitrogen source. To this end, in the present invention, pre-cultivation is carried out by a conventional culture method to measure the consumption of nitrogen and carbon sources, calculate the consumption ratio (R) of nitrogen and carbon sources, and then use the same to determine the carbon source from the additional amount of nitrogen source in real culture. The additional amount of the carbon source can be calculated from the additional amount of the nitrogen source based on the consumption amount (R _{c / n} ) of the nitrogen source and the carbon source shown in Equation 1 (see Example 1) (to be described later).

Equation 1 may be described as follows by exemplifying a case of producing threonine by culturing E. coli. Specifically, when culturing E. coli to produce threonine, as shown in Scheme 1, the carbon source and nitrogen source are used for the growth of E. coli and the production of threonine, and some carbon sources are discharged in the form of carbon dioxide.

A (C ₆ H ₁₂ O6) + B (O ₂ ) + C (NH ₃ ) + D (P0 ₄ ) →

E (C _1.0 H _1.73 N _{0.22 O0.30} P _0.06 ) + F (CO ₂ ) + G (H ₂ O) + H (C ₄ H ₉ O ₃ N)

A: number of moles of carbon source consumed B: number of moles of oxygen consumed

C: moles of nitrogen source consumed D: moles of phosphoric acid consumed

E: number of moles of grown cells F: number of moles of carbon dioxide produced

G: Mole of produced water H: Mole of produced product

Using the stoichiometric equation to calculate the consumption ratio of the nitrogen source to the carbon source R _{c / n} at a specific incubation time is the following equation (2).

Δ CDW: Changed cell mass per unit time

CER: CO2 emission factor (mole / l / h)

Δ threonine: the amount of threonine produced per unit of time

119.1: Threonine Molecular Weight

23.48: molecular weight of dry cells

Δt: Culture time to be measured

However, in order to calculate the consumption ratio R _{c / n} of the nitrogen source and the carbon source using Equation 1, the cell mass, carbon dioxide, and threonine produced at a specific incubation time should be measured precisely. These can be measured using methods and devices known in the art.

Meanwhile, even when the nitrogen source and the carbon source are precisely measured for each incubation time while performing the preliminary culture as described above, the consumption ratio R of the nitrogen source and the carbon source for each incubation time can be calculated.

By using the consumption ratio of the nitrogen source and the carbon source calculated by this process, it is possible to calculate the additional amount of the carbon source from the nitrogen source addition amount in the real culture, and supply the corresponding addition amount of the nitrogen source and the carbon source thus calculated to the culture tank.

The fed-batch culture method according to the present invention may be implemented using a culture device consisting of the following configuration.

The present invention control unit; Culture tank; A pH meter for measuring the pH of the culture solution; A nitrogen source chamber for storing a nitrogen source; A nitrogen source pump for supplying a nitrogen source from the nitrogen source chamber to the culture tank; A carbon source chamber for storing a carbon source; The present invention relates to a fed-batch cultivation apparatus in which a nitrogen source and a carbon source are automatically supplied according to a pH change including a carbon source pump for supplying a carbon source to a culture tank from a carbon source chamber.

In one aspect, the fed-batch culture apparatus 100 of the present invention is a control unit 110 for controlling the overall operation such as data signal and control signal transmission and reception, an input / output device (not shown) such as a monitor, a button, a culture tank in which the culture is performed ( 120), a pH meter 130 for measuring the pH of the culture solution; A nitrogen source chamber 140 storing a nitrogen source; A pump 150 for supplying a nitrogen source from the nitrogen source chamber to the culture tank according to the signal received from the controller; A carbon source chamber 160 storing a carbon source; It comprises a pump 170 for supplying a carbon source from the carbon source chamber to the culture tank in accordance with the signal received from the control unit.

Here, before incubation, a suitable pH value, a consumption ratio of a nitrogen source and a carbon source, is input for each incubation time through an input device. As the culture proceeds, the pH meter 130 immersed in the culture solution of the culture vessel 120 measures the pH of the culture solution, and the measured pH value is transmitted to the controller 110.

The controller 110 adds a nitrogen source when the measured pH value is out of the previously input pH value. To this end, an operating signal corresponding to the additional amount of the nitrogen source is transmitted to the nitrogen source supply pump 150. The nitrogen source supply pump 150 operates according to a signal received from the controller 110 to supply the nitrogen source stored in the nitrogen source chamber 140 to the culture tank 130. In the present invention, it is preferable to use a feedback control method when adding a nitrogen source to adjust the pH.

In addition, the controller 110 determines the additional amount of the carbon source by using the consumption ratio of the nitrogen source and the carbon source previously input from the additional amount of the nitrogen source. The carbon source feed pump 170 then sends an operating signal corresponding to the additional amount of carbon source. The carbon source supply pump 170 operates according to the signal received from the control unit 110 to supply the nitrogen source stored in the carbon source chamber 160 to the culture tank 120.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

<Example 1>

Preparation method of threonine by conventional glucose addition method (preliminary culture)

A. Preculture

Five 500 ml flasks each contain 40 ml of pre-culture medium [4% glucose (Sunday glucose), 0.1% trypto peptone (Difco), 0.1% yeast extract (Difco), 0.2% potassium phosphate (Cheil Chemistry), 0.1% magnesium sulfate (Cheil Chem), 0.1% ammonium sulfate (Cheil Chem), 0.0025% Isoleucine (Sigma), pH 7.0] were dispensed and autoclaved at 121 ° C. for 20 minutes. After the medium was cooled, L-threonine-producing E. coli (KBM-4501) was inoculated each with platinum teeth, followed by reciprocating shaking cultures 180 times per minute at 37 ° C. for 16 hours.

B. Production Culture

2 liters of production medium [4% glucose (Sunday glucose), 0.1% Cheil Phosphate (Cheil Chemistry), 0.1% Ammonium Sulfate (Cheil Chemistry), 0.1% Magnesium Sulfate (Cheil Chemistry), pH 7.0] Autoclaved at 121 ° C. for 20 minutes. After cooling the medium, the seed culture solution was inoculated at 10% and incubated at 37 ° C. for 60 hours at agitation speed of 40 to 1000 rpm and aeration condition of 1.0 to 1.5 VVM. In order to maintain the concentration of glucose at 1-3% in culture, 10-40 ml of 8% glucose solution was added every 1-3 hours from 14 hours after the start of the culture, and the pH was 6.3-6.4 using 25% ammonia water. Was maintained. At this time, the consumption of ammonia water and glucose during fermentation was calculated by precisely measuring on-line using an electronic balance connected to a computer. After fermentation was completed, the accumulation amount of L-threonine in the culture was 62.3 mg / ml (FIG. 2).

The ammonia water consumption and glucose consumption obtained in FIG. 2 were compared to obtain the same result as in FIG. 3. Glucose consumption compared to ammonia water consumption in the first half of the culture was calculated as 2.12, and 3.37 in the second half of the culture.

Example 2: Preparation of Threonine by the Culture Method of the Present Invention

A. Preculture

The preculture was carried out according to the same procedure as the preculture in Example 1-A.

B. Production Culture

2 L of production medium (same as the production medium of Example 1-B) was placed in a 5 L fermenter and autoclaved at 121 ° C. for 20 minutes. After cooling the medium, the seed culture solution was inoculated at 10% and incubated at 37 ° C. for 60 hours at agitation speed of 400 to 1000 rpm and aeration condition of 1.0 to 1.5 VVM. From 4 hours to 27 hours after the start of the culture, glucose was added twice as much as the ammonia consumption to adjust the pH to 6.3, and after 27 hours, 3.3 times as much glucose as the ammonia consumption was set. After completion of the fermentation, the accumulation amount of L-threonine in the culture was 105 mg / ml (FIG. 4).

Example 3: Preparation of threonine by the culturing method of the present invention

A. Preculture

The preculture was carried out according to the same procedure as the preculture in Example 1-A.

B. Preculture with 5 L Fermenter

2 L of the pre-culture medium (same as the pre-culture medium of Example 1-A) was put into a 5 L fermenter and sterilized under pressure at 121 ° C. for 20 minutes. After cooling the medium, the seed culture solution was inoculated with 1% and incubated at 37 ° C. for 12 hours at agitation speed of 100 to 400 rpm and aeration condition of 1.0 to 1.5 VVM.

C. Preculture with 50 L Fermenter

20 L of the pre-culture medium (same as the pre-culture medium of Example 1-A) was put into a 50-L fermenter and sterilized under pressure at 121 ° C. for 20 minutes. After cooling the medium, the seed culture solution was inoculated with 1% and incubated at 37 ° C. for 12 hours at agitation speed of 100 to 400 rpm and aeration condition of 1.0 to 1.5 VVM.

D. Production Culture with 500 L Fermenter

200 L of production medium (same as the production medium of Example 1-B) was placed in a 500 L fermenter and autoclaved at 121 ° C. for 20 minutes. After the medium was cooled, the seed culture solution was inoculated at 10% and incubated at 37 ° C. for 60 hours at a stirring speed of 100 to 400 rpm and aeration conditions of 1.0 to 1.5 VVM. From 4 hours to 27 hours after the start of the culture, glucose was added twice as much as the ammonia water consumption to adjust the pH to 6.3, and after 27 hours, 3.3 times as much glucose as the ammonia water consumption was set. After completion of the fermentation, the accumulation amount of L-threonine in the culture was 105 mg / ml (FIG. 5).

By supplying a nitrogen source while simultaneously supplying a nitrogen source to adjust the pH by the culturing of the present invention, not only can the product be obtained in high yield, but also the cultivation process is simplified.

Claims (7)

(1) measuring the pH of the culture solution; (2) determining an additional amount of a nitrogen source from the measured pH value and supplying it to the culture solution; (3) fed-batch culture method for automatically supplying the nitrogen source and the carbon source according to the pH change, including the step of determining the additional amount of the carbon source from the additional amount of the nitrogen source and supplying it to the culture solution. The method of claim 1, The cultivation is fed-batch culture method, characterized in that for producing amino acids using microorganisms. The method of claim 1, The nitrogen source is a fed-batch culture method characterized in that the ammonia water or ammonia gas. The method of claim 1, The fed-batch culture method, characterized in that the carbon source is glucose. The method of claim 1, Step (2) is fed-batch culture method, characterized in that made by a feedback control system. The method of claim 1, The fed-batch culture method, characterized in that the added amount of the carbon source from the additional amount of the nitrogen source is calculated by the equation (1).

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