Method for producing high-content lysine using automated gas introduction system

Abstract

The present disclosure relates to a method for producing lysine in which ammonium nitrogen in a medium and pH are monitored in real time, and carbon dioxide and / or a nitrogen source is introduced on the basis thereof to significantly increase the concentration of lysine while reducing the concentration of by-products during culture, thereby enhancing fermentation purity and improving the quality of lysine granules.

Description

Method for producing high-content lysine using a gas automated injection system

[0001] Cross-citation with related application(s)

[0002] This disclosure claims the benefit of priority to Republic of Korea Patent Application No. 10-2024-0016196, dated February 1, 2024, the entire contents of which are incorporated herein by reference.

[0003] Numerous papers and patents are referenced and cited throughout this disclosure. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety, thereby providing a clearer understanding of the state of the art and the content of this disclosure.

[0004] The present disclosure relates to a method for producing lysine, and more specifically, to a method for producing lysine at a high content using a gas automation injection system.

[0005]

[0006] Lysine (L-Lysine) is an essential amino acid used in various fields, including as a feed additive, food additive, and pharmaceutical raw material. It is a bulk chemical with a market size of 2.94 million tons as of 2022. Currently, most lysine is mass-produced through direct fermentation using a medium containing a carbon source (raw sugar, sucrose, glucose) and a nitrogen source (yeast extract, soybean meal decomposition, corn steep liquor).

[0007] In the medium components supplied for lysine fermentation using coryneform microorganisms, the sulfate in the ammonium sulfate acts as a counter ion to maintain a neutral pH condition for lysine biosynthesized during fermentation, and ammonium serves as a nitrogen source necessary for lysine biosynthesis. As a result, lysine sulfate exists in the culture medium, and in order to commercialize it in the form of lysine hydrochloride (Lysine HCl), a representative powdered formulation ingredient, the process of adsorbing and eluting lysine through an ion exchange resin process is essential. However, since this process requires an additional purification process and the input of auxiliary materials, it significantly increases the manufacturing cost.

[0008] To overcome the above problems, a fermentation process that eliminates ammonium sulfate added during fermentation has been developed (Chinese Patent Publication No. 110484575). This research relates to a method for manufacturing lysine containing carbonate, wherein the carbonate is generated during fermentation or supplied via an external carbonate aqueous solution and carbon dioxide gas. The fermentation solution obtained through this method can be granulated (Korean Patent No. 10-0838200) to significantly reduce manufacturing costs. The low hygroscopicity and good flowability of granulated products allow for formulation diversification, overcoming process issues and significantly improving cost competitiveness. However, in the granule manufacturing process using lysine carbonate fermentation, if granulation is performed without ion exchange resin treatment, byproducts other than lysine generated during fermentation are a major cause of product purity reduction. Therefore, it is essential to reduce organic acid and amino acid byproducts and improve product yield.

[0009]

[0010] The purpose of the present disclosure is to provide a method for producing lysine at a high content by utilizing a gas automation injection system.

[0011] The inventors of the present disclosure monitored the concentration of ammonium nitrogen (hereinafter also referred to as “AN”) in a medium during a fermentation process using a microorganism capable of producing lysine, and when the pH condition reaches a specific range and the ammonium nitrogen decreases below a specific value, a nitrogen source and / or carbon dioxide is added to the medium to adjust the ammonium nitrogen to an appropriate range, thereby confirming that the concentration of byproducts during culture is reduced while the concentration of lysine is greatly improved, thereby improving the fermentation purity and enhancing the quality of lysine granules, thereby completing the invention of the lysine production method of the present disclosure.

[0012]

[0013] This is specifically explained as follows. Meanwhile, each description and embodiment disclosed in this disclosure can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this disclosure fall within the scope of this disclosure. Furthermore, the scope of this disclosure is not limited by the specific descriptions described below. Furthermore, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated into this specification in their entirety by reference to more clearly explain the level of the technical field to which this disclosure belongs and the contents of this disclosure.

[0014]

[0015] According to one aspect of the present disclosure, the present disclosure provides a method for producing lysine by a fermentation process using a microorganism having the ability to produce lysine,

[0016] By monitoring the concentration of ammonium nitrogen in the medium,

[0017] A method for producing lysine is provided, characterized by adding a nitrogen source to a medium.

[0018] The inventors of the present disclosure have found that in a method for producing lysine by a fermentation process using a microorganism capable of producing lysine, by monitoring the concentration of ammonium nitrogen (hereinafter also referred to as “AN”) in a medium, when the pH condition reaches a specific range and / or the ammonium nitrogen decreases below a specific value, a nitrogen source and / or carbon dioxide is added to the medium to adjust the ammonium nitrogen to an appropriate range, the concentration of by-products during culture is reduced while the concentration of lysine is greatly improved, thereby improving the fermentation purity and enhancing the quality of lysine granules, and thus invented a method for producing lysine according to the present disclosure.

[0019] In the present disclosure, the nitrogen source and / or carbon dioxide may be automatically added to the medium. More specifically, the nitrogen source and / or carbon dioxide may be automatically added to the medium through an automated gas injection system.

[0020] In the present disclosure, the term "automated gas injection system" means a system that monitors in real time the concentration of ammonium nitrogen in a medium and / or the pH of the medium during a fermentation process using a microorganism capable of producing lysine, automatically injects ammonia gas and / or ammonia water as a nitrogen source into the medium under specific conditions, and / or automatically injects carbon dioxide into the medium to control the ammonium nitrogen and / or pH within an appropriate range. When utilizing such an automated gas injection system, the concentration of byproducts during cultivation is reduced while the concentration of lysine is significantly improved, thereby improving fermentation purity and enhancing the quality of lysine granules.

[0021]

[0022] The microorganism capable of producing lysine used in the lysine production method of the present disclosure is not particularly limited, and any microorganism capable of producing lysine through fermentation may be used. Examples of such microorganisms include coryneform bacteria and bacteria belonging to the genera Escherichia, Serratia, and Bacillus. While coryneform bacteria and bacteria belonging to the genus Escherichia are described below, the microorganisms used in the method of the present disclosure are not limited to these bacteria.

[0023] Coryneform bacteria (also referred to as "coryneform microorganisms" hereinafter) are incorporated into the genus Corynebacterium and include bacteria belonging to the genus Brevibacterium, which is closely related to Corynebacterium. Examples of such coryneform bacteria include:

[0024] Corynebacterium acetoacidophilum,

[0025] Corynebacterium acetoglutamicum,

[0026] Corynebacterium alkanolyticum,

[0027] Corynebacterium callunae,

[0028] Corynebacterium glutamicum,

[0029] Corynebacterium lilium (Corynebacterium glutamicum),

[0030] Corynebacterium melassecola,

[0031] Corynebacterium thermoaminogenes,

[0032] Corynebacterium herculis,

[0033] Brevibacterium divaricatum,

[0034] Brevibacterium flavum (Corynebacterium glutamicum),

[0035] Brevibacterium immariophilum,

[0036] Brevibacterium lactofermentum (Corynebacterium glutamicum),

[0037] Brevibacterium roseum,

[0038] Brevibacterium saccharolyticum,

[0039] Brevibacterium thiogenitalis,

[0040] Brevibacterium album,

[0041] Brevibacterium cerinum,

[0042] Microbacterium ammoniaphilum.

[0043] Examples of bacteria belonging to the genus Escherichia include, but are not limited to, Escherichia coli.

[0044] A microorganism capable of producing lysine may be one that originally possesses this ability, or may have been modified to have this ability. A microorganism capable of producing lysine can be acquired, for example, by imparting lysine production ability to such a microorganism, or by enhancing the lysine production ability of such a microorganism.

[0045] In one embodiment, the microorganism having the ability to produce lysine may be Corynebacterium glutamicum, and more specifically, may be Corynebacterium glutamicum CJ3P, which has L-lysine production ability by introducing the mutations pyc (P458S), hom (V59A), and lysC (T311I) that are known to be present in three genes using Corynebacterium glutamicum ATCC13032 as a parent strain (US 9556463 ​​B2), but is not limited thereto.

[0046] In the present disclosure, the culture method is not limited, but may be performed, for example, as batch culture, fed-batch culture, or continuous culture. In a specific embodiment, in the present disclosure, the seed culture is performed as batch culture, and in the present culture, the batch culture is followed by fed-batch culture, but the present disclosure is not limited thereto.

[0047] The medium used in the present disclosure may be a liquid medium, and such liquid medium is not particularly limited and may be any commonly known medium containing organic or inorganic nutrients such as carbon sources, nitrogen sources, and other trace nutrients that may be used depending on the microorganism used.

[0048] Any carbon source can be utilized by microorganisms, as long as they are available. Examples include sugars such as saccharose, glucose, fructose, molasses, and starch hydrolysates; organic acids such as acetic acid; and alcohols such as ethanol. Nitrogen sources include inorganic substances such as ammonium ions, protein hydrolysates, and yeast extracts. Trace nutrients include amino acids, vitamins, and trace metal elements.

[0049] Additionally, the pH of the medium can be adjusted by appropriately using basic compounds such as sodium hydroxide, potassium hydroxide, or ammonia, or acid compounds such as phosphoric acid or sulfuric acid. Furthermore, foaming can be suppressed by using antifoaming agents such as fatty acid polyglycol esters.

[0050] Meanwhile, the appropriate culture temperature, which can be selected depending on the microorganism used, is generally 20 to 45°C, preferably 25 to 40°C. Furthermore, sufficient stirring is performed during fermentation, and sufficient oxygen is supplied. Oxygen or an oxygen-containing gas (e.g., air) is injected into the culture medium to maintain an aerobic state.

[0051]

[0052] In one embodiment, ammonium sulfate may be further included in the medium to serve as a nitrogen source and counter-anion source. More specifically, the ammonium sulfate may be included in the medium at the beginning of the culture, more specifically at the start of the culture. In ammonium sulfate, sulfate acts as a counter ion for lysine biosynthesized during fermentation, and ammonium acts as a nitrogen source supply necessary for lysine biosynthesis. The present disclosure is characterized by using ammonium sulfate to a minimum. Specifically, the ammonium sulfate used in the present disclosure has a molar ratio of ammonium sulfate to the molar of the carbon source of 0.2 (mol / mol) or more, 0.3 (mol / mol) or more, 0.4 (mol / mol) or more, 0.45 (mol / mol) or more, 1.0 (mol / mol) or less, 0.8 (mol / mol) or less, 0.7 (mol / mol) or less, 0.6 (mol / mol) or less, 0.5 (mol / mol) or less, 0.2 (mol / mol) to 1.0 (mol / mol), 0.3 (mol / mol) to 1.0 (mol / mol), 0.4 to 1.0 (mol / mol), 0.45 (mol / mol) to 1.0 (mol / mol), 0.46 (mol / mol) to 1.0 (mol / mol), 0.2 (mol / mol) to 0.8 (mol / mol), 0.3 (mol / mol) to 0.8 (mol / mol), 0.4 to 0.8 (mol / mol), 0.45 (mol / mol) to 0.8 (mol / mol), 0.46 (mol / mol) to 0.8 (mol / mol), 0.2 (mol / mol) to 0.7 (mol / mol), 0.3 (mol / mol) to 0.7 (mol / mol), 0.4 (mol / mol) to 0.7 (mol / mol), 0.45 (mol / mol) to 0.7 (mol / mol), 0.46 (mol / mol) to 0.7 (mol / mol), 0.2 (mol / mol) to 0.6 (mol / mol), 0.3 (mol / mol) to 0.6 (mol / mol), 0.4 (mol / mol) to 0.6 (mol / mol), 0.45 (mol / mol) to 0.6 (mol / mol), 0.46 (mol / mol) to 0.6 (mol / mol), 0.2 (mol / mol) to 0.5 (mol / mol), 0.3 (mol / mol) to 0.5 (mol / mol), 0.4 (mol / mol) to 0.5 (mol / mol), 0.45 (mol / mol) to 0.5 (mol / mol), or 0.46 (mol / mol) to 0.5 (mol / mol), but is not limited thereto. Therefore, when the method for producing lysine of the present disclosure is used, an expensive process of adsorbing and eluting lysine by an ion exchange resin process that must be used when a large amount of ammonium sulfate is used is not used.

[0053] Specifically, in the present disclosure, ammonium sulfate is included in the medium at the initial stage of cultivation (more specifically, at the start of cultivation) to somewhat increase the concentration of ammonium nitrogen in the medium at the beginning of cultivation, and ammonium sulfate may not be additionally added to the medium during the fermentation process (during cultivation).

[0054]

[0055] In one embodiment, monitoring the concentration of ammonium nitrogen in the medium may be performed using a near infrared (NIR) spectrometer.

[0056] The above NIR can analyze multiple components simultaneously and continuously in a short period of time without any pretreatment of the sample, so it can analyze all components simultaneously without any pretreatment of the sample, unlike the existing wet analysis method that analyzes each component individually, such as the high-performance liquid chromatography (HPLC) method for analyzing sugars, amino acids, and nucleic acids, or the Kjeldahl analysis method for analyzing nitrogen components. Therefore, real-time analysis is possible by directly attaching a sensor or probe to the fermentation liquid. Therefore, not only can the analysis time, cost, and labor be significantly reduced, but it is a new real-time quantitative analysis method that is attracting attention as a method that is being applied to various processes as a method that enables rapid quantitative analysis in seconds or tens of seconds without the use of harmful chemicals used for pretreatment, etc.

[0057] By means of this NIR, unlike the conventional Kjeldahl analysis method, which is cumbersome and takes a considerable amount of time to obtain a portion of the fermentation solution by sampling during cultivation, the concentration of ammonium nitrogen (AN) in the medium can be analyzed online in real time.

[0058] Therefore, in a specific embodiment, the concentration of ammonium nitrogen in the medium can be monitored in real time, and the real-time monitoring of the concentration of ammonium nitrogen can be performed using a near infrared (NIR) spectrometer.

[0059] As demonstrated in the examples described below, the inventors of the present disclosure have developed a model capable of monitoring the concentration of ammonium nitrogen (AN) in real time with high accuracy using NIR spectroscopy.

[0060]

[0061] In one embodiment, the manufacturing method of the present disclosure described above can additionally monitor the pH of the medium, and more specifically, can monitor the pH in real time.

[0062] In one embodiment, the pH of the medium can be measured and recorded in real time with a pH meter.

[0063]

[0064] In one embodiment, in the manufacturing method of the present disclosure described above, when the concentration of ammonium nitrogen in the medium is less than 1.5 g / kg, a nitrogen source may be added to the medium.

[0065] In the present disclosure, when the concentration of ammonium nitrogen in the medium is less than 1.5 g / kg, it may mean when the concentration of ammonium nitrogen in the medium is less than 1.5 g / kg, less than 1.45 g / kg, less than 1.4 g / kg, less than 1.35 g / kg, less than 1.3 g / kg, less than 1.25 g / kg, less than 1.2 g / kg, less than 1.15 g / kg, less than 1.1 g / kg, less than 1.05 g / kg or less than 1.0 g / kg, but is not limited thereto, and a small amount less than 1.5 g / kg is included in the scope of the present disclosure.

[0066] In one embodiment, the nitrogen source may be at least one selected from the group consisting of ammonia gas and ammonia water.

[0067] In one embodiment, in the manufacturing method of the present disclosure described above, the concentration of ammonium nitrogen in the medium may be controlled to be maintained at 1.0 g / kg to 3.0 g / kg, and more specifically, the concentration of ammonium nitrogen in the medium may be 1.0 g / kg to 3.0 g / kg, 1.0 g / kg to 2.5 g / kg, 1.0 g / kg to 2.0 g / kg, 1.0 g / kg to 1.75 g / kg, 1.0 g / kg to 1.5 g / kg, 1.0 g / kg to 1.25 g / kg, 1.25 g / kg to 3.0 g / kg, 1.25 g / kg to 2.5 g / kg, 1.25 g / kg to 2.0 g / kg, 1.25 g / kg to 1.75 g / kg, 1.25 g / kg to 1.5 g / kg, 1.5 g / kg to 3.0 g / kg, 1.5 g / kg to 2.5 g / kg, 1.5 g / kg to 2.0 g / kg, or 1.5 g / kg to 1.75 g / kg, but is not limited thereto. More specifically, when the concentration of ammonium nitrogen in the medium is less than 1.5 g / kg as described above, by adding a nitrogen source to the medium, the concentration of ammonium nitrogen in the medium is increased to 1.0 g / kg to 3.0 g / kg, 1.0 g / kg to 2.5 g / kg, 1.0 g / kg to 2.0 g / kg, 1.0 g / kg to 1.75 g / kg, 1.0 g / kg to 1.5 g / kg, 1.0 g / kg to 1.25 g / kg, 1.25 g / kg to 3.0 g / kg, 1.25 g / kg to 2.5 g / kg, 1.25 g / kg to 2.0 g / kg, 1.25 g / kg to 1.75 g / kg, 1.25 g / kg to 1.5 g / kg, 1.5 g / kg to 3.0 g / kg, 1.5 g / kg to 2.5 g / kg, 1.5 g / kg to 2.0 g / kg, or 1.5 g / kg to 1.75 g / kg. The concentration of ammonium nitrogen in the medium can be adjusted to be maintained at 1.0 g / kg to 3.By controlling it to be maintained at 0 g / kg, the metabolic activity of microorganisms capable of producing lysine can be maintained, thereby improving the fermentation rate and increasing the fermentation yield.

[0068] In the present disclosure, the introduction of the nitrogen source into the medium may be automatic introduction of the nitrogen source. Specifically, the nitrogen source may be automatically introduced by an HMI (Human-Machine Interface) automation program, but is not limited thereto.

[0069] In one embodiment, adding the nitrogen source to the medium may increase the pH target value (set-point) of the medium, and adding the nitrogen source to the medium so that the pH of the medium can reach the pH target value.

[0070] Increasing the above pH target value (set-point) can be controlled by, but is not limited to, a human-machine interface (HMI) automation program.

[0071] In one implementation, when the concentration of ammonium nitrogen in the medium is less than 1.5 g / kg, the pH set-point may be slightly increased. More specifically, the pH set-point can be increased by about 0.05, for example, by 0.01 to 0.1, 0.01 to 0.075, 0.01 to 0.06, 0.01 to 0.05, 0.025 to 0.1, 0.025 to 0.075, 0.025 to 0.06, 0.025 to 0.05, 0.03 to 0.1, 0.03 to 0.075, 0.03 to 0.06, 0.03 to 0.05, 0.04 to 0.1, 0.04 to 0.075, 0.04 to 0.06, or 0.04 to 0.05.

[0072] More specifically, when the concentration of ammonium nitrogen in the medium is less than 1.5 g / kg, the pH set-point is adjusted upward to about 0.05 by an HMI (Human-Machine Interface) automation program, and the nitrogen source is added so that the pH of the medium can reach the pH target value, thereby increasing the concentration of ammonium nitrogen in the medium.

[0073] In one embodiment, the pH of the medium is additionally monitored, and when the pH of the medium is greater than or equal to pH 7.5 and less than pH 8.0 and the concentration of ammonium nitrogen in the medium is less than 1.5 g / kg, the pH set-point can be increased, and the nitrogen source can be added so as to reach the pH set-point.

[0074] In the present disclosure, when the pH of the medium is pH 7.5 or more but less than pH 8.0, it may mean, but is not limited to, a case where the pH of the medium is pH 7.5 or more but less than pH 8.0, pH 7.6 or more but less than pH 8.0, pH 7.7 or more but less than pH 8.0, pH 7.8 or more but less than pH 8.0, pH 7.9 or more but less than pH 8.0, pH 7.5 or more but less than pH 7.9, pH 7.6 or more but less than pH 7.9, pH 7.7 or more but less than pH 7.9, or pH 7.8 or more but less than pH 7.9.

[0075] In the present disclosure, when the concentration of ammonium nitrogen is less than 1.5 g / kg as described above, by gradually increasing the pH of the medium by adding a nitrogen source, the pH of the medium increases from pH 7.5 or more to pH 8.0 or more to pH 8.0 or less.

[0076] In the present disclosure, when the pH of the medium is pH 8.0 or more and less than pH 8.5, it may mean, but is not limited to, a case where the pH of the medium is pH 8.0 or more and less than pH 8.5, pH 8.0 or more and less than pH 8.4, pH 8.0 or more and less than pH 8.3, pH 8.0 or more and less than pH 8.2, or pH 8.0 or more and less than pH 8.1.

[0077] In one embodiment, the manufacturing method of the present disclosure may further monitor the pH of the medium, and when the pH of the medium is greater than or equal to pH 8.0 and less than pH 8.5, and the concentration of ammonium nitrogen in the medium is less than 1.5 g / kg, carbon dioxide is added to the medium.

[0078] In the present disclosure, the introduction of carbon dioxide into the medium may be automatic introduction of carbon dioxide. Specifically, the carbon dioxide may be automatically introduced by a human-machine interface (HMI) automation program, but is not limited thereto.

[0079] As described above, when the pH of the medium rises to a range of pH 8.0 or higher but lower than pH 8.5, the growth and metabolism of microorganisms capable of producing lysine may be inhibited due to the elevated pH, which may reduce the fermentation rate and yield, and may result in the generation of byproducts. Therefore, when the pH of the medium is pH 8.0 or higher but lower than pH 8.5 and the concentration of ammonium nitrogen in the medium is lower than 1.5 g / kg, carbon dioxide may be added to the medium to lower the pH of the medium.

[0080] In one embodiment, the carbon dioxide may be added until the pH of the medium is reduced by, but not limited to, 0.1 to 0.4, more specifically, 0.1 to 0.4, 0.1 to 0.3, 0.1 to 0.2, 0.2 to 0.4, or 0.2 to 0.3.

[0081] In one embodiment, the carbon dioxide may be introduced at 0.5 vvm to 3.0 vvm. More specifically, the ventilation amount at the time of carbon dioxide injection can be appropriately controlled by those skilled in the art, and can be injected, for example, at 0.5 to 3.0 vvm, more specifically, 0.5 to 3.0 vvm, 0.5 to 2.0 vvm, 0.5 to 1.7 vvm, 0.5 to 1.5 vvm, 0.7 to 3.0 vvm, 0.7 to 3.0 vvm, 0.7 to 2.0 vvm, 0.7 to 1.7 vvm, 0.7 to 1.5 vvm, 1.0 to 3.0 vvm, 1.0 to 2.0 vvm, 1.0 to 1.7 vvm, or 1.0 to 1.5 vvm (gas injection amount L / medium volume L / minute), but is not limited thereto.

[0082] In one embodiment, a nitrogen source may be added to the medium so that the pH of the medium, which has been reduced by the addition of carbon dioxide, can be increased. More specifically, the nitrogen source may be added to the medium so that the pH of the medium, which has been reduced by the addition of carbon dioxide, can again reach a previously set pH target value. The previously set pH target value may be pH 8.0 or higher and less than pH 8.5. The nitrogen source may be added simultaneously with or sequentially with the addition of carbon dioxide to the medium. The method of adding the nitrogen source, etc., is as described above.

[0083] In one embodiment, when the pH of the medium is 8.0 or more but less than 8.5 and the concentration of ammonium nitrogen in the medium is less than 1.5 g / kg, the process of adding carbon dioxide to the medium and adding a nitrogen source simultaneously or sequentially may be repeated. More specifically, when the pH of the medium is 8.0 or more but less than 8.5 and the concentration of ammonium nitrogen in the medium is less than 1.5 g / kg, when carbon dioxide is added to the medium, the pH of the medium decreases. When a nitrogen source is added to the medium so that the decreased pH of the medium can reach the previously set pH target value again, the pH increases again to 8.0 or more but less than 8.5. In this way, when the pH becomes 8.0 or more but less than 8.5 and the concentration of ammonium nitrogen in the medium becomes less than 1.5 g / kg again as fermentation progresses, the above process may be repeated. By repeating the above process, the concentration of ammonium nitrogen in the medium can be maintained at 1.0 g / kg or more, specifically in the range of 1.0 g / kg to 3.0 g / kg, and the pH of the medium can be controlled to less than 8.5, specifically in the range of pH 7.5 or more to pH 8.5 or less.

[0084]

[0085] In one embodiment, in the lysine production method of the present disclosure described above, the pH of the medium may be increased from pH 6.5 or more to less than pH 7.0 to pH 7.5 or more to less than pH 8.5, and then carbon dioxide may be added to adjust the pH of the medium to be maintained at pH 7.5 or more to less than pH 8.5.

[0086] In a specific embodiment, the pH of the medium may be maintained at a value of pH 6.5 or more and pH 7.0 or less before increasing the pH from pH 6.5 or more and pH 7.0 or less to pH 7.5 or more and pH 8.5 or less.

[0087] Specifically, at the beginning of fermentation, the pH of the medium can be maintained at a value of pH 6.5 or higher and less than pH 7.0. Without separate control, the pH at the beginning of fermentation gradually decreases due to the accumulation of organic acids and the emission of carbon dioxide gas as sugar is consumed. Therefore, in order to maintain the optimal pH for fermentation, the pH can be maintained at pH 6.5 or higher and less than pH 7.0 by adding ammonia gas and / or ammonia water, and more specifically, the pH can be maintained at pH 6.5, pH 6.6, pH 6.7, pH 6.8, or pH 6.9, but is not limited thereto, and all minor amounts included in the range of pH 6.5 or higher and less than pH 7.0 are included in the scope of the present disclosure. Maintaining the pH of the above medium at a value of pH 6.5 or higher and less than pH 7.0 may be achieved by setting the pH target value (set-point) to a value within the range of pH 6.5 or higher and less than pH 7.0, and injecting a nitrogen source, more specifically, ammonia gas and / or ammonia water, through an HMI (Human-Machine Interface) automation program so as to maintain the pH target value, but is not limited thereto. The above 'early stage of fermentation' may mean a period from the start of fermentation until the carbon source of the early stage of fermentation medium is exhausted, but is not limited thereto.

[0088] In a specific embodiment, when the carbon source of the initial fermentation medium is depleted, a feeding medium may be added, and the pH of the medium may be increased from pH 6.5 or more and less than pH 7.0 to pH 7.5 or more and less than pH 8.0. The increase in pH may be intended to increase the supply of a nitrogen source after the feeding medium is added. Increasing the pH as described above may mean increasing the pH set-point at a rate of 0.2 to 0.8 per hour, and more specifically, may mean increasing at a rate of 0.2 to 0.8, 0.2 to 0.8, 0.2 to 0.6, 0.2 to 0.5, 0.3 to 0.8, 0.3 to 0.8, 0.3 to 0.6, 0.3 to 0.5, 0.4 to 0.8, 0.4 to 0.8, 0.4 to 0.6, or 0.4 to 0.5 per hour. More specifically, the pH of the medium may be increased at a constant rate. Specifically, increasing the pH may mean injecting a nitrogen source, more specifically, ammonia gas and / or ammonia water, by an HMI (Human-Machine Interface) automation program so as to maintain the pH target value, but is not limited thereto.

[0089] Through the above process, when the pH of the medium reaches pH 7.5 or more and less than pH 8.0, and when the ammonium nitrogen concentration of the medium as described above is less than 1.5 g / kg, a process of adding a nitrogen source to the medium can be performed. Through the above process, the pH of the medium can be increased to pH 8.0 or more and less than pH 8.5. When the pH of the medium reaches pH 8.0 or more and less than pH 8.5, and when the ammonium nitrogen concentration of the medium as described above is less than 1.5 g / kg, carbon dioxide is added, and a process of adding a nitrogen source simultaneously or sequentially is performed, so that the pH of the medium is controlled to be maintained at pH 7.5 or more and less than pH 8.5.

[0090] As such, the method for producing lysine of the present disclosure described above may include the following steps:

[0091] (a) a step of increasing the pH of the medium from pH 6.5 or more and less than pH 7.0 to pH 7.5 or more and less than pH 8.0;

[0092] (b) When the pH of the medium is pH 7.5 or higher but lower than pH 8.0 and the concentration of ammonium nitrogen in the medium is lower than 1.5 g / kg, a step of increasing the pH set-point and adding a nitrogen source to the medium so that the pH of the medium can reach the pH set-point;

[0093] (c) When the pH reaches pH 8.0 or higher but lower than pH 8.5, a step of injecting carbon dioxide gas to lower the pH and injecting a nitrogen source.

[0094] In a specific embodiment, prior to step (a), a step of maintaining the pH of the medium at a value of pH 6.5 or higher and less than pH 7.0 may be included. In this case, the method for maintaining the pH value of the medium, etc., is as described above.

[0095] The method for increasing the pH in steps (a) and (b) above, etc. are as described above.

[0096] The method for reducing pH in the above step (c), the method for introducing a nitrogen source, etc. are as described above.

[0097] In a specific embodiment, the process of step (c) may be performed repeatedly. Specifically, when the pH of the medium is pH 8.0 or more and less than pH 8.5, and the concentration of ammonium nitrogen in the medium is less than 1.5 g / kg, carbon dioxide is automatically added to the medium, thereby decreasing the pH of the medium. When a nitrogen source is automatically added to the medium so that the pH of the decreased medium can reach the previously set pH target value again, the pH increases again to pH 8.0 or more and less than pH 8.5. In this way, when the pH becomes pH 8.0 or more and less than pH 8.5, and the concentration of ammonium nitrogen in the medium becomes less than 1.5 g / kg again as fermentation progresses, the above process may be performed repeatedly. By repeating the above process, the concentration of ammonium nitrogen in the medium is maintained at 1.0 g / kg or more, specifically in the range of 1.0 g / kg to 3.0 g / kg, and the pH of the medium can be controlled to less than 8.5, specifically in the range of pH 7.5 or more to pH 8.5 or less.

[0098] In this way, according to the present disclosure, the ammonium nitrogen concentration in the medium and the pH of the medium are managed to the optimal level range for lysine biosynthesis, thereby improving the purity of the process solution through reduction of fermentation byproducts and improvement of yield, thereby significantly reducing the manufacturing cost.

[0099]

[0100] In this disclosure, “cultivation” means a process of growing a specific microorganism or cell under conditions in which a suitable environment (nutrients, temperature, pH, oxygen concentration, etc.) is provided or inducing the microorganism or cell to produce a desired metabolite, and may be used interchangeably with “fermentation” for the purposes of this disclosure.

[0101] In the present disclosure, the culture is continued until the desired lysine production is maximized. For this purpose, the culture may be performed for, but is not limited to, 10 to 160 hours, more specifically, 10 to 160 hours, 10 to 100 hours, 10 to 50 hours, 10 to 35 hours, 20 to 160 hours, 20 to 100 hours, 20 to 50 hours, 20 to 35 hours, 30 to 160 hours, 30 to 100 hours, 30 to 50 hours, or 30 to 35 hours. The lysine may be released into the culture medium or may be contained within the cells.

[0102] In the case of the lysine production method of the present disclosure, when the concentration of ammonium nitrogen in the medium is controlled to be maintained at 1.0 g / kg to 3.0 g / kg by supplying a nitrogen source according to the method of the present disclosure, and when the culture is performed without the carbon dioxide injection process according to the method of the present disclosure, by-products can be reduced to about 30% to about 80%, and more specifically, about 30% to about 80%, about 30% to about 75%, about 30% to about 72%, about 30% to about 71.5%, about 45% to about 80%, about 45% to about 75%, about 45% to about 72%, about 45% to about 71.5%, about 60% to about 80%, about 60% to about 75%, about 60% to about 72%, about 60% to about 71.5%, about It may be reduced to, but is not limited to, a level of about 65% to about 80%, about 65% to about 75%, about 65% to about 72%, about 65% to about 71.5%, about 70% to about 80%, about 70% to about 75%, about 70% to about 72%, or about 70% to about 71.5%.

[0103] In the case of performing the culture by adding the nitrogen source while adding carbon dioxide according to the method of the present disclosure and adding the nitrogen source according to the method of the present disclosure, but the concentration of ammonium nitrogen in the medium is less than 0.3 g / kg rather than less than 1.5 g / kg, by-products can be reduced to about 1% to about 30%, and more specifically, about 1% to about 30%, about 1% to about 20%, about 1% to about 17.5%, about 5% to about 30%, about 5% to about 20%, about 5% to about 17.5%, about 10% to about 30%, about 10% to about 20%, about 10% to about 17.5%, about 12.5% ​​to about 30%, about 12.5% ​​to about 20%, about 12.5% ​​to about 17.5%, It may be reduced to, but is not limited to, about 15% to about 30%, about 15% to about 20%, or about 15% to about 17.5%.

[0104] According to the lysine production method of the present disclosure, compared to the case where the culture is performed without carbon dioxide input and ammonium nitrogen concentration control in the medium (maintained at less than 1.5 g / kg) according to the method of the present disclosure, the lysine production (concentration of produced lysine) is about 5% to about 60%, more specifically, about 5% to about 60%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 39.5%, about 15% to about 60%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 39.5%, about 30% to about 60%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about It may increase by, but is not limited to, 30% to about 39.5%, about 39% to about 60%, about 39% to about 50%, about 39% to about 45%, about 39% to about 40%, or about 39% to about 39.5%.

[0105] In the case of the lysine production method of the present disclosure, when the concentration of ammonium nitrogen in the medium is controlled to be maintained at 1.0 g / kg to 3.0 g / kg by supplying a nitrogen source according to the method of the present disclosure, but the culture is performed without the carbon dioxide injection process according to the method of the present disclosure, the lysine production (concentration of produced lysine) may be increased by about 1% to about 20%, more specifically, about 1% to about 20%, about 1% to about 10%, about 1% to about 4%, about 2% to about 20%, about 2% to about 10%, about 2% to about 4%, about 3.5% to about 20%, about 3.5% to about 10%, or about 3.5% to about 4%, but is not limited thereto.

[0106] In the case of performing culture by adding carbon dioxide according to the method of the present disclosure and adding a nitrogen source according to the method of the present disclosure, but when the concentration of ammonium nitrogen in the medium is less than 0.3 g / kg rather than less than 1.5 g / kg, the lysine production (concentration of produced lysine) may be increased by about 10% to about 40%, about 10% to about 30%, about 10% to about 25%, about 15% to about 40%, about 15% to about 30%, about 15% to about 25%, about 20% to about 40%, about 20% to about 30%, or about 20 to 22.5%, but is not limited thereto.

[0107] According to the lysine production method of the present disclosure, the purity of the lysine produced may be increased by about 1% to about 30%, more specifically, about 1% to about 30%, about 1% to about 20%, about 1% to about 14%, about 5% to about 30%, about 5% to about 20%, about 5% to about 14%, about 10% to about 30%, about 10% to about 20%, about 10% to about 14%, about 12.5% ​​to about 30%, about 12.5% ​​to about 20%, or about 12.5% ​​to about 14%, but is not limited thereto.

[0108] In the case of the lysine production method of the present disclosure, when the concentration of ammonium nitrogen in the medium is controlled to be maintained at 1.0 g / kg to 3.0 g / kg by supplying a nitrogen source according to the method of the present disclosure, and when the culture is performed without the carbon dioxide injection process according to the method of the present disclosure, the purity of the lysine produced is about 0.1% to about 20%, more specifically, about 0.1% to about 20%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.1% to about 2.5%, about 0.1% to about 1.5%, about 0.5% to about 20%, about 0.5% to about 10%, about 0.5% to about 5%, about 0.5% to about 2.5%, about 0.5% to about 1.5%, about 1.0% to about 20%, about It may increase by, but is not limited to, 1.0% to about 10%, about 1.0% to about 5%, about 1.0% to about 2.5%, or about 1.0% to about 1.5%.

[0109] In the case of performing the lysine production method of the present disclosure by adding carbon dioxide according to the method of the present disclosure and adding a nitrogen source according to the method of the present disclosure, but when the concentration of ammonium nitrogen in the medium is less than 0.3 g / kg rather than less than 1.5 g / kg, the purity of the lysine produced may be increased by about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 12.5% ​​to about 30%, about 12.5% ​​to about 25%, or about 12.5% ​​to about 20%, but is not limited thereto. The term “about” above means A range that includes all values ​​such as ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values ​​in a range equal to or similar to the value following the term "about," but is not limited thereto.

[0110]

[0111] The lysine production method of the present disclosure may further include a step of recovering lysine from a culture medium (a culture medium in which culture is performed) or a Corynebacterium genus microorganism. The recovering step may be additionally included after the culturing step.

[0112] The above recovery may be performed by collecting the desired amino acid using a suitable method known in the art according to the culture method of the microorganism of the present disclosure, such as a batch, continuous or fed-batch culture method. For example, various chromatographies such as centrifugation, filtration, treatment with a crystallized protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC or a combination thereof may be used, and the desired amino acid may be recovered from the medium or microorganism using a suitable method known in the art.

[0113] Additionally, the lysine production method of the present disclosure may additionally include a purification step. The purification may be performed using any suitable method known in the art. In one example, when the L-lysine production method of the present disclosure includes both a recovery step and a purification step, the recovery step and the purification step may be performed sequentially or discontinuously, regardless of order, or may be performed simultaneously or integrated into a single step, but is not limited thereto.

[0114] In one embodiment, the lysine produced by the above method can be obtained in the form of a fermentation liquid containing lysine. This fermentation liquid containing lysine can be further subjected to a decarboxylation process and a concentration process, and then granulated and dried to ultimately obtain high-content lysine granules.

[0115]

[0116] According to the method for producing lysine according to the present disclosure, when the ammonium nitrogen concentration is less than 1.5 g / kg, by supplying a nitrogen source, the ammonium nitrogen in the medium is managed to be within the optimal level range for lysine biosynthesis, thereby improving the purity of the process solution through reduction of fermentation by-products and improvement of yield, thereby significantly reducing the manufacturing cost.

[0117]

[0118] Figure 1 is a process diagram of a real-time monitoring model based on near-infrared spectroscopy (NIR spectroscopy) used in the present disclosure.

[0119] Figure 2 is a graph showing the results of cross validation of measured and predicted values ​​of ammonium nitrogen (AN). The x-axis represents the true AN value obtained by analyzing the spectral sample collected with NIR equipment using Kjeldahl analysis for the prediction model, and the y-axis represents the predicted AN value corresponding to the true AN value during preprocessing for creating a calibration curve.

[0120] Figure 3 is a drawing comparing a conventionally used lysine powder process and a lysine granule process used in the present disclosure.

[0121] Figure 4 is a schematic diagram of a culture process according to a specific embodiment of the present disclosure.

[0122] Figure 5 is a graph showing the concentration of AN in a medium over time according to the lysine manufacturing method according to Example 2, Comparative Example 1, Comparative Example 2, and Comparative Example 3.

[0123]

[0124] The present disclosure will be described in more detail below with examples. However, these are merely illustrative and are not intended to limit the scope of the present disclosure. It will be apparent to those skilled in the art that modifications to the embodiments described below may be made without departing from the essential spirit of the invention.

[0125]

[0126] [Example]

[0127]

[0128] Example 1) Method for creating an AN real-time monitoring model using NIR spectroscopy

[0129] The seed culture of Corynebacterium glutamicum CJ3P strain (US 9556463 ​​B2), a coryneform microorganism with lysine production ability, was conducted through solid plate medium and flask culture, and the seed culture stage was conducted in a 5 L fermenter, and the main culture stage was conducted in a 30 L fermenter.

[0130] Corynebacterium glutamicum CJ3P strain, stored in a -80°C deep freezer in the form of glycerol stock (GS), was inoculated into a solid plate medium prepared based on the following medium composition at a level of 50 μL by streaking, and cultured in a 30°C incubator for approximately 24 hours.

[0131] To perform flask shaking culture, 100 mL of liquid medium prepared based on the flask shaking culture medium composition below was prepared and placed in a 500 mL shake flask containing a baffle, and sterilized using a small autoclave at 121°C for 30 minutes. To perform shake flask inoculation, work was performed on a clean bench. Colonies were appropriately picked from the cultured solid plate using a disposable platinum tooth (10 μL loop) and aseptically inoculated into the shake culture medium. The inoculated 500 mL shake flask was cultured at 200 rpm and 30°C for approximately 10 hours.

[0132] The spawn obtained from the flask shaking culture was inoculated at a ratio of 3.0% (v / v) into a 5 L fermenter containing a medium containing ammonium sulfate 0.46 and phosphate 0.03, based on the molar ratio (target substance mol / glucose mol) of the glucose used in the seed culture medium composition. The culture was performed for approximately 20 hours under the conditions of pH 6.5–8.5, agitation speed 450 rpm, and aeration volume 1 vvm in batch fermentation mode. The pH was measured in real time with a pH meter (InPro3253i / SG / 120, Mettler Toledo). When the pH dropped by 0.05 to 0.1 in the range of 6.5 to 8.5, the pH was maintained at an appropriate range of 6.5 to 8.5 by injecting ammonia gas and / or ammonia water as a nitrogen source using an HMI (Human-Machine Interface) automation program (AVEVA InTouch HMI (AVEVA)). Samples were obtained at intervals of approximately 2 to 3 hours, and the cultivation was terminated when the residual sugar was depleted.

[0133] The seedlings obtained in the above seed culture step were used to inoculate a 30 L fermenter containing a medium containing 0.46 ammonium sulfate and 0.03 phosphate in a molar ratio based on glucose in the following main culture medium composition at a ratio of 20.0% (v / v). In this cultivation step, the culture was performed under the conditions of pH 6.5 to 8.5, stirring speed 450 rpm, and aeration amount 1 vvm in fed-batch fermentation mode. In the same manner as above, the pH was measured in real time with a pH meter, and the pH was maintained in an appropriate range of pH 6.5 to pH 8.5 by adding ammonia gas and / or ammonia water.

[0134] A small amount of broth was obtained through sampling from the fermenter, and the residual sugar level was checked using a Biochemistry Analyzer YSI2900 analysis device. When the initial carbon source of the medium was depleted, a feeding medium containing 40.0 wt% glucose was fed from a feeding tank at a variable flow rate so that the residual sugar could be maintained at the level of 0.5 wt% to 1.0 wt%. Samples were obtained at intervals of approximately 2 to 3 hours, and the ammonium nitrogen concentration (AN: ammonium nitrogen, g / kg) was measured using a Kjeldahl analyzer (Foss Kjeltec 8400). When the value was less than 1.5 g / kg, the nitrogen source was maintained constant by adding ammonia gas and / or ammonia water.

[0135] A calibration model was developed using OPUS software (Bruker) using the AN values ​​of the fermentation broth obtained by sampling at 2-3 hour intervals and the NIR spectrum data of the same time. The NIR spectrum was obtained using Bruker Matrix-F NIR with wavelength conditions of 3995-11987 cm -1 , and was analyzed at a scanner velocity of 40 kHz. Among the preprocessing techniques used to create the calibration curve model, vector and first derivative were selected and used. The probe conditions used for real-time monitoring were a resolution of 16 cm. -1 , scan time 32 scans, preamplifier Gain A mode was used, and the cross validation results comparing the predicted and measured values ​​are shown in Table 1 and Fig. 2, and there is a high positive correlation (R 2 >0.99), RMSECV (Root Mean Square Error of Cross-Validation) 0.06, and RPD (Residual Predictive Deviation) 10.1 were confirmed.

[0136] The calibration curve equation was as follows.

[0137] y = 0.9854x + 0.0264

[0138]

[0139] 1) Composition of solid plate medium

[0140] Glucose 10.0 g / L, bacto tryptone 5.0 g / L, bacto yeast extract 5.0 g / L, NaCl 2.5 g / L, urea 2.0 g / L

[0141]

[0142] 2) Composition of flask shaking culture medium

[0143] Glucose 10.0 g / L, bacto tryptone 5.0 g / L, bacto yeast extract 5.0 g / L, ammonium sulfate 10.0 g / L, urea 2.0 g / L, KH2PO4 5.0 g / L, K2HPO4 10.0 g / L, MgSO4 7H2O 0.5 g / L

[0144]

[0145] 3) Composition of culture medium for fermentation tank (5 L)

[0146] 1 mL / L antifoam, 10.0 g / L corn steep liquor, 1.0 mg / L biotin, 10.0 mg / L thiamine, 10.0 mg / L pantothenic acid, 10.0 mg / L niacinamide

[0147]

[0148] 4) Fermentation tank (30 L) Main culture medium composition

[0149] 1 mL / L antifoam, 10.0 g / L corn steep liquor, 1.0 mg / L biotin, 10.0 mg / L thiamine, 10.0 mg / L pantothenic acid, 10.0 mg / L niacinamide

[0150]

[0151] Item Fermentation tank 1 (D1) Fermentation tank 2 (D2) Fermentation tank 3 (D3) Fermentation tank 4 (D4) Average (n=4) R2 99.199.299.098.699.0RMSECV0.060.050.070.070.06RPD10.811.110.08.410.1

[0152] As described above, a model capable of monitoring AN values ​​in real time with high accuracy was established.

[0153]

[0154] Example 2) Step for manufacturing lysine using carbon dioxide gas automation injection technology

[0155] Using the seedlings obtained in the same manner as the seed culture step of Example 1, the seedlings were inoculated at a ratio of 20.0% (v / v) into a 30 L fermenter containing a medium containing ammonium sulfate 0.46 and phosphate 0.03 in a molar ratio based on glucose in the main culture medium composition. In the main culture step, the culture was performed in a fed-batch fermentation mode under the conditions of pH 6.5 to pH 8.5, a stirring speed of 450 rpm, and an aeration amount of 1 vvm. The pH was measured in real time with a pH meter (InPro3253i / SG / 120, Mettler Toledo). The main culture was performed as follows, and a schematic diagram of the culture process is shown in Fig. 4.

[0156] 1) In batch fermentation mode, in order to maintain a pH close to neutrality within the optimal pH of culture from the start of culture until the initial depletion of the carbon source in the medium, the pH target value (set-point) was set to pH 6.5 or higher and less than pH 7.0, and ammonia gas and / or ammonia water as a nitrogen source were supplied by an HMI (Human-Machine Interface) automation program (AVEVA InTouch HMI (AVEVA)) so that the pH of the medium could be maintained at the target value. At this time, since the consumption amount is greater than the supply amount of the nitrogen source, the AN value gradually decreases (section ① of Fig. 4).

[0157] 2) After the initial carbon source was exhausted, a feeding medium containing 40.0 wt% of glucose was added in the same manner as in Example 1 to maintain the residual sugar at 0.5 wt% to 1.0 wt%, and the fermentation was conducted in fed-batch mode. After the feeding medium was added, in order to increase the amount of ammonia sufficient for lysine biosynthesis, the pH set-point was raised at a rate of 0.2 to 0.8 / hr through the HMI automation program until it reached pH 7.5 or higher and pH 8.0 or lower, and ammonia gas and / or ammonia water, which are nitrogen sources, were continuously added to maintain the pH of the medium at the target value, thereby maintaining the AN value at 1.0 g / kg to 3.0 g / kg, which is the optimal range for lysine biosynthesis. At this time, since the ammonia supply and consumption amounts are similar, AN gradually decreases within the optimal AN range (section ② of FIG. 4).

[0158] 3) From the point where the pH of the medium first reached pH 7.5 or higher but lower than pH 8.0, the AN real-time monitoring technique using NIR spectroscopy established in Example 1 was utilized to perform the following process for more detailed culture condition management. When the pH of the medium was pH 7.5 or higher but lower than pH 8.0 and the AN value fell below 1.5 g / kg, the target value (set-point) of the fermentation pH was slightly raised (within 0.05) through the HMI program, and ammonia gas and / or ammonia water as a nitrogen source were automatically injected so as to reach the target value, thereby maintaining the AN value in the range of 1.0 g / kg to 3.0 g / kg (section ③ of Fig. 4). The above process was repeated to maintain the AN value in the range of 1.0 g / kg to 3.0 g / kg, but since the growth rate and metabolic efficiency of Corynebacterium microorganisms may decrease when the fermentation pH exceeds pH 8.0 or more but less than pH 8.5, the process was repeated only until the fermentation pH reached pH 8.0 or more but less than pH 8.5. Ammonia gas and / or ammonia water were introduced by controlling the speed with on / off time (1 sec / 10 sec) through the HMI automation program until the present value of pH reached the pH target value.

[0159] 4) From the time when the pH of the medium first reached pH 8.0 or higher but lower than pH 8.5, the AN real-time monitoring technique utilizing NIR spectroscopy established in Example 1 was used to automatically inject carbon dioxide gas to reduce the pH when the pH of the medium was pH 8.0 or higher but lower than pH 8.5 and the AN value in the medium fell below 1.5 g / kg due to a lack of nitrogen sources due to lysine biosynthesis. Specifically, carbon dioxide gas was injected at a rate of about 1.5 vvm (gas injection amount L / medium volume L / minute) through the HMI automation program, and the speed was controlled by on / off time (1 sec / 10 sec), thereby reducing the pH of the medium by 0.1 to 0.4 (about 0.2). When the pH decreased in this way, ammonia gas and / or ammonia water as a nitrogen source were automatically injected so that the original target pH value that was finally set in the process 3) above could be reached, and the AN value was maintained in the range of 1.0 g / kg to 3.0 g / kg. After performing the above process, when the pH of the medium reached pH 8.0 or higher to pH 8.5 or lower, and the AN value fell below 1.5 g / kg due to a lack of nitrogen source due to lysine biosynthesis, the process of decreasing the medium pH by introducing carbon dioxide gas as described above and injecting ammonia gas and / or ammonia water to reach the original target pH value was performed. In this way, the above process was repeated until the end of the culture to maintain the AN in the range of 1.0 g / kg to 3.0 g / kg (section ④ of FIG. 4).

[0160]

[0161] At the end of the cultivation, there was no residual sugar and the fermentation time was 30.3 hours. The maximum pH that could be increased through the system by automatically injecting carbon dioxide gas during fermentation was 8.5, and compared to the results of Comparative Example 2 described below, a 4.0% improvement in lysine concentration and a 28.6% decrease in byproduct concentration were confirmed, confirming that the standardized purity obtained from the fermentation process was improved by 1.3%.

[0162]

[0163] Comparative Example 1) Step for manufacturing lysine at AN less than 1.5 g / kg

[0164] Using the seedlings obtained in the same manner as in the seed culture step of Example 1, the seedlings were inoculated at a ratio of 20.0% (v / v) into a 30 L fermenter containing a medium containing 0.46 wt% ammonium sulfate and 0.03 phosphate based on the glucose molar ratio in the main culture medium composition. In the main culture step, the culture was performed under the conditions of a stirring speed of 450 rpm and an aeration amount of 1 vvm in a fed-batch fermentation mode. When the initial carbon source of the medium was exhausted, a feeding medium containing 40.0 wt% glucose was added in the same manner as in Example 1 to maintain the residual sugar at the level of 0.5 to 1.0 wt%.

[0165] Although there was no residual sugar at the end of the culture, the AN value was maintained below 1.5 g / kg due to the lack of AN value management, as shown in Fig. 5. The fermentation time was 39.9 hours due to delay, and compared to the results of Comparative Example 2 described below, the lysine concentration decreased by 25.4%, and the standardized purity decreased by 10.4%, confirming a significant decrease.

[0166]

[0167] Comparative Example 2) Step for manufacturing lysine using only ammonia without adding carbon dioxide

[0168] Using the seedlings obtained in the same manner as in the seed culture step of Example 1, the seedlings were inoculated at a ratio of 20.0% (v / v) into a 30 L fermenter containing a medium containing ammonium sulfate 0.46 and phosphate 0.03 in a glucose-based molar ratio in the main culture medium composition. In the main culture step, the culture was performed under the conditions of pH 6.5 to 8.5, agitation speed 450 rpm, and aeration amount 1 vvm in fed-batch fermentation mode. When the pH dropped by 0.05 to 0.1 in the range of 6.5 to pH 8.5, ammonia gas and / or ammonia water as a nitrogen source were added using an HMI (Human-Machine Interface) automation program (AVEVA InTouch HMI (AVEVA)) to maintain the pH in an appropriate range of pH 6.5 to pH 8.5.

[0169] In this comparative example, steps 1) to 3) of Example 2 were performed as is, but step 4) of Example 2, which reduces the pH by automatically adding carbon dioxide gas when the fermentation pH reaches the upper maximum (pH 8.0 or more to pH 8.5 or less) and AN becomes less than 1.5 g / kg, was not performed, so that the pH of the medium can increase to pH 8.5 or more.

[0170] At the end of the culture, there was no residual sugar, the fermentation time was 28.7 hours, and the lysine concentration was 192.4 g / L.

[0171]

[0172] Comparative Example 3) Step for manufacturing lysine at AN less than 0.5 g / kg using carbon dioxide gas automation injection technology

[0173] Using the seedlings obtained in the same manner as the seed culture step of Example 1, the seedlings were inoculated at a ratio of 20.0% (v / v) into a 30 L fermenter containing a medium containing ammonium sulfate 0.46 and phosphate 0.03 in a molar ratio based on glucose in the main culture medium composition. In the main culture step, the culture was performed under the conditions of pH 6.5-8.5, agitation speed 450 rpm, and aeration volume 1 vvm in fed-batch fermentation mode.

[0174] In this comparative example, the processes 1) to 3) of the above Example 2 were performed as is, but in Example 2, when the fermentation pH reached the upper maximum (pH 8.0 or more to pH 8.5 or less) and AN was less than 1.5 g / kg, the pH was reduced by automatic injection of carbon dioxide gas, whereas in this comparative example, when the fermentation pH reached the upper maximum (pH 8.0 or more to pH 8.5 or less) and AN was less than 0.3 g / kg, the pH of the medium was reduced by 0.1 to 0.4 (about 0.2) by automatic injection of carbon dioxide gas, and ammonia gas and / or ammonia water as a nitrogen source were automatically injected, so that the AN value was maintained in the range of up to 0.5 g / kg. After performing the above process, when the pH of the medium reached pH 8.0 or higher but lower than pH 8.5 and the AN value fell below 0.3 g / kg due to a lack of nitrogen source due to lysine biosynthesis, the process of decreasing the medium pH by adding carbon dioxide gas and adding ammonia gas and / or ammonia water to reach the original target pH value was performed. In this way, the above process was repeated until the end of the culture to maintain the AN in the range of less than 0.5 g / kg.

[0175] As the supply of AN, an essential nitrogen source for lysine biosynthesis, was limited to less than 0.5 g / kg, the lysine concentration decreased by 18.0% to 164.1 g / L compared to the results of Example 2 described above, and the standardized purity was confirmed to have decreased significantly to 13.3% as the by-product increased by 660.0%. In addition, as the fermentation time elapsed, the sugar consumption rate rapidly decreased, and the fermentation time was 31.7 hours, and the residual sugar at the end of the culture was approximately 4.8 g / L.

[0176] The culture results of Example 2, Comparative Example 1, and Comparative Example 2 are compared in Table 2 below, and the culture results of Example 2 and Comparative Example 3 are compared in Table 3 below. The AN concentration in the medium is shown in Figure 5.

[0177] Item Unit Example 2 Comparative Example 1 Comparative Example 2 Fermentation Time Hr 30.3 39.9 28.7 Residual Sugar g / L 0.00.00.0 By-Product*% 71.4-100.0 Lysine g / L 200.0 143.5 192.4 Purity*% 101.3 89.6 100.0

[0178] * Comparative Example 2 Normalization result value based on 100% of the results

[0179] Item Unit Example 2 Comparative Example 3 Fermentation Time Hr 30.3 31.7 Residual Sugar g / L 0.0 4.8 By-Product *% 100.0 66 0.0 Lysine g / L 200.0 16 4.1 Purity *% 100.0 8 6.7

[0180] * Example 2 Normalization result value based on 100% of the results

[0181] If we summarize the experimental results shown in Tables 2 and 3 above,

[0182] Example 2 utilized the real-time AN ​​monitoring technique established in Example 1 during the main cultivation stage, and when the AN was less than 1.5 g / kg during fermentation, the fermentation pH was gradually increased to a maximum of pH 8.0 or more but less than pH 8.5 to supply a nitrogen source, and after reaching pH 8.0 or more but less than pH 8.5, carbon dioxide gas and ammonia gas and / or ammonia water were injected to limit the fermentation pH to pH 7.5 or more but less than pH 8.5. Therefore, compared to the results of Comparative Example 2, it was confirmed that a 28.6% reduction in standardized by-products and a 4.0% improvement in lysine were observed at the same level of fermentation time, confirming a 1.3% improvement based on the final standardized process solution purity.

[0183] In Comparative Example 1, lysine fermentation was conducted without managing AN according to the real-time AN ​​monitoring technique established in Example 1 during the main cultivation stage, and since the AN level was not managed, it was maintained below 1.5 g / kg AN for most of the fermentation period conducted as a fed-batch. It was confirmed that the fermentation time was delayed by approximately 39.0% and lysine production decreased by 25.4% compared to Comparative Example 2 due to the absence of management of AN, the most important factor in lysine production.

[0184] Comparative Example 3 utilized the real-time AN ​​monitoring technique established in Example 1 during the main cultivation stage, and when the AN was less than 0.3 g / kg during fermentation, the fermentation pH was gradually increased to a maximum of pH 8.0 or more but less than pH 8.5 to supply a nitrogen source, and after reaching pH 8.0 or more but less than pH 8.5, carbon dioxide gas and ammonia gas and / or ammonia water were injected to limit the fermentation pH to pH 7.5 or more but less than pH 8.5. Therefore, compared to the results of Example 2, as the maximum AN was limited to 0.5 g / kg, the lysine concentration was reduced by 18.0% to 164.1 g / L compared to the results of Example 2 described above, and as by-products increased by 660.0%, the standardized purity decreased by 13.3%, which was confirmed to be a significant decrease. In addition, as the fermentation time elapsed, the sugar consumption rate decreased rapidly, and the fermentation time was 31.7 hours, and the residual sugar at the end of the culture was at the level of 4.8 g / L. Therefore, it was confirmed that maintaining AN below 0.5 g / kg during fermentation has a negative effect on the fermentation index, and when AN decreases to a value below 1.5 g / kg, adding a nitrogen source and / or carbon dioxide to the medium to maintain AN in an appropriate range of 1.0 g / kg to 3.0 g / kg is an essential condition for lysine production.

[0185] The ion exchange resin process significantly increases manufacturing costs due to the additional purification process and the input of auxiliary raw materials. In the granule manufacturing process utilizing lysine carbonate, reducing byproducts and improving their concentration within the resulting fermentation broth is essential to improving the purity of the final product. In the lysine granule process without a separate ion exchange resin process, the purity of the product declines due to impurities in the fermentation broth. However, using the carbon dioxide fermentation method introduced by this technology reduces the concentration of byproducts during cultivation and significantly improves the concentration of lysine, thereby improving fermentation purity and enhancing the quality of the granule product.

Claims

1. A method for producing lysine by a fermentation process using a microorganism having the ability to produce lysine, By monitoring the concentration of ammonium nitrogen in the medium, A method for producing lysine, characterized in that a nitrogen source is added to the medium when the concentration of ammonium nitrogen in the medium is less than 1.5 g / kg.

2. In paragraph 1, A method for producing lysine, wherein the concentration of ammonium nitrogen in the medium is controlled to be maintained at 1.0 g / kg to 3.0 g / kg.

3. In paragraph 1, A method for producing lysine, wherein the nitrogen source is at least one selected from the group consisting of ammonia gas and ammonia water.

4. In paragraph 1, Additional monitoring of the pH of the medium, A method for producing lysine, wherein when the pH of the medium is pH 7.5 or higher and less than pH 8.0 and the concentration of ammonium nitrogen in the medium is less than 1.5 g / kg, the pH target value (set-point) is increased and a nitrogen source is added to the medium so that the pH of the medium can reach the pH target value.

5. In paragraph 1, Additional monitoring of the pH of the medium, A method for producing lysine, wherein carbon dioxide is added to the medium when the pH of the medium is pH 8.0 or higher and less than pH 8.5 and the concentration of ammonium nitrogen in the medium is less than 1.5 g / kg.

6. In paragraph 5, A method for producing lysine, wherein the carbon dioxide is added until the pH of the medium is reduced by 0.1 to 0.

4.

7. In paragraph 5, A method for producing lysine, wherein the carbon dioxide is introduced at 0.5 vvm to 3.0 vvm.

8. A method for producing lysine, wherein a nitrogen source is added to the medium so that the pH of the medium, which has been reduced by automatic addition of carbon dioxide, can be increased in the fifth paragraph.

9. In paragraph 8, A method for producing lysine, wherein a nitrogen source is added simultaneously or sequentially with the addition of the carbon dioxide to the medium.

10. In paragraph 5, A method for producing lysine, wherein the pH of a medium is increased from pH 6.5 or more to pH 7.0 or less to pH 7.5 or more to pH 8.5 or less, and then carbon dioxide is added to adjust the pH of the medium to maintain the pH of the medium at pH 7.5 or more to pH 8.5 or less.

11. In paragraph 1, A method for producing lysine, wherein monitoring the concentration of ammonium nitrogen in a medium is performed using a near infrared (NIR) spectrometer.

12. In paragraph 1, A method for producing lysine, wherein the above medium contains ammonium sulfate as a nitrogen source.

13. In paragraph 1, A method for producing lysine, wherein ammonium sulfate is not additionally added to the medium during the above fermentation process.

Images