Method for preparing high-content lysine by using automatic gas introduction system

Abstract

The present disclosure relates to a method for preparing lysine, which monitors ammonium nitrogen and pH in a culture medium in real time, and introduces carbon dioxide and / or a nitrogen source accordingly, thereby significantly increasing the concentration of lysine while reducing the concentration of by-products, and improving fermentation purity and enhancing the quality of lysine particles.

Description

Technical Field

[0001] Cross-reference to related applications

[0002] This disclosure claims the benefit based on priority of Korean Patent Application No. 10-2024-0016196, filed on February 1, 2024, and all the contents disclosed in the relevant Korean patent application are incorporated herein as part of this disclosure.

[0003] Throughout this disclosure, numerous papers and patent documents are cited, and their citations are indicated. The contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which this disclosure pertains and the content of this disclosure.

[0004] This disclosure relates to a method for preparing lysine, and more specifically, to a method for preparing high-content lysine using an automated gas introduction system. Background Technology

[0005] Lysine (L-lysine) is an essential amino acid used in various fields, primarily as a feed additive, food additive, and pharmaceutical raw material. It is a major chemical, with a market size of 2.94 million tons as of 2022. Currently, most lysine is produced in large quantities through direct fermentation using a culture medium containing carbon sources (raw sugar, sucrose, glucose) and nitrogen sources (yeast extract, soybean meal hydrolysate, corn steep liquor).

[0006] In the ammonium sulfate added to the culture medium components for lysine fermentation using Corynebacterium-shaped microorganisms, the sulfate ion acts as an counterion to maintain neutral pH conditions for lysine biosynthesis during fermentation, while ammonium provides the nitrogen source required for lysine biosynthesis. Therefore, to exist in the culture medium as lysine sulfate and to commercialize it as lysine hydrochloride (Lysine HCl) (a representative powdered formulation component), an adsorption and elution method using ion exchange resins is necessary. However, the purification process and the addition of auxiliary materials required in this method significantly increase production costs.

[0007] To overcome the aforementioned problems, a fermentation method for removing ammonium sulfate added during fermentation has been developed (Chinese Patent Publication No. 110484575). Corresponding research involves a method for preparing lysine containing carbonate, where the carbonate is generated during fermentation or supplied via an external carbonate aqueous solution and carbon dioxide gas. The production cost of the fermentation solution obtained by this method can be significantly reduced by introducing granular formulations (Korean Patent No. 10-0838200). Granular products have advantages such as low hygroscopicity and high flowability; by diversifying the formulation, process problems can be overcome, and cost competitiveness can be significantly improved. However, when granulation is performed during the fermentation preparation of lysine carbonate without ion exchange resin treatment, other byproducts generated during fermentation besides lysine are the main cause of reduced product purity. Therefore, it is necessary to reduce organic acid and amino acid byproducts and increase product yield. Summary of the Invention

[0008] Technical issues

[0009] The purpose of this disclosure is to provide a method for preparing high-content lysine using an automated gas introduction system.

[0010] The inventors of this disclosure have demonstrated that, during the fermentation process using microorganisms capable of producing lysine, by monitoring the concentration of ammonium nitrogen (hereinafter also referred to as "AN") in the culture medium, the concentration of lysine is significantly increased while the concentration of byproducts decreases, thereby improving the fermentation purity and the quality of lysine particles. Furthermore, when the pH conditions reach a specific range and the ammonium nitrogen decreases to a specific value or less, a nitrogen source and / or carbon dioxide are introduced into the culture medium to control the ammonium nitrogen within a suitable range, thus completing the invention of the method for preparing lysine disclosed herein.

[0011] Technical solution

[0012] A detailed explanation follows. On the other hand, the various descriptions and embodiments disclosed in this application can also be applied to other various descriptions and embodiments. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application should not be considered limited to the specific descriptions below. In addition, numerous papers and patent documents are cited throughout this specification, and their citations are indicated. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which this disclosure pertains and the content of this disclosure.

[0013] According to one aspect of this disclosure, this disclosure provides a method for preparing lysine using microorganisms capable of producing lysine through a fermentation process, characterized in that...

[0014] Monitor the concentration of ammonium nitrogen in the culture medium, and

[0015] Introduce a nitrogen source into the culture medium.

[0016] The inventors of this disclosure have demonstrated that in the method for preparing lysine using microorganisms capable of producing lysine through a fermentation process, by monitoring the concentration of ammonium nitrogen (hereinafter also referred to as "AN") in the culture medium, the concentration of lysine is significantly increased while the concentration of byproducts is reduced, thereby improving the fermentation purity and the quality of lysine particles. Furthermore, when the pH conditions reach a specific range and the ammonium nitrogen decreases to a specific value or less, a nitrogen source and / or carbon dioxide are introduced into the culture medium to control the ammonium nitrogen within a suitable range, thus completing the invention of the method for preparing lysine disclosed herein.

[0017] In this disclosure, nitrogen sources and / or carbon dioxide can be automatically introduced into the culture medium. More specifically, nitrogen sources and / or carbon dioxide can be automatically introduced into the culture medium via an automatic gas introduction system.

[0018] In this disclosure, the "automatic gas introduction system" refers to the system that, during the fermentation process using microorganisms capable of producing lysine, monitors in real time the concentration of ammonium nitrogen and / or the pH of the culture medium, and automatically introduces ammonia gas and / or ammonia solution as a nitrogen source under specific conditions, and / or automatically introduces carbon dioxide into the culture medium to adjust the ammonium nitrogen and / or pH within a suitable range. When using such an automatic gas introduction system, the concentration of byproducts decreases during cultivation, and the concentration of lysine is significantly increased; therefore, fermentation purity can be improved, and the quality of lysine particles can be enhanced.

[0019] The microorganisms capable of producing lysine used in the method for preparing lysine disclosed herein are not particularly limited; all microorganisms can be used as long as they are capable of producing lysine through fermentation. Examples of such microorganisms include corynebacterial bacteria and bacteria belonging to the genera *Escherichia*, *Serratia*, *Bacillus*, etc. While corynebacterial bacteria and bacteria belonging to the genus *Escherichia* are described below, the microorganisms used in the method of this disclosure are not limited to these bacteria.

[0020] Corynebacterial morphology bacteria (or, hereinafter, also referred to as "corynebacterial morphological microorganisms") are integrated into the genus *Corynebacterium*, and include bacteria belonging to the genus *Brevibacterium*, which is closely related to *Corynebacterium*. Examples of corynebacterial morphology bacteria are described below:

[0021] Corynebacterium acetoacidophilum

[0022] Corynebacterium acetoglutamicum

[0023] Corynebacterium alkanolyticum

[0024] Corynebacterium callunae

[0025] Corynebacterium glutamicum

[0026] Corynebacterium lilium (Corynebacterium glutamicum)

[0027] Corynebacterium melassecola

[0028] Corynebacterium thermoaminogenes

[0029] Corynebacterium herculis

[0030] Brevibacterium divaricatum

[0031] Brevibacterium flavum (Corynebacterium glutamicum)

[0032] Brevibacterium immariophilum

[0033] Brevibacterium lactofermentum (a short, lactic acid-fermenting bacterium)

[0034] (Corynebacterium glutamicum)),

[0035] Brevibacterium roseum

[0036] Brevibacterium saccharolyticum,

[0037] Brevibacterium thiogenitalis,

[0038] Brevibacterium album, white short bacillus

[0039] Brevibacterium cerinum,

[0040] Microbacterium ammoniaphilum.

[0041] Examples of bacteria belonging to the genus Escherichia include, but are not limited to, Escherichia coli.

[0042] Microorganisms capable of producing lysine can possess this capability naturally or be modified to achieve it. Lysine-producing microorganisms can be obtained by conferring or enhancing their lysine-producing capacity.

[0043] In one implementation, the microorganism capable of producing lysine may be Corynebacterium glutamicum, more specifically, it may be Corynebacterium glutamicum CJ3P, which has the ability to produce L-lysine by introducing three genes with conventionally known mutants pyc (P458S), hom (V59A), and lysC (T311I) using Corynebacterium glutamicum ATCC13032 as a parent strain (US 9556463 ​​B2), but is not limited thereto.

[0044] In this disclosure, the cultivation method is not limited, but can be carried out by, for example, batch culture, fed-batch culture, or continuous culture. In one specific example, in this disclosure, seed culture is carried out by batch culture, and master culture is carried out by batch culture and subsequent fed-batch culture, but is not limited thereto.

[0045] The culture medium used in this disclosure may be a liquid culture medium, and there are no particular limitations on the liquid culture medium, and it is any culture medium generally known to contain organic or inorganic nutrients such as carbon and nitrogen sources, as well as other micronutrients that may be used depending on the microorganisms used.

[0046] All carbon sources can be used as long as microorganisms can utilize them. For example, sugars (such as sucrose, glucose, fructose, molasses, and starch hydrolysates), organic acids (such as acetates), and alcohols (such as ethanol) can be mentioned. As nitrogen sources, inorganic substances (such as ammonium ions), protein hydrolysates, and yeast extracts can be mentioned. As micronutrients, amino acids, vitamins, and trace metals can be mentioned.

[0047] In addition, the pH of the culture medium can be adjusted appropriately using alkaline compounds (such as sodium hydroxide, potassium hydroxide, and ammonia) or acidic compounds (such as phosphoric acid or sulfuric acid). Furthermore, antifoaming agents such as fatty acid polyethylene glycol esters can be used to suppress bubble formation.

[0048] On the other hand, the culture temperature, which can be appropriately selected depending on the microorganisms used, is typically between 20 and 45°C, preferably between 25 and 40°C. Furthermore, thorough stirring and sufficient oxygen are provided during fermentation. To maintain an aerobic environment, oxygen or an oxygen-containing gas (such as air) is introduced into the culture.

[0049] In one embodiment, ammonium sulfate may be further included for use as a nitrogen source and counterion source in the culture medium. More specifically, ammonium sulfate may be included in the culture medium at the start of the culture, more specifically, at the initial moment of the culture. In ammonium sulfate, the sulfate ion acts as a counterion for lysine, and the ammonium acts as a nitrogen source required for lysine biosynthesis. This disclosure is characterized by the use of at least this type of ammonium sulfate. Specifically, the ammonium sulfate used in this disclosure may contain 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... 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) The values ​​are 0.45 to 0.6 mol / mol, 0.46 to 0.6 mol / mol, 0.2 to 0.5 mol / mol, 0.3 to 0.5 mol / mol, 0.4 to 0.5 mol / mol, 0.45 to 0.5 mol / mol, or 0.46 to 0.5 mol / mol, but not limited to these.Therefore, when using the method for preparing lysine disclosed herein, the high-cost method of adsorbing and eluting lysine via ion exchange resin processes is not used, which is essentially used when large amounts of ammonium sulfate are employed.

[0050] Specifically, in this disclosure, in order to slightly increase the concentration of ammonium nitrogen in the culture medium at the start of the culture, ammonium sulfate is included in the culture medium in the initial step of the culture (more specifically, at the start of the culture), and thereafter, ammonium sulfate is not added to the culture medium during the fermentation process (during the culture).

[0051] In one implementation, a near-infrared (NIR) spectrometer can be used to monitor the concentration of ammonium nitrogen in the culture medium.

[0052] NIR can simultaneously and continuously analyze various components in a short time without sample pretreatment. It can also be performed simultaneously using conventional wet methods (i.e., solid-liquid chromatography (HPLC) for analyzing sugars, amino acids, nucleic acids, etc.) or component-by-component analysis methods such as the Kjeldahl method for nitrogen component analysis, without sample pretreatment. Therefore, by directly connecting sensors or probes to the fermentation solution, real-time analysis becomes possible. This is a promising technology for real-time quantitative analysis, already being explored for various processes. It significantly reduces analysis time, cost, and labor, enabling rapid quantitative analysis within seconds or tens of seconds without the use of hazardous chemicals required for pretreatment.

[0053] In this disclosure, unlike the conventional Kjeldahl analysis method (which is cumbersome and time-consuming to analyze a portion of the fermentation solution obtained by sampling during culture), this NIR method allows for online, real-time analysis of the concentration of ammonium nitrogen (AN) in the culture medium.

[0054] Therefore, in a specific implementation, the concentration of ammonium nitrogen in the culture 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 spectroscopy analyzer.

[0055] As illustrated in the embodiments described below, the inventors of this disclosure have developed a model that can use NIR spectroscopy to monitor the concentration of ammonium nitrogen (AN) in real time with high precision.

[0056] In one embodiment, in the method for preparing lysine according to the present disclosure described above, the pH of the culture medium can also be monitored, and more specifically, the pH can be monitored in real time.

[0057] In one implementation, a pH meter can be used to measure and record the pH of the culture medium in real time.

[0058] In one embodiment, in the method for preparing lysine disclosed above, when the concentration of ammonium nitrogen in the culture medium is less than 1.5 g / kg, a nitrogen source can be introduced into the culture medium.

[0059] In this disclosure, when the concentration of ammonium nitrogen in the culture medium is less than 1.5 g / kg, it can refer to the case where the concentration of ammonium nitrogen in the culture 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 decimals less than 1.5 g / kg are also included within the scope of this invention.

[0060] In one embodiment, the nitrogen source may be at least one selected from the group consisting of ammonia gas and ammonia solution.

[0061] In one embodiment, in the method for preparing lysine according to the present disclosure described above, the concentration of ammonium nitrogen in the culture medium can be controlled to be maintained at 1.0 g / kg to 3.0 g / kg, more specifically, the concentration of ammonium nitrogen in the culture medium can be controlled to be maintained at 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, 1.5 g / kg to 1.75 g / kg, but not limited to these. More specifically, as described above, when the concentration of ammonium nitrogen in the culture medium is less than 1.5 g / kg, the concentration of ammonium nitrogen in the culture medium can be adjusted to maintain at 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, and 1.0 g / kg to 1.25 g / kg by adding a nitrogen source to the culture medium. The concentrations of ammonium nitrogen in the culture medium can be maintained at 1.0 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. By maintaining the concentration of ammonium nitrogen in the culture medium at 1.0 g / kg to 3.0 g / kg, the metabolic activity of microorganisms capable of producing lysine can be maintained, thereby increasing the fermentation rate and yield.

[0062] In this disclosure, introducing a nitrogen source into the culture medium may include automated nitrogen source introduction. Specifically, the nitrogen source may be introduced automatically via an HMI (Human-Machine Interface) automated program, but is not limited thereto.

[0063] In one embodiment, introducing a nitrogen source into the culture medium may include raising the pH set point of the culture medium and then introducing the nitrogen source into the culture medium such that the pH of the culture medium reaches the pH set point.

[0064] The pH set point can be increased through HMI (Human Machine Interface) automated programs, but is not limited to this.

[0065] In one embodiment, when the concentration of ammonium nitrogen in the culture medium is less than 1.5 g / kg, the pH set point can be slightly increased. More specifically, the pH set point can be increased by approximately 0.05, for example, 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.

[0066] More specifically, when the concentration of ammonium nitrogen in the culture medium is less than 1.5 g / kg, the pH set point can be adjusted up by about 0.05 through an automated HMI (human-machine interface) program. Furthermore, the concentration of ammonium nitrogen in the culture medium can be increased by introducing a nitrogen source, so that the pH of the culture medium can reach the pH set point.

[0067] In one implementation, the pH of the culture medium can also be monitored, and when the pH of the culture medium is pH 7.5 or higher and less than pH 8.0, and the concentration of ammonium nitrogen in the culture medium is less than 1.5 g / kg, the pH set point can be increased, and a nitrogen source can be introduced to achieve the pH set point.

[0068] In this disclosure, when the pH of the culture medium is pH 7.5 or higher and less than pH 8.0, it can refer to the following situations: pH 7.5 or higher and less than pH 8.0, pH 7.6 or higher and less than pH 8.0, pH 7.7 or higher and less than pH 8.0, pH 7.8 or higher and less than pH 8.0, pH 7.9 or higher and less than pH 8.0, pH 7.5 or higher and less than pH 7.9, pH 7.6 or higher and less than pH 7.9, pH 7.7 or higher and less than pH 7.9, or pH 7.8 or higher and less than pH 7.9, but is not limited to these situations.

[0069] In this disclosure, when the concentration of ammonium nitrogen is less than 1.5 g / kg as described above, the pH of the culture medium increases from pH 7.5 or higher and less than pH 8.0 to pH 8.0 or higher and less than pH 8.5 as the pH of the culture medium is gradually and slightly increased by adding a nitrogen source.

[0070] In this disclosure, when the pH of the culture medium is pH 8.0 or higher and less than pH 8.5, it can refer to the cases where the pH of the culture medium is pH 8.0 or higher and less than pH 8.5, pH 8.0 or higher and less than pH 8.4, pH 8.0 or higher and less than pH 8.3, pH 8.0 or higher and less than pH 8.2, or pH 8.0 or higher and less than pH 8.1, but is not limited thereto.

[0071] In one embodiment, the pH of the culture medium can also be monitored, and carbon dioxide can be introduced into the culture medium when the pH of the culture medium is pH 8.0 or higher and less than pH 8.5, and the concentration of ammonium nitrogen in the culture medium is less than 1.5 g / kg.

[0072] In this disclosure, introducing carbon dioxide into the culture medium can include automated carbon dioxide introduction. Specifically, carbon dioxide can be automatically introduced via an HMI (Human-Machine Interface) automation program, but is not limited thereto.

[0073] As mentioned above, when the pH of the culture medium increases to pH 8.0 or higher but less than pH 8.5, the fermentation rate and yield may decrease, and byproducts may be generated, as the growth and metabolism of microorganisms capable of producing lysine are inhibited by the increased pH. Therefore, when the pH of the culture medium is pH 8.0 or higher but less than pH 8.5, and the concentration of ammonium nitrogen in the culture medium is less than 1.5 g / kg, carbon dioxide can be introduced into the culture medium to lower its pH.

[0074] In one implementation, carbon dioxide may be introduced until the pH of the culture medium decreases by 0.1 to 0.4, more specifically, by 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, but is not limited thereto.

[0075] In one embodiment, carbon dioxide may be introduced at a rate of 0.5 vvm to 3.0 vvm. More specifically, those skilled in the art can appropriately adjust the gas flow rate when introducing carbon dioxide, for example, at a rate of 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 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 introduction rate L / culture medium volume L / min), but are not limited thereto.

[0076] In one embodiment, a nitrogen source can be introduced into the culture medium, thereby raising the pH of the medium that has decreased due to the introduction of carbon dioxide. More specifically, a nitrogen source can be introduced into the culture medium so that the pH of the medium, which has decreased due to the introduction of carbon dioxide, can return to a previously set pH set point. The previously set pH set point can be pH 8.0 or higher and lower than pH 8.5. The nitrogen source can be introduced simultaneously with or sequentially with the introduction of carbon dioxide into the culture medium. The method for introducing the nitrogen source, etc., is as described above.

[0077] In one implementation, when the pH of the culture medium is pH 8.0 or higher and less than pH 8.5, and the concentration of ammonium nitrogen in the culture medium is less than 1.5 g / kg, the process of introducing a nitrogen source simultaneously with or sequentially introducing carbon dioxide into the culture medium can be repeated. More specifically, when the pH of the culture medium is pH 8.0 or higher and less than pH 8.5, and the concentration of ammonium nitrogen in the culture medium is less than 1.5 g / kg, introducing carbon dioxide into the culture medium lowers the pH of the culture medium. When a nitrogen source is introduced into the culture medium such that the lowered pH of the culture medium reaches a previously set pH set point, the pH rises again to a range of pH 8.0 or higher and less than pH 8.5. In this way, when the pH reaches pH 8.0 or higher and less than pH 8.5, and as fermentation proceeds, the concentration of ammonium nitrogen in the culture medium again becomes less than 1.5 g / kg, the above process can be repeated. When the above process is repeated, the concentration of ammonium nitrogen in the culture medium can be maintained in the range of 1.0 g / kg or higher, specifically from 1.0 g / kg to 3.0 g / kg, and the pH of the culture medium can be controlled to be less than pH 8.5, specifically pH 7.5 or higher and less than pH 8.5.

[0078] In one embodiment, the pH of the culture medium can be increased from pH 6.5 or higher and less than pH 7.0 to pH 7.5 or higher and less than pH 8.5, and then carbon dioxide can be introduced to control the pH of the culture medium to be maintained at pH 7.5 or higher and less than pH 8.5.

[0079] In one specific implementation, the pH of the culture medium can be maintained within the range of pH 6.5 or higher and lower than pH 7.0 before the pH is increased from pH 6.5 or higher and lower than pH 7.0 to pH 7.5 or higher and lower than pH 8.5 as described above.

[0080] Specifically, at the start of fermentation, the pH of the culture 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 start of fermentation will gradually decrease due to the accumulation of organic acids and the emission of carbon dioxide gas as sugar is depleted. Therefore, 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 introducing ammonia gas and / or ammonia solution, more specifically, at pH 6.5, pH 6.6, pH 6.7, pH 6.8, or pH 6.9, but not limited thereto, and all decimals included in pH 6.5 or higher and less than pH 7.0 are included within the scope of this disclosure. Maintaining the pH of the culture medium at pH 6.5 or higher and less than pH 7.0 can be achieved by setting the pH setpoint to a value within the range of pH 6.5 or higher and less than pH 7.0 and introducing a nitrogen source, more specifically, ammonia gas and / or ammonia solution, through an HMI (Human Machine Interface) automated program to maintain the pH of the culture medium at the pH setpoint, but not limited thereto. "Fermentation start" can refer to the period from the beginning of fermentation until the carbon source in the initial fermentation medium is depleted, but it is not limited to this.

[0081] In one specific implementation, when the carbon source in the initial fermentation medium is depleted, a feed medium can be introduced, and the pH of the medium can be increased to pH 6.5 or higher and less than pH 7.0 to pH 7.5 or higher and less than pH 8.0. The pH increase can be to increase the nitrogen source supply after the addition of the feed medium. As described above, the pH increase can be a rate of 0.2 to 0.8 per hour from the set point, more specifically, at rates 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. Specifically, the pH of the medium can be increased at a constant rate. Specifically, increasing pH can be achieved by introducing a nitrogen source, or more specifically, by introducing ammonia gas and / or ammonia solution through an automated HMI (Human-Machine Interface) program, so that the pH of the culture medium can reach the pH set point, but is not limited to this.

[0082] When the pH of the culture medium reaches pH 7.5 or higher but less than pH 8.0 through the above process, and when the concentration of ammonium nitrogen in the culture medium becomes less than 1.5 g / kg, the process of introducing a nitrogen source into the culture medium as described above can be carried out. Through the above process, the pH of the culture medium can be raised to pH 8.0 or higher but less than pH 8.5. When the pH of the culture medium reaches pH 8.0 or higher but less than pH 8.5, and when the concentration of ammonium nitrogen in the culture medium becomes less than 1.5 g / kg, the process of introducing carbon dioxide and simultaneously or sequentially introducing a nitrogen source into it as described above can be carried out, and the pH of the culture medium can be controlled to be maintained at pH 7.5 or higher but less than pH 8.5.

[0083] In this way, the method for preparing lysine disclosed herein may include the following steps:

[0084] (a) Increase the pH of the culture medium from pH 6.5 or higher and less than pH 7.0 to pH 7.5 or higher and less than pH 8.0;

[0085] (b) When the pH of the culture medium is pH 7.5 or higher and less than pH 8.0, and the concentration of ammonium nitrogen in the culture medium is less than 1.5 g / kg, increase the pH set point and introduce a nitrogen source into the culture medium so that the pH of the culture medium can reach the pH set point.

[0086] (c) When the pH reaches pH 8.0 or higher but is lower than pH 8.5, carbon dioxide gas is introduced to lower the pH and a nitrogen source is introduced.

[0087] In one specific embodiment, prior to step (a), the method for preparing lysine disclosed herein may include maintaining the pH of the culture medium at a value in the range of pH 6.5 or higher and less than pH 7.0. In this case, the method for maintaining the pH of the culture medium, etc., is as described above.

[0088] In steps (a) and (b), the methods for increasing pH are described as described above.

[0089] In step (c), methods such as lowering pH and introducing a nitrogen source are described above.

[0090] In one specific implementation, step (c) can be repeated. Specifically, when the pH of the culture medium is pH 8.0 or higher and less than pH 8.5, and the concentration of ammonium nitrogen in the culture medium is less than 1.5 g / kg, carbon dioxide is automatically introduced into the culture medium to lower the pH. When a nitrogen source is automatically introduced into the culture medium, such that the pH value of the culture medium lowered due to the introduction of carbon dioxide can again reach the preset pH set point, the pH is raised again to pH 8.0 or higher and less than pH 8.5. In this way, when the pH reaches pH 8.0 or higher and less than pH 8.5, and as fermentation proceeds, the concentration of ammonium nitrogen in the culture medium becomes less than 1.5 g / kg again, the above process can be repeated. When the above process is repeated, the concentration of ammonium nitrogen in the culture medium can be maintained in the range of 1.0 g / kg or higher, specifically, 1.0 g / kg to 3.0 g / kg, and the pH of the culture medium can be controlled to be less than pH 8.5, specifically, pH 7.5 or higher and less than pH 8.5.

[0091] Therefore, according to this disclosure, the concentration of ammonium nitrogen in the culture medium and the pH of the culture medium are controlled within the optimal range for lysine biosynthesis. Thus, by reducing fermentation byproducts and increasing yield, the purity of the process solution can be improved, thereby significantly reducing production costs.

[0092] In this disclosure, “culture” refers to the process of proliferating a specific microorganism or cell under conditions that provide a suitable environment (nutrients, temperature, pH, oxygen concentration, etc.) or inducing it to produce a desired metabolite. Depending on the purpose of this disclosure, it may be used interchangeably with “fermentation”.

[0093] In this disclosure, culturing continues until the maximum amount of lysine is obtained. This can typically be carried out for 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, but is not limited thereto. Lysine may be released into the culture medium or contained within the cells.

[0094] Compared to culturing while maintaining the concentration of ammonium nitrogen in the culture medium at 1.0 g / kg to 3.0 g / kg by introducing a nitrogen source as disclosed herein, but without the carbon dioxide introduction process of this disclosure, the byproducts in the method for preparing lysine disclosed herein can be reduced to a level of about 30% to about 80%, more specifically, to 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 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%, but not limited thereto.

[0095] Compared to cultivation by introducing carbon dioxide as in this disclosure and introducing a nitrogen source according to this disclosure, but introducing the nitrogen source when the concentration of ammonium nitrogen in the culture medium becomes less than 0.3 g / kg instead of less than 1.5 g / kg, in the case of the method for preparing lysine of this disclosure, the byproducts can be reduced to a level of about 1% to about 30%, more specifically, to 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%, about 15% to about 30%, about 15% to about 20%, or about 15% to about 17.5%, but not limited thereto.

[0096] Compared to culturing without introducing carbon dioxide and without controlling the concentration of ammonium nitrogen in the culture medium (maintaining it to less than 1.5 g / kg), in the method for preparing lysine disclosed herein, the yield of lysine (the concentration of lysine produced) can be increased by about 5% to about 60%, more specifically, by 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%, and 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 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%, but not limited thereto.

[0097] Compared to culturing while maintaining the concentration of ammonium nitrogen in the culture medium at 1.0 g / kg to 3.0 g / kg by introducing a nitrogen source as disclosed herein, but without the carbon dioxide introduction process of this disclosure, the production of lysine (the concentration of lysine produced) can 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 not limited thereto.

[0098] Compared to cultivation by introducing carbon dioxide as in this disclosure and introducing a nitrogen source according to this disclosure, but introducing the nitrogen source when the concentration of ammonium nitrogen in the culture medium becomes less than 0.3 g / kg instead of less than 1.5 g / kg, in the case of the method for preparing lysine disclosed herein, the production of lysine (the concentration of lysine produced) can 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.

[0099] Compared to culturing without introducing carbon dioxide and without controlling the concentration of ammonium nitrogen in the culture medium (keeping it less than 1.5 g / kg), the purity of the lysine produced in the method of preparing lysine disclosed herein can 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 not limited thereto.

[0100] Compared to culturing while maintaining the concentration of ammonium nitrogen in the culture medium at 1.0 g / kg to 3.0 g / kg by introducing a nitrogen source as disclosed herein, but without the carbon dioxide introduction process of this disclosure, the purity of the lysine produced in the method of preparing lysine disclosed herein can be increased by 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 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%, but not limited thereto.

[0101] Compared to cultivation by introducing carbon dioxide as in this disclosure and introducing a nitrogen source according to this disclosure, but introducing the nitrogen source when the concentration of ammonium nitrogen in the culture medium becomes less than 0.3 g / kg instead of less than 1.5 g / kg, in the case of the method for preparing lysine of this disclosure, the purity of the produced lysine can 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 not limited thereto.

[0102] The term “about” includes, but is not limited to, all ranges including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and all values ​​in the range that are equivalent to or similar to the values ​​following the term “about”.

[0103] The method for producing lysine disclosed herein may further include recovering lysine from a culture medium (in which the culture is carried out) or from a Corynebacterium spp. microorganism. Recovery may also be included after culturing.

[0104] Recovery can be performed using suitable methods known in the art to collect the target amino acids according to the microbial culture methods disclosed herein, such as batch, continuous, or fed-batch culture methods. For example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatography methods (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC, or combinations of these methods can be used to recover the target amino acids from the culture medium or microorganisms using suitable methods known in the art.

[0105] Furthermore, the method for producing lysine disclosed herein may additionally include a purification step. Purification can be performed using suitable methods known in the art. In one embodiment, when the method for producing L-lysine disclosed herein includes a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously without regard to the order, or may be performed simultaneously or integrated into a single step, but are not limited thereto.

[0106] In one embodiment, the lysine produced by the method can be obtained in the form of a fermentation solution containing lysine. This lysine-containing fermentation solution can be further subjected to decarboxylation and concentration processes, followed by granulation and drying, ultimately yielding high-lysine granules.

[0107] Beneficial effects

[0108] According to the method for preparing lysine disclosed herein, by supplying a nitrogen source with an ammonium nitrogen concentration of less than 1.5 g / kg, the ammonium nitrogen in the culture medium is controlled within the optimal range for lysine biosynthesis, thereby improving the purity of the process solution by reducing fermentation byproducts and increasing yield, thus significantly reducing production costs. Attached Figure Description

[0109] Figure 1 A process diagram relating to the real-time monitoring model based on near-infrared spectroscopy (NIR spectroscopy) used in this disclosure.

[0110] Figure 2 This is a graph showing the cross-validation results of the actual measured and predicted values ​​of ammonium nitrogen (AN). The x-axis represents the actual AN value (actual AN) analyzed from spectral samples collected by the NIR instrument using Kjeldahl analysis, and the y-axis represents the predicted AN value (predicted AN) corresponding to the actual AN value during preprocessing to create a calibration curve.

[0111] Figure 3 This is a diagram comparing the conventionally used lysine powder method and the lysine particle method used in this disclosure.

[0112] Figure 4 A schematic diagram of a cultivation process according to a specific embodiment of the present disclosure is shown.

[0113] Figure 5 This is a graph showing the change in the concentration of AN in the culture medium over time, confirming the method for preparing lysine according to Example 2, Comparative Example 1, and Comparative Example 2. Detailed Implementation

[0114] The present disclosure will be described in more detail below by way of examples, but these are merely illustrative and not intended to limit the scope of the disclosure. It will be apparent to those skilled in the art that modifications can be made to the examples described below without departing from the essential principles of the invention.

[0115] [Example]

[0116] Example 1) Method for creating a real-time monitoring model using NIR spectroscopy

[0117] Seed culture of Corynebacterium glutamicum CJ3P strain (US 9556463 ​​B2) (a Corynebacterium morphology microorganism with lysine production capacity) was carried out by solid plate culture and flask culture. The seed culture step was carried out in a 5L fermenter, and the main culture step was carried out in a 30L fermenter.

[0118] A 50 μL sample of Corynebacterium glutamicum CJ3P strain, stored in glycerol stock (GS) at 80°C, was taken out and inoculated into a solid plate medium prepared based on the following culture medium composition by streak plating and incubated at 30°C for approximately 24 hours.

[0119] To perform shake-flask culture, prepare 100 mL of liquid culture medium based on the following shake-flask culture medium composition and add it to a 500 mL shake flask containing a baffle. Sterilize the flask using a small autoclave at 121°C for 30 minutes. For shake-flask inoculation, perform the procedure on a clean bench. Using a disposable platinum loop (10 μL), appropriately remove colonies from the cultured solid plate and aseptically inoculate them into the shake-flask culture medium. Incubate the inoculated 500 mL shake flask at 30°C and 200 rpm for approximately 10 hours.

[0120] Using seeds obtained from shake-flask cultures, they were inoculated at a rate of 3.0% (v / v) into 5L fermenters containing a medium in which ammonium sulfate and phosphate were added at a molar ratio (target substance mol / glucose mol) of 0.46 and 0.03 based on the glucose used in the seed culture composition. Culture was carried out in batches for approximately 20 hours at pH 6.5–8.5, agitation speed of 450 rpm, and gas flow rate of 1 vvm. pH was measured in real time using a pH meter (InPro3253i / SG / 120, Mettler Toledo). When the pH decreased by 0.05 to 0.1 within the pH range of 6.5 to 8.5, ammonia gas and / or ammonia solution as a nitrogen source was introduced via an automated HMI (Hyperson-Machine Interface) program (AVEVA InTouch HMI of AVEVA) to maintain the pH within the appropriate range of pH 6.5 to 8.5. Samples were taken at intervals of approximately 2–3 hours, and the culture was terminated when residual sugars were depleted.

[0121] Using seeds obtained from the seed culture step, they were inoculated at a rate of 20.0% (v / v) into a 30L fermenter containing a medium in which ammonium sulfate and phosphate were added at a molar ratio of 0.46 and 0.03 based on glucose in the following main culture medium composition. In the main culture step, cultivation was carried out in fed-batch fermentation mode under conditions of pH 6.5–8.5, agitation speed of 450 rpm, and gas flow rate of 1 vvm. The pH was measured in real time with a pH meter using the same method as described above, and the pH was maintained within a suitable range of pH 6.5 to pH 8.5 by introducing ammonia gas and / or ammonia solution.

[0122] A small amount of fermentation broth was obtained from the fermenter by sampling, and the level of residual sugar was examined using a YSI2900 biochemical analyzer. To maintain the residual sugar at a level of 0.5 wt% to 1.0 wt%, a feed medium containing 40.0 wt% glucose was introduced from the feed tank at a variable flow rate when the initial carbon source of the medium was depleted. Samples were obtained at intervals of approximately 2 to 3 hours, and when the ammonium nitrogen concentration (AN: ammonium nitrogen, g / kg) was measured by a Kjeldahl analyzer (Foss Kjeltec 8400) and the value was less than 1.5 g / kg, the nitrogen source was maintained constant by introducing ammonia gas and / or ammonia solution.

[0123] Using AN values ​​and corresponding NIR spectral data from fermentation broth samples collected at 2- to 3-hour intervals, a calibration model was developed using OPUS software (Bruker) through simultaneous NIR spectral data obtained from sampling at 2- to 3-hour intervals. A Bruker Matrix-F NIR spectrometer was used, with wavelengths ranging from 3995 to 11987 cm⁻¹. -1 Near-infrared spectra were analyzed at a scan rate of 40 kHz. For the preprocessing technique used to generate the calibration model, the vector method (i.e., the first derivative) was selected and used among various methods. The probe conditions for real-time monitoring were a resolution of 16 cm⁻¹. -1 The scan time was 32 scans, the preamplifier was in Gain A mode, and the cross-validation results comparing the predicted values ​​and the actual measured values ​​are shown in Table 1 and 2. Figure 2 As shown, and highly positively correlated (R 2 >0.99), confirming the values ​​of RMSECV (Root Mean Square Error of Cross-Validation) of 0.06 and RPD (Residual Predictive Deviation) of 10.1.

[0124] The formula for the calibration curve is as follows.

[0125] y = 0.9854x + 0.0264

[0126] 1) Components of solid plate culture medium

[0127] 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

[0128] 2) Shake flask culture medium components

[0129] 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

[0130] 3) Seed culture medium composition for fermenter (5L)

[0131] Defoamer 1 mL / L, corn steep liquor 10.0 g / L, biotin 1.0 mg / L, thiamine 10.0 mg / L, pantothenic acid 10.0 mg / L, nicotinamide 10.0 mg / L

[0132] 4) Main culture medium components for fermenter (30L)

[0133] Defoamer 1 mg / L, corn steep liquor 10.0 g / L, biotin 1.0 mg / L, thiamine 10.0 mg / L, pantothenic acid 10.0 mg / L, nicotinamide 10.0 mg / L

[0134] Table 1

[0135]

[0136] As mentioned above, a model can be created that can monitor the AN value in real time with high accuracy.

[0137] Example 2) Method for preparing lysine using automatic carbon dioxide gas introduction technology

[0138] Seeds obtained using the same method as the seed culture steps in Example 1 were inoculated at a rate of 20.0% (v / v) into a 30L fermenter containing a medium in which ammonium sulfate and phosphate were added at a molar ratio of 0.46 and 0.03 based on glucose in the main culture composition. In the main culture, the culture was carried out in fed-batch fermentation mode at pH 6.5 to pH 8.5, agitation speed of 450 rpm, and a gas flow rate of 1 vvm. pH was measured in real time using a pH meter (InPro3253i / SG / 120, Mettler Toledo). The main culture was carried out as follows, and a schematic diagram of the culture process is shown below. Figure 4 As shown.

[0139] 1) In batch fermentation, to maintain a near-neutral pH within the optimal pH range from the start of cultivation until the initial carbon source in the medium is depleted, the pH setpoint is set to pH 6.5 or higher but lower than pH 7.0. To maintain the pH at the setpoint, ammonia gas and / or ammonia solution are introduced as a nitrogen source via an automated HMI (Human-Machine Interface) program (AVEV's AAVEVA InTouchHMI). At this point, because the nitrogen source consumption exceeds its supply, the AN value gradually decreases. Figure 4 (Part ①).

[0140] 2) After the initial carbon source was depleted, a fed-batch culture medium containing 40.0 wt% glucose was added using the same method as in Example 1 to maintain the residual sugar at a level of 0.5 wt% to 1.0 wt%, and this process was carried out in a fed-batch fermentation mode. After adding the fed-batch culture medium, in order to increase the ammonia supply sufficient for lysine biosynthesis, the pH set point was increased at a rate of 0.2 to 0.8 / hour using an HMI automated program until it reached pH 7.5 or higher but less than pH 8.0. To maintain the pH of the culture medium at the set point, ammonia gas and / or ammonia solution were continuously introduced as a nitrogen source, thereby keeping the AN value within the optimal range for lysine biosynthesis (from 1.0 g / kg to 3.0 g / kg). At this point, due to the similarity between the ammonia supply and consumption, the AN value slowly decreased within the optimal range. Figure 4 (Part ②).

[0141] 3) From the moment the pH of the culture medium first reaches pH 7.5 or higher but less than pH 8.0, for more refined management of culture conditions, the following procedures are performed using the real-time AN ​​monitoring method with NIR spectroscopy established in Example 1. When the pH of the culture medium is pH 7.5 or higher but less than pH 8.0, and the AN value decreases to less than 1.5 g / kg, the fermentation pH setpoint is slightly increased (approximately 0.05), and to reach the setpoint, a nitrogen source (ammonia gas and / or ammonia solution) is automatically introduced to maintain the AN value within the range of 1.0 g / kg to 3.0 g / kg. Figure 4 (Part ③). Repeat the above process to maintain the AN value in the range of 1.0 g / kg to 3.0 g / kg. However, since the growth rate and metabolic efficiency of Corynebacterium microorganisms may decrease when the fermentation pH exceeds pH 8.0 or higher but is less than pH 8.5, repeat the process only until the fermentation pH reaches pH 8.0 or higher but less than pH 8.5. Introduce ammonia gas and / or ammonia solution via an HMI automation program, controlling the on / off time ratio (1 second / 10 seconds) until the current pH value reaches the pH set point.

[0142] 4) Starting when the pH of the culture medium first reaches pH 8.0 or higher but less than pH 8.5, and when the pH of the culture medium is pH 8.0 or higher but less than pH 8.5, and the AN value in the culture medium becomes less than 1.5 g / kg due to the lack of nitrogen source for lysine biosynthesis, carbon dioxide gas is automatically introduced to lower the pH using the real-time AN ​​monitoring method with NIR spectroscopy created in Example 1. Specifically, carbon dioxide gas is introduced at approximately 1.5 vvm (gas introduction rate L / culture medium volume L / min) via an HMI automation program to lower the pH of the culture medium by 0.1 to 0.4 (approximately 0.2). As the pH decreases in this manner, ammonia gas and / or ammonia solution as a nitrogen source are automatically introduced to allow the pH of the culture medium to reach the final pH set point preset in process 3), ensuring that the AN value remains within the range of 1.0 g / kg to 3.0 g / kg. After the above process, when the pH of the culture medium again reaches pH 8.0 or higher but less than pH 8.5, and the AN value again becomes less than 1.5 g / kg due to nitrogen source consumption caused by lysine biosynthesis, the process of introducing carbon dioxide gas to lower the pH and introducing ammonia gas and / or ammonia solution to restore the pH to the pre-set set point is performed as described above. In this way, the above process is repeated until the end of the culture to maintain AN within the range of 1.0 g / kg to 3.0 g / kg. Figure 4 (Part ④).

[0143] At the end of the culture, no residual sugar was found, and the fermentation time was 30.3 hours. During fermentation, the maximum pH that could be increased was less than pH 8.5 due to the automated carbon dioxide introduction system. Compared with the results of Comparative Example 2 described later, a 4.0% increase in lysine concentration and a 28.6% decrease in byproduct concentration were confirmed, thus confirming a 1.3% increase in the normalized purity obtained from the fermentation process.

[0144] Comparative Example 1) Method for preparing lysine with AN less than 1.5 g / kg

[0145] Seeds obtained using the same method as in the seed culture step of Example 1 were inoculated at a rate of 20.0% (v / v) into a 30 L fermenter containing a medium in which ammonium sulfate and phosphate were added at a molar ratio of 0.46 and 0.03 based on glucose in the main culture composition. In the main culture step, they were cultured in fed-batch fermentation mode at a stirring speed of 450 rpm and a gas flow rate of 1 vvm. When the initial carbon source of the medium was depleted, a feed medium containing 40.0 wt% glucose was added using the same method as in Example 1 to maintain residual sugars at a level of 0.5–1.0 wt%.

[0146] At the end of the culture, there was no residual sugar, but as Figure 5 As shown, due to the lack of control over the AN value, the AN value remained below 1.5 g / kg. Due to the delay, the fermentation time was 39.9 hours, and compared to the results of Comparative Example 2 described later, it can be confirmed that the lysine concentration decreased by 25.4% and the normalized purity decreased by 10.4%, thus showing a significant decrease.

[0147] Comparative Example 2) A method for preparing lysine using only ammonia without introducing carbon dioxide.

[0148] Seeds obtained using the same method as the seed culture steps in Example 1 were inoculated at a rate of 20.0% (v / v) into a 30L fermenter containing a medium in which ammonium sulfate and phosphate were added at a molar ratio of 0.46 and 0.03 based on glucose in the main culture composition. In the main culture step, they were cultured in fed-batch fermentation mode at pH 6.5–8.5, agitation speed of 450 rpm, and gas flow rate of 1 vvm. When the pH decreased by 0.05 to 0.1 within the pH range of 6.5 to 8.5, ammonia and / or ammonia solution were introduced as a nitrogen source via an HMI (Human-Machine Interface) automation program (AVEVA InTouchHMI of AVEVA) to maintain the pH within the appropriate range of pH 6.5 to 8.5.

[0149] In this comparative example, processes 1) to 3) as described in Example 2 were performed, but process 4) of Example 2 was not performed. This process involves automatically introducing carbon dioxide gas to lower the pH when the fermentation pH reaches its upper limit (pH 8.0 or higher and less than pH 8.5) and the AN value becomes less than 1.5 g / kg. As a result, the pH of the culture medium can be increased to pH 8.5 or higher.

[0150] 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.

[0151] Comparative Example 3) Method for preparing lysine with an AN level below 0.5 g / kg using an automated carbon dioxide gas introduction system.

[0152] Seeds obtained using the same method as the seed culture steps in Example 1 were inoculated at a rate of 20.0% (v / v) into a 30 L fermenter containing a medium in which ammonium sulfate and phosphate were added at a molar ratio of 0.46 and 0.03 based on glucose in the main culture composition. In the main culture step, they were cultured in fed-batch fermentation mode at pH 6.5–8.5, a stirring speed of 450 rpm, and a gas flow rate of 1 vvm.

[0153] In this comparative example, processes 1) to 3) as described in Example 2 were performed. However, in process 4) of Example 2, when the fermentation pH reached its upper limit (pH 8.0 or higher and less than pH 8.5) and the AN value became less than 1.5 g / kg, the pH was lowered by automatically introducing carbon dioxide gas. In this comparative example, when the fermentation pH reached its upper limit (pH 8.0 or higher and less than pH 8.5) and the AN value became less than 0.3 g / kg, carbon dioxide gas was automatically introduced to lower the pH of the culture medium by 0.1 to 0.4 (approximately 0.2). Additionally, ammonia gas and / or ammonia solution as a nitrogen source were automatically introduced to maintain the AN value within the range of less than 0.5 g / kg.

[0154] Because the supply of AN, the essential nitrogen source for lysine biosynthesis, was limited to below 0.5 g / kg, the lysine concentration was 164.1 g / L, a decrease of 18.0% compared to the results of Example 2 above. Furthermore, the normalized purity decreased significantly to 13.3% due to a 660.0% increase in byproducts. Additionally, the sugar consumption rate decreased sharply as fermentation progressed, resulting in a fermentation time of 31.7 hours and approximately 4.8 g / L of residual sugar remaining at the end of the culture.

[0155] The culture results of Example 2, Comparative Example 1, and Comparative Example 2 are shown in Table 2 below for comparison, while the culture results of Example 2 and Comparative Example 3 are shown in Table 3 below for comparison. The AN concentration in their culture media is as follows: Figure 5 As shown.

[0156] Table 2

[0157] project unit Example 2 Comparative Example 1 Comparative Example 2 Fermentation time Hour 30.3 39.9 28.7 Residual sugar g / L 0.0 0.0 0.0 Byproducts* ％ 71.4 - 100.0 Lysine g / L 200.0 143.5 192.4 purity* ％ 101.3 89.6 100.0

[0158] *Based on 100% standardized results from Comparative Example 2

[0159] Table 3

[0160] project unit Example 2 Comparative Example 3 Fermentation time Hour 30.3 31.7 Residual sugar g / L 0.0 4.8 Byproducts* ％ 100.0 660.0 Lysine g / L 200.0 164.1 purity* ％ 100.0 86.7

[0161] * 100% standardized result value based on the results of Example 2

[0162] Summarize the experimental results in Tables 2 and 3.

[0163] Example 2 used the real-time AN ​​monitoring method established in Example 1 in the main culture step. When the AN concentration fell below 1.5 g / kg during fermentation, the fermentation pH was gradually increased to a maximum range of pH 8.0 or higher but less than pH 8.5 to supply nitrogen. Once the culture medium pH reached pH 8.0 or higher but less than pH 8.5, carbon dioxide gas and ammonia gas and / or ammonia solution were introduced to maintain the fermentation pH at pH 7.5 or higher but less than pH 8.5. Therefore, compared to the results of Comparative Example 2, a 28.6% reduction in standardized byproducts and a 4.0% increase in lysine were observed at the same fermentation duration, resulting in a 1.3% increase in the purity of the final standardized process solution.

[0164] Comparative Example 1 involved lysine fermentation without controlling the AN (acetic acid) level during the main culture step according to the real-time AN ​​monitoring method established in Example 1, and without controlling the AN value. Therefore, AN remained less than 1.5 g / kg for most of the fermentation time in fed-batch mode. Due to the lack of control over AN, the most crucial factor in lysine production, a fermentation time delay of approximately 39.0% and a 25.4% reduction in lysine production were observed compared to Comparative Example 2.

[0165] In Comparative Example 3, the real-time AN ​​monitoring method established in Example 1 was applied during the main culture step. When the AN level fell below 0.3 g / kg during fermentation, the fermentation pH was gradually increased to a maximum range of pH 8.0 or higher but less than pH 8.5 to supply the nitrogen source. Once this range was reached, carbon dioxide gas, ammonia gas, and / or ammonia solution were introduced to maintain the fermentation pH at a range of pH 7.5 or higher but less than pH 8.5. Therefore, compared to the results of Example 2, the lysine concentration was reduced by 18.0% to 164.1 g / L due to limiting the maximum AN level to 0.5 g / kg. Furthermore, a significant reduction was confirmed by a substantial decrease in normalized purity of 13.3% due to a 660.0% increase in byproducts. Additionally, the sugar consumption rate decreased sharply as fermentation progressed, resulting in a fermentation time of 31.7 hours and approximately 4.8 g / L of residual sugar remaining at the end of the culture.

[0166] These results indicate that maintaining AN levels below 0.5 g / kg during fermentation has a negative impact on fermentation parameters. It was observed that when AN levels drop below 1.5 g / kg, the addition of a nitrogen source and / or carbon dioxide to the culture medium to maintain AN within an appropriate range of 1.0 g / kg to less than 3.0 g / kg is necessary for lysine production.

[0167] The ion exchange resin process significantly increases production costs due to the added purification process and the introduction of auxiliary raw materials. Furthermore, in the preparation of lysine granules using lysine carbonate, to improve the purity of the final product, it is necessary to reduce and increase the concentration of byproducts in the resulting fermentation solution. In lysine granulation methods without a separate ion exchange resin process, there is a problem of decreased product purity due to byproducts from the fermentation solution. However, when using the carbon dioxide fermentation method incorporating this technology, the concentration of byproducts decreases and the concentration of lysine significantly increases during cultivation. Therefore, fermentation purity can be improved, and the quality of the granule product can be enhanced.

Claims

1. A method for preparing lysine using microorganisms capable of producing lysine through a fermentation process, characterized in that... Monitor the concentration of ammonium nitrogen in the culture medium, and When the concentration of ammonium nitrogen in the culture medium is less than 1.5 g / kg, a nitrogen source is introduced into the culture medium.

2. The method for preparing lysine according to claim 1, wherein the concentration of ammonium nitrogen in the culture medium is controlled to be maintained at 1.0 g / kg to 3.0 g / kg.

3. The method for preparing lysine according to claim 1, wherein the nitrogen source is at least one selected from the group consisting of ammonia gas and ammonia solution.

4. The method for preparing lysine according to claim 1, wherein the pH of the culture medium is further monitored, and When the pH of the culture medium is 7.5 or higher and less than 8.0, and the concentration of ammonium nitrogen in the culture medium is less than 1.5 g / kg, Increase the pH set point, and The nitrogen source is introduced into the culture medium so that the pH of the culture medium can reach the pH set point.

5. The method for preparing lysine according to claim 1, wherein the pH of the culture medium is further monitored, and When the pH of the culture medium is pH 8.0 or higher and less than pH 8.5, and the concentration of ammonium nitrogen in the culture medium is less than 1.5 g / kg, Nitrogen dioxide is introduced into the culture medium.

6. The method for preparing lysine according to claim 5, wherein the carbon dioxide is introduced until the pH of the culture medium decreases by 0.1 to 0.

4.

7. The method for preparing lysine according to claim 5, wherein the carbon dioxide is introduced at a concentration of 0.5 vvm to 3.0 vvm.

8. The method for preparing lysine according to claim 5, wherein the nitrogen source is introduced into the culture medium, thereby increasing the pH of the culture medium, which has decreased due to the introduction of carbon dioxide.

9. The method for preparing lysine according to claim 8, wherein the nitrogen source is introduced into the culture medium simultaneously with or sequentially with the carbon dioxide.

10. The method for preparing lysine according to claim 5, wherein the pH of the culture medium is increased from pH 6.5 or higher and less than pH 7.0 to pH 7.5 or higher and less than pH 8.5, and then carbon dioxide is introduced to control the pH of the culture medium to be maintained at pH 7.5 or higher and less than pH 8.

5.

11. The method for preparing lysine according to claim 1, wherein the concentration of ammonium nitrogen in the culture medium is monitored using a near-infrared (NIR) spectrometer.

12. The method for preparing lysine according to claim 1, wherein the culture medium contains ammonium sulfate as a nitrogen source.

13. The method for preparing lysine according to claim 1, wherein ammonium sulfate is not added to the culture medium during the fermentation process.

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